Widespread enthusiasm about potential contributions of genome-edited crops to address climate change, food security, nutrition and health, environmental sustainability and diversification of agriculture is dampened by concerns about the associated risks.
Analysis of the top 7 risks of genome-edited crops finds that the scientific risks are comparable to those of accepted, past and current breeding methods, but failure to address regulatory, legal and trade framework, and the granting of social license, squanders the potential benefits.
…Here we focus on site-directed nuclease type 1 and 2 (SDN1 and SDN2)11,15-edited varieties. SDN1 produces a double-stranded DNA break that is repaired via nonhomologous end joining, which randomly deletes or adds nucleotides, often causing a frameshift mutation. In SDN2, the double-stranded break is repaired by homologous recombination, which uses a synthetic DNA template to add, delete or replace specific nucleotides. By contrast, SDN3 introduces a gene segment, or whole gene(s) at a specific site in the genome using homologous recombination, which could result in a transgenic product depending on the nature and origin of the introduced segment. CGIAR and its partners focus on SDN1 and SDN2 edits to address issues such as climate resilience in rice; disease resistance in banana, maize, potato, rice, wheat and yam; and nutrition improvement and consumer and environmental safety traits in cassava (Figure 1). Additional traits where CGIAR envisions using genome editing include brown streak virus resistance and haploid induction in cassava; nutritional quality and digestibility in bean; Striga resistance in sorghum; low phytate and high provitamin A in maize; reduced acrylamide, phytate and polyphenol oxidase in wheat; reduced aflatoxin in groundnut; delayed flour rancidity in pearl millet; reduced glycaemic index and apomixis in rice; and heat tolerance and apomixis in potato.
The daring Chinese biophysicist [He Jiankui] who created the world’s first gene-edited children has been set free after 3 years in a Chinese prison…Following international condemnation of the experiment, He was placed under home arrest and then detained. In December 2019, he was convicted by a Chinese court, which said the researcher had “deliberately violated” medical regulations and had “rashly applied gene editing technology to human assisted reproductive medicine.”
His release from prison was confirmed by people familiar with the situation and He answered his mobile phone when contacted early today. “It’s not convenient to talk right now”, he said before hanging up…It’s unclear whether He has plans to return to scientific research in China or another country. People who know him have described the biophysicist, who was trained at Rice University and Stanford, as idealistic, naïve, and ambitious.
…He, who has a wife and children, paid a steep price. He was fired from his university job and spent time in a prison distant from his hometown in Shenzhen…“It is extraordinary and unusual that [He Jiankui] and some of his colleagues were imprisoned for this experiment”, says Eben Kirksey, an associate professor at the Alfred Deakin Institute, in Australia, and the author of The Mutant Project, a book about He’s experiment that includes interviews with some of the participants. “At the same time many of [his] international collaborators—like Michael Deem and John Zhang—were never sanctioned or formally censured for involvement. In many ways justice has not been served”, says Kirksey.
…In February, according to a news report in Nature, 2 senior Chinese bioethicists called on China’s government to create a research program to oversee the health of the CRISPR children. They classified the children as a “vulnerable group” and called for genetic analyses to determine whether their bodies contain genetic errors they could pass to future generations.
Sexual reproduction evolved 1–2 billion years ago and underlies the biodiversity of our planet. Nevertheless, devolution of sexual into asexual reproduction can occur across all phyla of the animal kingdom. The genetic basis for how parthenogenesis can arise is completely unknown. To understand the mechanism and benefits of parthenogenesis, we have sequenced the genome of the facultative parthenogen, Drosophila mercatorum, and compared its organisation and expression pattern during parthenogenetic or sexual reproduction. We identified three genes, desat2, Myc, and polo in parthenogenetic D. mercatorum that when mis-regulated in a non-parthenogenetic species, D. melanogaster, enable facultative parthenogenetic reproduction. This simple genetic switch leads us to propose that sporadic facultative parthenogenesis could evolve as an 9escape route9 preserving the genetic lineage in the face of sexual isolation.
Two prominent bioethicists in China are calling on the government to set up a research centre dedicated to ensuring the well-being of the first children born with edited genomes. Scientists have welcomed the discussion, but many are concerned that the pair’s approach would lead to unnecessary surveillance of the children.
The document—which Qiu and Lei have shared with various scientists, several Chinese ministries and to Nature, but which has not yet been published—states that the children need special protections because they’re a “vulnerable group”. Gene editing could have created errors in the children’s genomes, which could be passed to their children. They recommend regular sequencing of the children’s genomes to check for “abnormalities”, including conducting genetic tests of their embryos in the future.
Qiu and Ruipeng also recommend that He contribute to the children’s medical expenses, and take primary financial, moral and legal responsibility for their health and well-being, along with the Southern University of Science and Technology in Shenzhen, with which He was affiliated, and the government.
But Joy Zhang, a sociologist at the University of Kent in Canterbury, UK, says it is difficult for scientists to know what recommendations to make because there is almost no information about the children’s current condition, and the circumstances of their conception. “China has kept everything so tight”, she says.
…Eben Kirksey, a medical anthropologist at Alfred Deakin Institute in Melbourne, Australia, who has written a book on human genome-editing, agrees that He should shoulder some responsibility for the children. He promised that they would receive health insurance for the first 18 years of their lives, but because the twins were born prematurely, they were initially denied coverage, which He initially stepped in to pay, according to Kirksey’s investigations. He and the university should make good on promises of medical assistance, Kirksey says.
Mitochondria host key metabolic processes vital for cellular energy provision and are central to cell fate decisions. They are subjected to unique genetic control by both nuclear DNA and their own multi-copy genome—mitochondrial DNA (mtDNA). Mutations in mtDNA often lead to clinically heterogeneous, maternally inherited diseases that display different organ-specific presentation at any stage of life.
For a long time, genetic manipulation of mammalian mtDNA has posed a major challenge, impeding our ability to understand the basic mitochondrial biology and mechanisms underpinning mitochondrial disease. However, an important new tool for mtDNA mutagenesis has emerged recently, namely double-stranded DNA deaminase (DddA)-derived cytosine base editor (DdCBE).
Here, we test this emerging tool for in vivo use, by delivering DdCBEs into mouse heart using adeno-associated virus (AAV) vectors and show that it can install desired mtDNA edits in adult and neonatal mice.
This work provides proof-of-concept for use of DdCBEs to mutagenize mtDNA in vivo in post-mitotic tissues and provides crucial insights into potential translation to human somatic gene correction therapies to treat primary mitochondrial disease phenotypes.
Although great progress has been achieved regarding wheat genetic transformation technology in the past decade, genotype dependency, the most impactful factor in wheat genetic transformation, currently limits the capacity for wheat improvement by transgenic integration and genome-editing approaches. The application of regeneration-related genes during in vitro culture could potentially contribute to enhancement of plant transformation efficiency.
In the present study, we found that overexpression of the wheat gene TaWOX5 from the WUSCHEL family dramatically increases transformation efficiency with less genotype dependency than other methods. The expression of TaWOX5 in wheat calli prohibited neither shoot differentiation nor root development. Moreover, successfully transformed transgenic wheat plants can clearly be recognized based on a visible botanic phenotype, relatively wider flag leaves. Application of TaWOX5 improved wheat immature embryo transformation and regeneration.
Background: Sickle cell disease is characterized by the painful recurrence of vaso-occlusive events. Gene therapy with the use of LentiGlobin for sickle cell disease (bb1111; lovotibeglogene autotemcel) consists of autologous transplantation of hematopoietic stem and progenitor cells transduced with the BB305 lentiviral vector encoding a modified β-globin gene, which produces an antisickling hemoglobin, HbAT87Q.
Methods: In this ongoing phase 1–2 study, we optimized the treatment process in the initial 7 patients in Group A and 2 patients in Group B with sickle cell disease. Group C was established for the pivotal evaluation of LentiGlobin for sickle cell disease, and we adopted a more stringent inclusion criterion that required a minimum of 4 severe vaso-occlusive events in the 24 months before enrollment. In this unprespecified interim analysis, we evaluated the safety and efficacy of LentiGlobin in 35 patients enrolled in Group C. Included in this analysis was the number of severe vaso-occlusive events after LentiGlobin infusion among patients with at least 4 vaso-occlusive events in the 24 months before enrollment and with at least 6 months of follow-up.
Results: As of February 2021, cell collection had been initiated in 43 patients in Group C; 35 received a LentiGlobin infusion, with a median follow-up of 17.3 months (range, 3.7 to 37.6). Engraftment occurred in all 35 patients. The median total hemoglobin level increased from 8.5 g per deciliter at baseline to 11 g or more per deciliter from 6 months through 36 months after infusion. HbAT87Q contributed at least 40% of total hemoglobin and was distributed across a mean (±SD) of 85±8% of red cells. Hemolysis markers were reduced. Among the 25 patients who could be evaluated, all had resolution of severe vaso-occlusive events, as compared with a median of 3.5 events per year (range, 2.0 to 13.5) in the 24 months before enrollment. 3 patients had a nonserious adverse event related or possibly related to LentiGlobin that resolved within 1 week after onset. No cases of hematologic cancer were observed during up to 37.6 months of follow-up.
Conclusions: One-time treatment with LentiGlobin resulted in sustained production of HbAT87Q in most red cells, leading to reduced hemolysis and complete resolution of severe vaso-occlusive events. (Funded by Bluebird Bio; HGB-206 ClinicalTrials.gov number, NCT02140554.)
Animals are essential genetic tools in scientific research and global resources in agriculture. In both arenas, a single sex is often required in surplus. The ethical and financial burden of producing and culling animals of the undesired sex is considerable.
Using the mouse as a model, we develop a synthetic lethal, bicomponent CRISPR-Cas9 strategy that produces male-only or female-only litters with 100% efficiency. Strikingly, we observe a degree of litter size compensation relative to control matings, indicating that our system has the potential to increase the yield of the desired sex in comparison to standard breeding designs. The bicomponent system can also be repurposed to generate postnatal sex-specific phenotypes.
Our approach, harnessing the technological applications of CRISPR-Cas9, may be applicable to other vertebrate species, and provides strides towards ethical improvements for laboratory research and agriculture.
Standard lipid nanoparticles (LNPs) deliver gene editing cargoes to hepatocytes through receptor-mediated uptake via the low-density lipoprotein receptor (LDLR). Homozygous familial hypercholesterolemia (HoFH) is a morbid genetic disease characterized by complete or near-complete LDLR deficiency, markedly elevated blood low-density lipoprotein cholesterol (LDL-C) levels, and premature atherosclerotic cardiovascular disease. In order to enable in vivo liver gene editing in HoFH patients, we developed a novel LNP delivery technology that incorporates a targeting ligand—N-acetylgalactosamine (GalNAc)—which binds to the asialoglycoprotein receptor (ASGPR). In a cynomolgus monkey (Macaca fascicularis) non-human primate (NHP) model of HoFH created by somatic knockout of the LDLR gene via CRISPR-Cas9, treatment with GalNAc-LNPs formulated with an adenine base editor mRNA and a guide RNA (gRNA) targeting the ANGPTL3 gene yielded ~60% whole-liver editing and ~94% reduction of blood ANGPTL3 protein levels, whereas standard LNPs yielded minimal editing. Moreover, in wild-type NHPs, the editing achieved by GalNAc-LNPs compared favorably to that achieved by standard LNPs, suggesting that GalNAc-LNP delivery technology may prove useful across a range of in vivo therapeutic applications targeting the liver.
Measurements of gene expression and signal transduction activity are conventionally performed with methods that require either the destruction or live imaging of a biological sample within the timeframe of interest.
Here we demonstrate an alternative paradigm, termed ENGRAM (ENhancer-driven Genomic Recording of transcriptional Activity in Multiplex), in which the activity and dynamics of multiple transcriptional reporters are stably recorded to DNA. ENGRAM is based on the prime editing-mediated insertion of signal-specific or enhancer-specific barcodes to a genomically encoded recording unit.
We show how this strategy can be used to concurrently genomically record the relative activity of at least hundreds of enhancers with high fidelity, sensitivity and reproducibility. Leveraging synthetic enhancers that are responsive to specific signal transduction pathways, we further demonstrate time-dependent and concentration-dependent genomic recording of Wnt, NF-κB, and Tet-On activity.
Finally, by coupling ENGRAM to sequential genome editing, we show how serially occurring molecular events can potentially be ordered.
Looking forward, we envision that multiplex, ENGRAM-based recording of the strength, duration and order of enhancer and signal transduction activities has broad potential for application in functional genomics, developmental biology and neuroscience.
DNA is naturally well-suited to serve as a digital medium for in vivo molecular recording. However, DNA-based memory devices described to date are constrained in terms of the number of distinct signals that can be concurrently recorded and/or by a failure to capture the precise order of recorded events.
Here we describe DNA Ticker Tape, a general system for in vivo molecular recording that largely overcomes these limitations. Blank DNA Ticker Tape consists of a tandem array of partial CRISPR-Cas9 target sites, with all but the first site truncated at their 59 ends, and therefore inactive. Signals of interest are coupled to the expression of specific prime editing guide RNAs. Editing events are insertional, and record the identity of the guide RNA mediating the insertion while also shifting the position of the “write head” by one unit along the tandem array, i.e. sequential genome editing.
In this proof-of-concept of DNA Ticker Tape, we demonstrate the recording and decoding of complex event histories or short text messages; evaluate the performance of dozens of orthogonal tapes; and construct “long tape” potentially capable of recording the order of as many as 20 serial events. Finally, we demonstrate how DNA Ticker Tape simplifies the decoding of cell lineage histories.
Programmable and multiplexed genome integration of large, diverse DNA cargo independent of DNA repair remains an unsolved challenge of genome editing. Current gene integration approaches require double-strand breaks that evoke DNA damage responses and rely on repair pathways that are inactive in terminally differentiated cells. Furthermore, CRISPR-based approaches that bypass double stranded breaks, such as Prime editing, are limited to modification or insertion of short sequences. We present Programmable Addition via Site-specific Targeting Elements, or PASTE, which achieves efficient and versatile gene integration at diverse loci by directing insertion with a CRISPR-Cas9 nickase fused to both a reverse transcriptase and serine integrase. Without generating double stranded breaks, we demonstrate integration of sequences as large as ~36 kb with rates between 10–50% at multiple genomic loci across three human cell lines, primary T cells, and quiescent non-dividing primary human hepatocytes. To further improve PASTE, we discover thousands of novel serine integrases and cognate attachment sites from metagenomes and engineer active orthologs for high-efficiency integration using PASTE. We apply PASTE to fluorescent tagging of proteins, integration of therapeutically relevant genes, and production and secretion of transgenes. Leveraging the orthogonality of serine integrases, we engineer PASTE for multiplexed gene integration, simultaneously integrating three different genes at three genomic loci. PASTE has editing efficiencies comparable to or better than those of homology directed repair or non-homologous end joining based integration, with activity in non-dividing cells and fewer detectable off-target events. For therapeutic applications, PASTE can be delivered as mRNA with synthetically modified guides to programmably direct insertion of DNA templates carried by AAV or adenoviral vectors. PASTE expands the capabilities of genome editing via drag-and-drop gene integration, offering a platform with wide applicability for research, cell engineering, and gene therapy.
Genomic insertions, duplications and insertion/deletions (indels), which account for ~14% of human pathogenic mutations, cannot be accurately or efficiently corrected by current gene-editing methods, especially those that involve larger alterations (>100 base pairs (bp)).
Here, we optimize prime editing (PE) tools for creating precise genomic deletions and direct the replacement of a genomic fragment ranging from ~1 kilobases (kb) to ~10 kb with a desired sequence (up to 60 bp) in the absence of an exogenous DNA template. By conjugating Cas9 nuclease to reverse transcriptase (PE-Cas9) and combining it with 2 PE guide RNAs (pegRNAs) targeting complementary DNA strands, we achieve precise and specific deletion and repair of target sequences via using this PE-Cas9-based deletion and repair (PEDAR) method.
PEDAR outperformed other genome-editing methods in a reporter system and at endogenous loci, efficiently creating large and precise genomic alterations. In a mouse model of tyrosinemia, PEDAR removed a 1.38-kb pathogenic insertion within the Fah gene and precisely repaired the deletion junction to restore FAH expression in liver.
Current methods to delete genomic sequences are based on clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 and pairs of single-guide RNAs (sgRNAs), but can be inefficient and imprecise, with errors including small indels as well as unintended large deletions and more complex rearrangements.
In the present study, we describe a prime editing-based method, PRIME-Del, which induces a deletion using a pair of prime editing sgRNAs (pegRNAs) that target opposite DNA strands, programming not only the sites that are nicked but also the outcome of the repair. PRIME-Del achieves markedly higher precision than CRISPR-Cas9 and sgRNA pairs in programming deletions up to 10 kb, with 1–30% editing efficiency. PRIME-Del can also be used to couple genomic deletions with short insertions, enabling deletions with junctions that do not fall at protospacer-adjacent motif sites. Finally, extended expression of prime editing components can substantially enhance efficiency without compromising precision.
We anticipate that PRIME-Del will be broadly useful for precise, flexible programming of genomic deletions, epitope tagging and, potentially, programming genomic rearrangements.
So surgeons at N.Y.U. Langone Health took an astonishing step: With the family’s consent, they attached the pig’s kidney to a brain-dead patient who was kept alive on a ventilator, and then followed the body’s response while taking measures of the kidney’s function. It is the first operation of its kind. The researchers tracked the results for just 54 hours, and many questions remained to be answered about the long-term consequences of such an operation. The procedure will not be available to patients any time soon, as there are substantial medical and regulatory hurdles to overcome.
Still, experts in the field hailed the surgery as a milestone. “This is a huge breakthrough”, said Dr. Dorry Segev, a professor of transplant surgery at Johns Hopkins School of Medicine who was not involved in the research. “It’s a big, big deal.”
A steady supply of organs from pigs—which could eventually include hearts, lungs and livers—would offer a lifeline to the more than 100,000 Americans currently on transplant waiting lists, including the 90,240 who need a kidney. 12 people on the waiting lists die each day.
…The transplanted kidney was obtained from a pig genetically engineered to grow an organ unlikely to be rejected by the human body. In a close approximation of an actual transplant procedure, the kidney was attached to blood vessels in the patient’s upper leg, outside the abdomen. The organ started functioning normally, making urine and the waste product creatinine “almost immediately”, according to Dr. Robert Montgomery, the director of the N.Y.U. Langone Transplant Institute, who performed the procedure in September.
…The group was involved in the selection and identification of the brain-dead patient receiving the experimental procedure. The patient was a registered organ donor, and because the organs were not suitable for transplantation, the patient’s family agreed to permit research to test the experimental transplant procedure.
…The combination of 2 new technologies—gene editing and cloning—has yielded genetically altered pig organs. Pig hearts and kidneys have been transplanted successfully into monkeys and baboons, but safety concerns precluded their use in humans. “The field up to now has been stuck in the preclinical primate stage, because going from primate to living human is perceived as a big jump”, Dr. Montgomery said.
The kidney used in the new procedure was obtained by knocking out a pig gene that encodes a sugar molecule that elicits an aggressive human rejection response. The pig was genetically engineered by Revivicor and approved by the Food and Drug Administration for use as a source for human therapeutics.
Dr. Montgomery and his team also transplanted the pig’s thymus, a gland that is involved in the immune system, in an effort to ward off immune reactions to the kidney.
After attaching the kidney to blood vessels in the upper leg, the surgeons covered it with a protective shield so they could observe it and take tissue samples over the 54-hour study period. Urine and creatinine levels were normal, Dr. Montgomery and his colleagues found, and no signs of rejection were detected during more than 2 days of observation. “There didn’t seem to be any kind of incompatibility between the pig kidney and the human that would make it not work”, Dr. Montgomery said. “There wasn’t immediate rejection of the kidney.”
[Twitter] IscB proteins are putative nucleases encoded in a distinct family of IS200/IS605 transposons and are likely ancestors of the RNA-guided endonuclease Cas9, but the functions of IscB and its interactions with any RNA remain uncharacterized.
Using evolutionary analysis, RNA-seq, and biochemical experiments, we reconstruct the evolution of CRISPR-Cas9 systems from IS200/IS605 transposons. We show that IscB utilizes a single non-coding RNA for RNA-guided cleavage of double-stranded DNA and can be harnessed for genome editing in human cells. We also demonstrate the RNA-guided nuclease activity of TnpB, another IS200/605 transposon-encoded protein and the likely ancestor of Cas12 endonucleases.
This work reveals a widespread class of transposon-encoded RNA-guided nucleases, which we name OMEGA (Obligate Mobile Element Guided Activity), with strong potential for developing as biotechnologies.
Eukaryotic genomes contain domesticated genes from integrating viruses and mobile genetic elements. Among these are homologs of the capsid protein (known as Gag) of long terminal repeat (LTR) retrotransposons and retroviruses. We identified several mammalian Gag homologs that form virus-like particles and one LTR retrotransposon homolog, PEG10, that preferentially binds and facilitates vesicular secretion of its own messenger RNA (mRNA).
We showed that the mRNA cargo of PEG10 can be reprogrammed by flanking genes of interest with Peg10’s untranslated regions. Taking advantage of this reprogrammability, we developed selective endogenous encapsidation for cellular delivery (SEND) by engineering both mouse and human PEG10 to package, secrete, and deliver specific RNAs. Together, these results demonstrate that SEND is a modular platform suited for development as an efficient therapeutic delivery modality.
CRISPR-based gene-drives targeting the gene doublesex in the malaria vector Anopheles gambiae effectively suppressed the reproductive capability of mosquito populations reared in small laboratory cages. To bridge the gap between laboratory and the field, this gene-drive technology must be challenged with vector ecology.
Here we report the suppressive activity of the gene-drive in age-structured An. gambiae populations in large indoor cages that permit complex feeding and reproductive behaviours.
The gene-drive element spreads rapidly through the populations, fully suppresses the population within one year and without selecting for resistance to the gene drive. Approximate Bayesian computation allowed retrospective inference of life-history parameters from the large cages and a more accurate prediction of gene-drive behaviour under more ecologically-relevant settings.
Generating data to bridge laboratory and field studies for invasive technologies is challenging. Our study represents a paradigm for the stepwise and sound development of vector control tools based on gene-drive.
Here, we show that transgenic expression of the human RNA demethylaseFTO in rice caused a more than 3× increase in grain yield under greenhouse conditions. In field trials, transgenic expression of FTO in rice and potato caused ~50% increases in yield and biomass.
We demonstrate that the presence of FTO stimulates root meristem cell proliferation and tiller bud formation and promotes photosynthetic efficiency and drought tolerance but has no effect on mature cell size, shoot meristem cell proliferation, root diameter, plant height or ploidy. FTO mediates substantial m6A demethylation (around 7% of demethylation in poly(A) RNA and around 35% decrease of m6A in non-ribosomal nuclear RNA) in plant RNA, inducing chromatin openness and transcriptional activation.
Therefore, modulation of plant RNA m6A methylation is a promising strategy to dramatically improve plant growth and crop yield.
Genetic improvement in aquaculture species has a major role in global food security. Advances in biotechnology provide new opportunities to support aquaculture breeding.
Advances in biotechnology provide new opportunities to support aquaculture breeding.
Donor-derived gametes can be produced from surrogate broodstock of several aquaculture species.
Surrogate broodstock technology provides new opportunities for application of genome editing.
Surrogate broodstock can accelerate genetic gain, and improve dissemination of elite germplasm.
Aquaculture is playing an increasingly important role in meeting global demands for seafood, particularly in low and middle income countries. Genetic improvement of aquaculture species has major untapped potential to help achieve this, with selective breeding and genome editing offering exciting avenues to expedite this process. However, limitations to these breeding and editing approaches include long generation intervals of many fish species, alongside both technical and regulatory barriers to the application of genome editing in commercial production.
Surrogate broodstock technology facilitates the production of donor-derived gametes in surrogate parents, and comprises transplantation of germ cells of donors into sterilised recipients. There are many successful examples of intra-species and inter-species germ cell transfer and production of viable offspring in finfish, and this leads to new opportunities to address the aforementioned limitations.
Firstly, surrogate broodstock technology raises the opportunity to improve genome editing via the use of cultured germ cells, to reduce mosaicism and potentially enable in vivo CRISPR screens in the progeny of surrogate parents. Secondly, the technology has pertinent applications in preservation of aquatic genetic resources, and in facilitating breeding of high-value species which are otherwise difficult to rear in captivity. Thirdly, it holds potential to drastically reduce the effective generation interval in aquaculture breeding programmes, expediting the rate of genetic gain. Finally, it provides new opportunities for dissemination of tailored, potentially genome edited, production animals of high genetic merit for farming.
This review focuses on the state-of-the-art of surrogate broodstock technology, and discusses the next steps for its applications in research and production. The integration and synergy of genomics, genome editing, and reproductive technologies have exceptional potential to expedite genetic gain in aquaculture species in the coming decades.
Background: Transthyretin amyloidosis, also called ATTR amyloidosis, is a life-threatening disease characterized by progressive accumulation of misfolded transthyretin (TTR) protein in tissues, predominantly the nerves and heart. NTLA-2001 is an in vivo gene-editing therapeutic agent that is designed to treat ATTR amyloidosis by reducing the concentration of TTR in serum. It is based on the clustered regularly interspaced short palindromic repeats and associated Cas9 endonuclease (CRISPR-Cas9) system and comprises a lipid nanoparticle encapsulating messenger RNA for Cas9 protein and a single guide RNA targeting TTR.
Methods: After conducting preclinical in vitro and in vivo studies, we evaluated the safety and pharmacodynamic effects of single escalating doses of NTLA-2001 in 6 patients with hereditary ATTR amyloidosis with polyneuropathy, 3 in each of the 2 initial dose groups (0.1 mg per kilogram and 0.3 mg per kilogram), within an ongoing phase 1 clinical study.
Results: Preclinical studies showed durable knockout of TTR after a single dose. Serial assessments of safety during the first 28 days after infusion in patients revealed few adverse events, and those that did occur were mild in grade. Dose-dependent pharmacodynamic effects were observed. At day 28, the mean reduction from baseline in serum TTR protein concentration was 52% (range, 47 to 56) in the group that received a dose of 0.1 mg per kilogram and was 87% (range, 80 to 96) in the group that received a dose of 0.3 mg per kilogram.
Conclusions: In a small group of patients with hereditary ATTR amyloidosis with polyneuropathy, administration of NTLA-2001 was associated with only mild adverse events and led to decreases in serum TTR protein concentrations through targeted knockout of TTR. (Funded by Intellia Therapeutics and Regeneron Pharmaceuticals; ClinicalTrials.gov number, NCT04601051.)
Sickle cell disease (SCD) is caused by a mutation in the β-globin gene HBB.
We used a custom adenine base editor (ABE8e-NRCH) to convert the SCD allele (HBBS) into Makassar β-globin (HBBG), a non-pathogenic variant. Ex vivo delivery of mRNA encoding the base editor with a targeting guide RNA into haematopoietic stem and progenitor cells (HSPCs) from patients with SCD resulted in 80% conversion of HBBS to HBBG. 16 weeks after transplantation of edited human HSPCs into immunodeficient mice, the frequency of HBBG was 68% and hypoxia-induced sickling of bone marrow reticulocytes had decreased 5×, indicating durable gene editing.
To assess the physiological effects of HBBS base editing, we delivered ABE8e-NRCH and guide RNA into HSPCs from a humanized SCD mouse and then transplanted these cells into irradiated mice. After 16 weeks, Makassar β-globin represented 79% of β-globin protein in blood, and hypoxia-induced sickling was reduced 3×. Mice that received base-edited HSPCs showed near-normal haematological parameters and reduced splenic pathology compared to mice that received unedited cells. Secondary transplantation of edited bone marrow confirmed that the gene editing was durable in long-term haematopoietic stem cells and showed that HBBS-to-HBBG editing of 20% or more is sufficient for phenotypic rescue. Base editing of human HSPCs avoided the p53 activation and larger deletions that have been observed following Cas9 nuclease treatment.
These findings point towards an one-time autologous treatment for SCD that eliminates pathogenic HBBS, generates benign HBBG, and minimizes the undesired consequences of double-strand DNA breaks.
Late one evening last March, just before the coronavirus pandemic shut down the country, Mingzheng Wu, a graduate student at Northwestern University, plopped 2 male mice into a cage and watched as they explored their modest new digs: sniffing, digging, fighting a little.
With a few clicks on a nearby computer, Mr. Wu then switched on a blue light implanted in the front of each animal’s brain. That light activated a tiny piece of cortex, spurring neurons there to fire. Mr. Wu zapped the 2 mice at the same time and at the same rapid frequency—putting that portion of their brains quite literally in sync. Within a minute or 2, any animus between the 2 creatures seemed to disappear, and they clung to each other like long-lost friends. “After a few minutes, we saw that those animals actually stayed together, and one animal was grooming the other”, said Mr. Wu, who works in the neurobiology lab of Yevgenia Kozorovitskiy.
…The experiment, published this month in Nature Neuroscience, was made possible thanks to an impressive new wireless technology that allows scientists to observe—and manipulate—the brains of multiple animals as they interact with one another.
…Weizhe Hong didn’t know about any of these human studies when, a few years ago, his team stumbled upon the same sort of synchrony while recording from brain cells of interacting mice. “For about 6 months, we were very puzzled by it”, said Dr. Hong, a neuroscientist at the University of California Los Angeles. “I just found it too good to be true, too surprising to me.”
In most social interactions, after all, the 2 interacting animals aren’t doing the same thing at the same time; in a conversation, one person may listen while the other talks. So it did not immediately make sense to him why his mice would show such robust neural synchrony. But after digging into the scientific literature, he said, “I realized, oh actually, there’s 15 years of history of studying human synchrony.”
In their experiments, Dr. Hong’s team recorded this synchrony in a part of the brain called the medial prefrontal cortex, which had been linked to a range of social behaviors. Certain neurons in each animal’s brain seemed to encode the animal’s own behavior, whereas other cells’ activity correlated with the behavior of the other animal. There was some overlap between the 2 groups, suggesting that certain cells were responsive to both animals. These findings could be related to previous studies of “mirror neurons”, which fire when an animal acts or when it observes that action in another animal, although that link is far from clear, Dr. Hong said. “Whether they’re mirror neurons or not is definitely something we’re very interested in”, he added.
…The Northwestern researchers who carried out the new study in Nature Neuroscience were familiar with these human and animal experiments on interbrain synchrony. “It seemed interesting and a little bit strange”, Dr. Kozorovitskiy said. She thought the phenomenon could be further probed with a new tool they had developed to manipulate the brains—and activities—of animals.
Their tool involves optogenetics, a technique that uses a tiny LED light, implanted into an animal’s brain, to activate discrete groups of neurons. (A gene that encodes a light-sensitive protein derived from algae is first inserted into the neurons of interest, to make them responsive.)
But studying social behavior with optogenetics had historically been difficult because the light source was typically attached to the animal’s head through fiber-optic cables, which interfered with the animal’s normal behavior. So John Rogers, a biomedical engineer at Northwestern who specializes in bioelectronics, developed tiny wireless devices that, once implanted, can be controlled remotely by a nearby computer.
“Because everything is implanted, mice can behave naturally and they can socially interact with one another naturally”, Dr. Rogers said. “You don’t have the cables that get tangled up, and there’s no head-mounted gear” for the mice to gnaw on.
The tool also allowed researchers to independently control multiple devices—and multiple animals—at once. Dr. Rogers and Dr. Kozorovitskiy began looking for a way to test it. Dr. Kozorovitskiy had seen the Cell study showing that interacting mice produce synchronies in the medial prefrontal cortex. Perhaps, she thought, the optogenetic device could test the converse relationship: If 2 animals’ brains were synchronized, would the animals become more social?
The answer, as Mr. Wu discovered that late night last spring, was yes. The results may suggest that brain synchrony is a causal driver of social behavior—and is more than just a byproduct of brains performing similar activities, or thinking similar thoughts, in a shared environment.
Somewhere in Paris, in a white room, seated at a white table, a man wearing a headset reminiscent of those worn by VR gamers reached out with his right hand and placed his fingers on a black notebook. This simple motion, which he executed with confidence, was notable for one very important reason: The man had been blind for close to 4 decades.
What was different now was that as part of a clinical trial, genes had been injected into one of his eyes, causing neurons in the retina to produce a light-sensing protein normally found in the slimy bodies of green algae. When the black goggles he was wearing projected video images of his surroundings as a pulsed light beam onto those now-light-sensitive cells, the neurons fired, and the signal traveled up the optic nerve and into the visual processing center of the brain. The genetically modified neurons had become stand-ins for the photoreceptors he had lost many years before to a genetic disease called retinitis pigmentosa.
The man’s progress identifying objects inside the lab and out in the world were reported Monday in Nature Medicine. While he couldn’t see colors or fine details, the case study describes the first time optogenetic therapy successfully restored partial vision to a blind patient.
…For this study, researchers injected one eye of a 58-year old patient with an adenovirus-associated vector carrying the genetic instructions for a protein called ChrimsonR. When amber light strikes it, the protein shape-shifts, allowing ions to flow in and out of cells. The vector targeted retinal ganglion cells, which in a healthy eye, would gather signals from cones and rods and shuttle that information up to the brain’s visual cortex. Even in patients with advanced retinitis pigmentosa, these ganglion cells are still alive, but left idling without any information coming in. The addition of ChrimsonR allows them to sense light themselves.
Sahel and his collaborators had previously tried a different protein, one that is activated by blue-green light. And in mice it worked great. But that end of the visual spectrum is very energetic, and when they moved to testing in primate models, they encountered problems.
In a normal mammalian retina, one photo-sensing protein would activate another and another and another, resulting in an cascade that amplifies the signal. One protein can open up to 1 million ion channels. With optogenetics, one protein equals one channel, so scientists need to amplify the signal another way—by adding more light. That’s what the goggles are for. But too much blue-green light can be toxic to the remaining cells (the reason why you shouldn’t stare directly into the sun). By switching to ChrimsonR and amber light, the researchers were able to strike the right balance between effectiveness and safety.
…In addition to the notebook, the first patient was able to locate and count other objects, like cups and a small bottle of light green liquid. The bigger the objects and the higher the contrast, the more consistently he was able to spot them. The patient also reported being able to see crosswalks outside on the street and even count the number of white stripes. During the lab-testing portion of the study, the researchers used an EEG to record the neuronal activity across the man’s visual cortex, which suggested that the ChrimsonR activation was indeed propagating up to the brain.
None of these changes were immediate. It took 4 to 6 months post-injection for the proteins to be expressed in sufficient quantities, and a few months of training with the goggles for the patient to be able to orient the beam of light directly onto those protein-expressing cells in the retina. To locate the objects, the patient used his whole head to scan the area back and forth. And the vision that was returned to him was a grainy world of black and white contrast. To do things like read or recognize faces would require much higher resolution than what the optogenetic approach could provide.
Optogenetics may enable mutation-independent, circuit-specific restoration of neuronal function in neurological diseases. Retinitis pigmentosa is a neurodegenerative eye disease where loss of photoreceptors can lead to complete blindness.
In a blind patient, we combined intraocular injection of an adeno-associated viral vector encoding ChrimsonR with light stimulation via engineered goggles. The goggles detect local changes in light intensity and project corresponding light pulses onto the retina in real time to activate optogenetically transduced retinal ganglion cells.
The patient perceived, located, counted and touched different objects using the vector-treated eye alone while wearing the goggles. During visual perception, multichannel electroencephalographic recordings revealed object-related activity above the visual cortex. The patient could not visually detect any objects before injection with or without the goggles or after injection without the goggles.
This is the first reported case of partial functional recovery in a neurodegenerative disease after optogenetic therapy.
Gene-editing technologies, which include the CRISPR-Cas nucleases and CRISPR base editors, have the potential to permanently modify disease-causing genes in patients. The demonstration of durable editing in target organs of nonhuman primates is a key step before in vivo administration of gene editors to patients in clinical trials.
Here we demonstrate that CRISPR base editors that are delivered in vivo using lipid nanoparticles can efficiently and precisely modify disease-related genes in living cynomolgus monkeys (Macaca fascicularis).
We observed a near-complete knockdown of PCSK9 in the liver after a single infusion of lipid nanoparticles, with concomitant reductions in blood levels of PCSK9 and low-density lipoprotein cholesterol of approximately 90% and about 60%, respectively; all of these changes remained stable for at least 8 months after a single-dose treatment.
In addition to supporting a ‘once-and-done’ approach to the reduction of low-density lipoprotein cholesterol and the treatment of atherosclerotic cardiovascular disease (the leading cause of death worldwide), our results provide a proof-of-concept for how CRISPR base editors can be productively applied to make precise single-nucleotide changes in therapeutic target genes in the liver, and potentially in other organs.
We report a methodology for the pooled construction of mutants bearing precise genomic sequence variations and multiplex phenotypic characterization of these mutants using next-generation sequencing (NGS). Unlike existing techniques depending on CRISPR-Cas-directed genomic breaks for genome editing, this strategy instead uses single-stranded DNA produced by a retron element for recombineering. This enables libraries of millions of elements to be constructed and offers relaxed design constraints which permit natural DNA or random variation to be used as inputs.
Creating and characterizing individual genetic variants remains limited in scale, compared to the tremendous variation both existing in nature and envisioned by genome engineers. Here we introduce retron library recombineering (RLR), a methodology for high-throughput functional screens that surpasses the scale and specificity of CRISPR-Cas methods.
We use the targeted reverse-transcription activity of retrons to produce single-stranded DNA (ssDNA) in vivo, incorporating edits at >90% efficiency and enabling multiplexed applications. RLR simultaneously introduces many genomic variants, producing pooled and barcoded variant libraries addressable by targeted deep sequencing.
We use RLR for pooled phenotyping of synthesized antibiotic resistance alleles, demonstrating quantitative measurement of relative growth rates. We also perform RLR using the sheared genomic DNA of an evolved bacterium, experimentally querying millions of sequences for causal variants, demonstrating that RLR is uniquely suited to utilize large pools of natural variation.
Using ssDNA produced in vivo for pooled experiments presents avenues for exploring variation across the genome.
After a decade of fighting for regulatory approval and public acceptance, a biotechnology firm has released genetically engineered mosquitoes into the open air in the United States for the first time. The experiment, launched this week in the Florida Keys—over the objections of some local critics—tests a method for suppressing populations of wild Aedes aegypti mosquitoes, which can carry diseases such as Zika, dengue, chikungunya and yellow fever.
Oxitec, the firm based in Abingdon, UK, that developed the mosquitoes, has previously field-tested the insects in Brazil, Panama, the Cayman Islands and Malaysia.
…In late April of this year, project researchers placed boxes containing Oxitec’s mosquito eggs at 6 locations in 3 areas of the Keys. The first males are expected to emerge within the first 2 weeks of May. About 12,000 males will exit the boxes each week over the next 12 weeks. In a second phase later this year, intended to collect even more data, nearly 20 million mosquitoes will emerge over a period of about 16 weeks, according to Oxitec…The biotech firm plans to present the results to the US Environmental Protection Agency (EPA), which gave the green light for the trial. The data will help the EPA to determine whether Oxitec can release the mosquitoes more broadly in the United States. The company is still testing them in Brazil and other countries.
…Residential pushback: Opposition to the Florida field trial has been fierce from some residents in the Keys. Worried about being bitten by the mosquitoes or that the insects will disrupt the Florida ecosystem—and generally unhappy about being chosen as a test site—some have threatened to derail the experiments by spraying insecticides near the release points. “As you can imagine, emotions run high, and there are people who feel really strongly either for or against it”, says molecular biologist Natalie Kofler, who lectures at Harvard Medical School in Cambridge, Massachusetts, and is the founder of Editing Nature, an organization that advocates for responsible development and oversight of gene-editing technologies. “And I can see how, if you didn’t agree to this, it could be really concerning to have mosquitoes released in your neighborhood.”
CRISPRoff is a single fusion protein that programs heritable epigenetic memory
CRISPRoff can heritably silence most genes, including genes without CpG islands
CRISPRoff is highly specific and has a broad targeting window across gene promoters
CRISPRoff epigenetic memory persists through differentiation of iPSCs into neurons
A general approach for heritably altering gene expression has the potential to enable many discovery and therapeutic efforts. Here, we present CRISPRoff—a programmable epigenetic memory writer consisting of a single dead Cas9 fusion protein that establishes DNA methylation and repressive histone modifications.
Transient CRISPRoff expression initiates highly specific DNA methylation and gene repression that is maintained through cell division and differentiation of stem cells to neurons. Pairing CRISPRoff with genome-wide screens and analysis of chromatin marks establishes rules for heritable gene silencing. We identify single guide RNAs (sgRNAs) capable of silencing the large majority of genes including those lacking canonical CpG islands (CGIs) and reveal a wide targeting window extending beyond annotated CGIs.
The broad ability of CRISPRoff to initiate heritable gene silencing even outside of CGIs expands the canonical model of methylation-based silencing and enables diverse applications including genome-wide screens, multiplexed cell engineering, enhancer silencing, and mechanistic exploration of epigenetic inheritance.
[Keywords: CRISPR, epigenetics, DNA methylation, cell therapy, dCas9]
China’s new Criminal Code, which came into effect four weeks ago on March 1st, has a new section dedicated to ‘illegal medical practices’, which makes it a punishable crime to create gene-edited babies, human clones and animal-human chimeras.
The new section is an amendment to Article 336 of China’s Criminal Law, and officially outlaws “the implantation of genetically-edited or cloned human embryos into human or animal bodies, or the implantation of genetically edited or cloned animal embryos into human bodies”—with penalties ranging from fines to 7 years imprisonment.
…Although Dr He had been sentenced for genetically modifying human embryos, China’s previous criminal code on ‘illegal medical practices’, under which he was sentenced, was extremely vague on the gene-editing of human embryos, and was mostly used to prosecute providers of dangerous medical procedures, and not researchers. The only official Chinese Government legal document that made a stipulation against genetically altering human embryos at the time of Dr He’s sentencing was a scientifically-outdated 2003 guideline by the Chinese Ministry of Health, which mostly addressed ethical issues on human embryonic stem cell research. And thus due to this legal vagueness on human gene-editing, legal experts in China found the court sentencing of Dr He to be very problematic…The new addition to the criminal code is meant to clear up these questions.
Brain organoids with Neanderthal genes: The genomes of Neanderthals and modern humans are overall very similar. To understand the impact of genetic variants that are specific to modern humans, Trujillo et al performed a genome-wide analysis to identify 61 coding variants in protein-coding genes. Identifying the gene encoding the RNA-binding protein NOVA1 as a top candidate for functional analyses, they introduced the archaic gene variant into human pluripotent stem cells and generated brain organoids. These organoids showed alterations in gene expression and splicing as well as morphology and synaptogenesis, suggesting that this method could be used to explore other genetic changes that underlie the phenotypic traits separating our species from extinct relatives.
The evolutionarily conserved splicing regulator neuro-oncological ventral antigen 1 (NOVA1) plays a key role in neural development and function. NOVA1 also includes a protein-coding difference between the modern human genome and Neanderthal and Denisovan genomes. To investigate the functional importance of an amino acid change in humans, we reintroduced the archaic allele into human induced pluripotent cells using genome editing and then followed their neural development through cortical organoids. This modification promoted slower development and higher surface complexity in cortical organoids with the archaic version of NOVA1. Moreover, levels of synaptic markers and synaptic protein co-associations correlated with altered electrophysiological properties in organoids expressing the archaic variant. Our results suggest that the human-specific substitution in NOVA1, which is exclusive to modern humans since divergence from Neanderthals, may have had functional consequences for our species’ evolution.
Introduction: Current views of human evolution, as supported by the fossil record, indicate that many hominin lineage branches arose, but only one survived to the present. Neanderthals and Denisovans, two of these extinct lineages, are our closest evolutionary relatives and therefore provide the most subtle genetic and phenotypic contrast to our species. Comparison of Neanderthal, Denisovan, and extant human genomes has shown that many humans today carry genes introduced through past admixture events and has allowed enumeration of human-specific genetic differences that may have been important for recent human evolution. Neuro-oncological ventral antigen 1 (NOVA1) includes one of the few protein-coding differences between modern human and archaic hominin genomes that could affect human neurodevelopment.
Rationale: NOVA1 regulates alternative splicing in the developing nervous system and is a master regulator of splicing genes responsible for synapse formation. Altered NOVA1 splicing activity in humans is associated with neurological disorders, underscoring the role of NOVA1 in neural function. Using CRISPR-Cas9 genome-editing technology in human induced pluripotent stem cells (iPSCs), we replaced the modern human allele of the NOVA1 gene with the ancestral allele found in Neanderthals and Denisovans, which contains a single-nucleotide substitution at position 200 that causes an isoleucine-to-valine change. To investigate the functional importance of this amino acid change in humans, we followed iPSC neural development through functional cortical organoids.
Results: The reintroduction of the archaic version of NOVA1 into a human genetic background causes changes in alternative splicing in genes involved in neurodevelopment, proliferation, and synaptic connectivity. These changes co-occur with differences in organoid morphology and neural network function, suggesting a functional role for the derived human-specific substitution in NOVA1. Furthermore, cortical organoids carrying the archaic NOVA1 displayed distinct excitatory synaptic changes, which may have led to the observed alterations in neural network development. Collectively, our data suggest that expression of the archaic NOVA1 leads to modified synaptic protein interactions, affects glutamatergic signaling, underlies differences in neuronal connectivity, and promotes higher heterogeneity of neurons regarding their electrophysiological profiles.
Conclusion: A subset of genetic changes may underlie the phenotypic traits that separate our species from these extinct relatives. We developed a platform to test the impact of human-specific genetic variants by reintroducing the archaic form found in Neanderthals and Denisovans and measuring its effects during neurodevelopment using human brain organoids. Our results suggest that the human-specific substitution in NOVA1, which became fixed in modern humans after divergence from Neanderthals, may have had functional consequences for our species’ evolution.
Reactivating neural crest pluripotency: Cranial neural crest cells (CNCCs) are a transient cell group with an extraordinary differentiation potential that extends beyond its ectodermal lineage to form the majority of facial mesenchyme. Zalc et al identified a neuroepithelial precursor population that transiently reactivates pluripotency factors to generate CNCCs. The pluripotency factor Oct4 is required for the expansion of CNCC developmental potential to form facial mesenchyme. Analysis of the chromatin landscape of Oct4+ CNCC precursors showed that these cells resemble those of epiblast stem cells, with additional features suggestive of future priming for neural crest programs. Thus, to expand their cellular potency, CNCC precursors undergo a natural in vivo reprogramming event.
During development, cells progress from a pluripotent state to a more restricted fate within a particular germ layer. However, cranial neural crest cells (CNCCs), a transient cell population that generates most of the craniofacial skeleton, have much broader differentiation potential than their ectodermal lineage of origin. Here, we identify a neuroepithelial precursor population characterized by expression of canonical pluripotency transcription factors that gives rise to CNCCs and is essential for craniofacial development. Pluripotency factor Oct4 is transiently reactivated in CNCCs and is required for the subsequent formation of ectomesenchyme. Furthermore, open chromatin landscapes of Oct4+ CNCC precursors resemble those of epiblast stem cells, with additional features suggestive of priming for mesenchymal programs. We propose that CNCCs expand their developmental potential through a transient reacquisition of molecular signatures of pluripotency.
Introduction: Cell differentiation is classically described as an unidirectional process that progresses through a series of lineage restriction events, with cellular potential being increasingly reduced as the embryo develops, a concept famously illustrated by Conrad Waddington in his epigenetic landscape. However, the vertebrate-specific transient cell population called cranial neural crest cells (CNCCs) challenges this paradigm. Although they originate in the ectoderm and are capable of differentiating into cell types typical of this germ layer, CNCCs can also give rise to mesenchymal cell types canonically associated with the mesoderm lineage, such as bone, cartilage, and smooth muscle. How CNCCs expand their differentiation potential beyond their germ layer of origin remains unresolved.
Rationale: We hypothesized that unbiased analysis of transcriptional heterogeneity during the early stages of mammalian CNCC development may identify a precursor population and provide clues as to how these specialized cells gain their extraordinary differentiation potential. To test this, we combined single-cell RNA-sequencing analysis of murine CNCCs from staged mouse embryos with follow-up lineage-tracing, loss-of-function, and epigenomic-profiling experiments.
Results: We found that premigratory CNCCs are heterogeneous and carry positional information reflective of their origin in the neuroepithelium, but this early positional information is subsequently erased, with delaminating CNCCs showing a relatively uniform transcriptional signature that later rediversifies as CNCCs undergo first commitment events. We identify an early precursor population that expresses canonical pluripotency transcription factors and gives rise to CNCCs and craniofacial structures. Rather than being maintained from the epiblast, pluripotency factor Oct4 is transiently reactivated in the prospective CNCCs after head-fold formation, and its expression shifts from the most anterior to the more posterior part of the cranial domain as development progresses. Oct4 is not required for the induction of CNCCs in the neuroepithelium, but instead is important for the specification and survival of facial mesenchyme, thus directly linking this pluripotency factor with the expansion of CNCC cellular potential. Open chromatin landscapes of Oct4+ CNCC precursors are consistent with their neuroepithelial origin while also broadly resembling those of pluripotent epiblast stem cells. In addition, we saw priming of distal regulatory regions at a subset of loci associated with future neural crest migration and mesenchyme formation.
Conclusion: Our results show that premigratory CNCCs first form as a heterogeneous population that rapidly changes its transcriptional identity during delamination, resulting in the formation of a transcriptionally (and likely also functionally) equivalent cell group capable of adapting to future locations during and after migration. Such functional equivalency and plasticity of CNCCs is consistent with previous embryological studies. Furthermore, the demonstration that CNCC precursors transiently reactivate pluripotency factors suggests that these cells undergo a natural in vivo reprogramming event that allows them to climb uphill on Waddington’s epigenetic landscape. Indeed, our results show that at least one of the pluripotency factors, Oct4, is required for the expansion of CNCC developmental potential to include formation of facial mesenchyme. Whether this mechanism is specific to CNCCs and if such expansion of cellular plasticity could be harnessed for regenerative medicine purposes remain interesting questions for future investigations.
Hutchinson-Gilford progeria syndrome (HGPS or progeria) is typically caused by a dominant-negative C•G-to-T•A mutation (c).1824 C>T; p.G608G) in LMNA, the gene that encodes nuclear lamin A. This mutation causes RNA mis-splicing that produces progerin, a toxic protein that induces rapid ageing and shortens the lifespan of children with progeria to ~14 years. Adenine base editors (ABEs) convert targeted A•T base pairs to G•C base pairs with minimal by-products and without requiring double-strand DNA breaks or donor DNA templates. Here we describe the use of an ABE to directly correct the pathogenic HGPS mutation in cultured fibroblasts derived from children with progeria and in a mouse model of HGPS. Lentiviral delivery of the ABE to fibroblasts from children with HGPS resulted in 87–91% correction of the pathogenic allele, mitigation of RNA mis-splicing, reduced levels of progerin and correction of nuclear abnormalities. Unbiased off-target DNA and RNA editing analysis did not detect off-target editing in treated patient-derived fibroblasts. In transgenic mice that are homozygous for the human LMNA c).1824 C>T allele, a single retro-orbital injection of adeno-associated virus 9 (AAV9) encoding the ABE resulted in substantial, durable correction of the pathogenic mutation (around 20–60% across various organs six months after injection), restoration of normal RNA splicing and reduction of progerin protein levels. In vivo base editing rescued the vascular pathology of the mice, preserving vascular smooth muscle cell counts and preventing adventitial fibrosis. A single injection of ABE-expressing AAV9 at postnatal day 14 improved vitality and greatly extended the median lifespan of the mice from 215 to 510 days. These findings demonstrate the potential of in vivo base editing as a possible treatment for HGPS and other genetic diseases by directly correcting their root cause.
During female germline development, oocytes become a highly specialized cell type and form a maternal cytoplasmic store of crucial factors. Oocyte growth is triggered at the transition from primordial to primary follicle and is accompanied by dynamic changes in gene expression1, but the gene regulatory network that controls oocyte growth remains unknown. Here we identify a set of transcription factors that are sufficient to trigger oocyte growth. By investigation of the changes in gene expression and functional screening using an in vitro mouse oocyte development system, we identified eight transcription factors, each of which was essential for the transition from primordial to primary follicle. Notably, enforced expression of these transcription factors swiftly converted pluripotent stem cells into oocyte-like cells that were competent for fertilization and subsequent cleavage. These transcription-factor-induced oocyte-like cells were formed without specification of primordial germ cells, epigenetic reprogramming or meiosis, and demonstrate that oocyte growth and lineage-specific de novo DNA methylation are separable from the preceding epigenetic reprogramming in primordial germ cells. This study identifies a core set of transcription factors for orchestrating oocyte growth, and provides an alternative source of ooplasm, which is an unique material for reproductive biology and medicine.
This report examines public perceptions of biotechnology, evolution and the relationship between science and religion. Data in this report come from a survey conducted in 20 publics from October 2019 to March 2020 across Europe, Russia, the Americas and the Asia-Pacific region. Surveys were conducted by face-to-face interview in Russia, Poland, the Czech Republic, India and Brazil. In all other places, the surveys were conducted by telephone. All surveys were conducted with representative samples of adults ages 18 and older in each survey public.
…A 20-public median of 63% say scientific research on gene editing is a misuse—rather than an appropriate use—of technology, according to the survey fielded in publics across Europe, the Asia-Pacific region, the United States, Canada, Brazil and Russia.
However, views on specific instances where gene editing might be used highlight the complex and contextual nature of public attitudes. Majorities say it would be appropriate to change a baby’s genetic characteristics to treat a serious disease the baby would have at birth (median of 70%), and somewhat smaller shares, though still about half or more, say using these techniques to reduce the risk of a serious disease that could occur over the course of the baby’s lifetime would be appropriate (60%). But a median of just 14% say it would be appropriate to change a baby’s genetic characteristics to make the baby more intelligent. A far larger share (median of 82%) would consider this to be a misuse of technology.
Ageing is a degenerative process that leads to tissue dysfunction and death. A proposed cause of ageing is the accumulation of epigenetic noise that disrupts gene expression patterns, leading to decreases in tissue function and regenerative capacity. Changes to DNA methylation patterns over time form the basis of ageing clocks, but whether older individuals retain the information needed to restore these patterns—and, if so, whether this could improve tissue function—is not known.
Over time, the central nervous system (CNS) loses function and regenerative capacity. Using the eye as a model CNS tissue, here we show that ectopic expression of Oct4 (also known as Pou5f1), Sox2 and Klf4 genes (OSK) in mouse retinal ganglion cells restores youthful DNA methylation patterns and transcriptomes, promotes axon regeneration after injury, and reverses vision loss in a mouse model of glaucoma and in aged mice. The beneficial effects of OSK-induced reprogramming in axon regeneration and vision require the DNA demethylases TET1 and TET2.
These data indicate that mammalian tissues retain a record of youthful epigenetic information—encoded in part by DNA methylation—that can be accessed to improve tissue function and promote regeneration in vivo.
The majority of polygenic risk scores (PRSs) have been developed and optimized in individuals of European ancestry and may have limited generalizability across other ancestral populations. Understanding aspects of PRSs that contribute to this issue and determining solutions is complicated by disease-specific genetic architecture and limited knowledge of sharing of causal variants and effect sizes across populations.
Motivated by these challenges, we undertook a simulation study to assess the relationship between ancestry and the potential bias in PRSs developed in European ancestry populations. Our simulations show that the magnitude of this bias increases with increasing divergence from European ancestry, and this is attributed to population differences in linkage disequilibrium and allele frequencies of European-discovered variants, likely as a result of genetic drift. Importantly, we find that including into the PRS variants discovered in African ancestry individuals has the potential to achieve unbiased estimates of genetic risk across global populations and admixed individuals.
We confirm our simulation findings in an analysis of hemoglobin A1c (HbA1c), asthma, and prostate cancer in the UK Biobank.
Given the demonstrated improvement in PRS prediction accuracy, recruiting larger diverse cohorts will be crucial—and potentially even necessary—for enabling accurate and equitable genetic risk prediction across populations.
Food crops produced by new technologies such as genetic engineering are widely opposed (Gaskell, Bauer, Durant, & Allum, 1999; Scott, Inbar, Wirz, Brossard, & Rozin, 2018). Here, we examine one reason for this opposition: recency. More recently-developed crops are evaluated less favorably, whether they are produced by artificial selection (ie. conventional breeding), natural or man-made irradiation, or genetic engineering. Negative effects of recency persist in a within-subjects design where people are able to explicitly compare crops developed at different times, and an internal meta-analysis shows that the negative effect of recency is robust across measures and stimuli. These results have implications for the evaluation of crops produced using new modification techniques, including the widespread opposition to genetic engineering.
Obesity and type 2 diabetes (T2D) are associated with poor tissue responses to insulin1,2, disturbances in glucose and lipid fluxes3–5 and comorbidities including steatohepatitis6 and cardiovascular disease7,8. Despite extensive efforts at prevention and treatment9,10, diabetes afflicts over 400 million people worldwide11. Whole body metabolism is regulated by adipose tissue depots12–14, which include both lipid-storing white adipocytes and less abundant “brown” and “brite/beige” adipocytes that express thermogenic uncoupling protein UCP1 and secrete factors favorable to metabolic health15–18. Application of clustered regularly interspaced short palindromic repeats (CRISPR) gene editing19,20 to enhance “browning” of white adipose tissue is an attractive therapeutic approach to T2D. However, the problems of cell-selective delivery, immunogenicity of CRISPR reagents and long term stability of the modified adipocytes are formidable. To overcome these issues, we developed methods that deliver complexes of SpyCas9 protein and sgRNA ex vivo to disrupt the thermogenesis suppressor gene NRIP121,22 with near 100% efficiency in human or mouse adipocytes. NRIP1 gene disruption at discrete loci strongly ablated NRIP1 protein and upregulated expression of UCP1 and beneficial secreted factors, while residual Cas9 protein and sgRNA were rapidly degraded. Implantation of the CRISPR-enhanced human or mouse brown-like adipocytes into high fat diet fed mice decreased adiposity and liver triglycerides while enhancing glucose tolerance compared to mice implanted with unmodified adipocytes. These findings advance a therapeutic strategy to improve metabolic homeostasis through CRISPR-based genetic modification of human adipocytes without exposure of the recipient to immunogenic Cas9 or delivery vectors.
Emmanuelle Charpentier and Jennifer A. Doudna have discovered one of gene technology’s sharpest tools: the CRISPR / Cas9 genetic scissors. Using these, researchers can change the DNA of animals, plants and microorganisms with extremely high precision. This technology has had a revolutionary impact on the life sciences, is contributing to new cancer therapies and may make the dream of curing inherited diseases come true.
Researchers need to modify genes in cells if they are to find out about life’s inner workings. This used to be time-consuming, difficult and sometimes impossible work. Using the CRISPR/Cas9 genetic scissors, it is now possible to change the code of life over the course of a few weeks.
“There is enormous power in this genetic tool, which affects us all. It has not only revolutionised basic science, but also resulted in innovative crops and will lead to ground-breaking new medical treatments”, says Claes Gustafsson, chair of the Nobel Committee for Chemistry.
As so often in science, the discovery of these genetic scissors was unexpected. During Emmanuelle Charpentier’s studies of Streptococcus pyogenes, one of the bacteria that cause the most harm to humanity, she discovered a previously unknown molecule, tracrRNA. Her work showed that tracrRNA is part of bacteria’s ancient immune system, CRISPR/Cas, that disarms viruses by cleaving their DNA.
Charpentier published her discovery in 2011. The same year, she initiated a collaboration with Jennifer Doudna, an experienced biochemist with vast knowledge of RNA. Together, they succeeded in recreating the bacteria’s genetic scissors in a test tube and simplifying the scissors’ molecular components so they were easier to use.
In an epoch-making experiment, they then reprogrammed the genetic scissors. In their natural form, the scissors recognise DNA from viruses, but Charpentier and Doudna proved that they could be controlled so that they can cut any DNA molecule at a predetermined site. Where the DNA is cut it is then easy to rewrite the code of life.
Since Charpentier and Doudna discovered the CRISPR/Cas9 genetic scissors in 2012 their use has exploded. This tool has contributed to many important discoveries in basic research, and plant researchers have been able to develop crops that withstand mould, pests and drought. In medicine, clinical trials of new cancer therapies are underway, and the dream of being able to cure inherited diseases is about to come true. These genetic scissors have taken the life sciences into a new epoch and, in many ways, are bringing the greatest benefit to humankind.
The National Academy of Sciences arranged summits and reports in an attempt to set some boundaries. In 2017 the academy concluded that using Crispr for human genetic enhancement was a hard no. But they stopped short of a full moratorium. What about gene editing to address serious, incurable diseases? Well, that could maybe one day be fine, provided it was proven safe and effective. But that 2017 report didn’t spell out exactly how one might prove those things…Still, the revelation that someone had gotten as far as he [He Jiankui] had sent scientists and policymakers scrambling to lay down some firmer ground rules. China formed a national ethics committee tasked with enforcing the country’s new clinical research guidelines. The World Health Organization assembled a panel to establish global regulatory standards for governments to follow. (Its first order of business was to urge all nations to put a hold on any experiments that would lead to the births of more gene-edited humans until the implications of such work could be more fully examined.) And another National Academies commission was formed. This one was international—with 18 members from 10 nations—and was assigned a less sprawling task: to set clear, explicit, scientific standards for heritable gene editing in humans.
On Thursday, after more than a year of work, the commission finally released its 225-page report—the most comprehensive and highly technical such document to date. It describes in great detail the types and quality of evidence that scientists must provide to show they’ve correctly edited an embryo, before they can attempt to try it out in humans. It is, in essence, a road map for how to safely and responsibly get to clinical trials. But importantly, say the report’s authors, it’s not an endorsement.
“No attempt to establish a pregnancy with a human embryo that has undergone genome editing should proceed unless and until it has been clearly established that it is possible to efficiently and reliably make precise genomic changes without undesired changes in human embryos”, the report states. “These criteria have not yet been met and further research and review would be necessary to meet them.”
…For that reason, the authors of the report lay out exactly how many and what kind of off-target effects might be acceptable. They put that threshold at no more than the average rate of new mutations an embryo spontaneously acquires. DNA replication isn’t perfect, and most people are born with a few dozen mutations that don’t exist in either of their biological parents’ genomes. Gene editing shouldn’t introduce any more genetic variations than occur naturally, the authors concluded, and the types of changes should be carefully studied in the lab to make sure they don’t lead to adverse outcomes.
The trouble is, though, that right now there aren’t any good methods for assessing off-target effects in embryos. Doing so requires collecting large amounts of DNA, which can only be done by sacrificing a number of cells from the embryo for genetic sequencing. In addition to being unreliable, these methods harm the viability of the embryo, making it less likely to result in a pregnancy. It could take years for better methods of evaluation to be developed, says commission member Haoyi Wang, a reproductive biologist at the Institute of Zoology and Institute for Stem Cell and Regeneration at the Chinese Academy of Sciences. “From the genome editing to the genome sequencing of a single embryo, there are still many gaps to be filled”, Wang told reporters at a press briefing Thursday.
The commission was more narrowly focused on addressing these sorts of scientific gaps, while other authorities, like the WHO, will look more broadly at how societies might decide to accept human germline editing and how governments will regulate the technology. Developing ethical frameworks can’t just be about autonomy, privacy, and justice, says commission member Bartha Maria Knoppers, who directs the Centre for Genomics and Policy and serves as the Canada Research Chair in Law and Medicine at McGill University in Montreal. “For me, scientific quality and safety are primordial ethical considerations; they’re not peripheral”, she says. “I think this report reflects the emphasis on getting those aspects right.”
Heritable human genome editing—making changes to the genetic material of eggs, sperm, or any cells that lead to their development, including the cells of early embryos, and establishing a pregnancy—raises not only scientific and medical considerations but also a host of ethical, moral, and societal issues. Human embryos whose genomes have been edited should not be used to create a pregnancy until it is established that precise genomic changes can be made reliably and without introducing undesired changes—criteria that have not yet been met, says Heritable Human Genome Editing.
From an international commission of the U.S. National Academy of Medicine, U.S. National Academy of Sciences, and the U.K.’s Royal Society, the report considers potential benefits, harms, and uncertainties associated with genome editing technologies and defines a translational pathway from rigorous preclinical research to initial clinical uses, should a country decide to permit such uses. The report specifies stringent preclinical and clinical requirements for establishing safety and efficacy, and for undertaking long-term monitoring of outcomes. Extensive national and international dialogue is needed before any country decides whether to permit clinical use of this technology, according to the report, which identifies essential elements of national and international scientific governance and oversight.
The State of the Science
Potential Applications of Heritable Human Genome Editing
A Translational Pathway to Limited and Controlled Clinical Applications of Heritable Human Genome Editing
National and International Governance of Heritable Human Genome Editing
Turning up the heat: Uncoupling protein 1 (UCP1) is the major player in the energy-siphoning thermogenesis that primarily occurs in brown adipose tissue (BAT). Wang et al generated UCP1-overexpressing human white adipocytes so that they more resembled their brown counterparts. Transplantation of the modified white adipocytes prevented diet-induced obesity and glucose intolerance and increased energy expenditure in the recipient mice. These metabolic benefits resulted from increased nitric oxide signaling in the transplanted human cells, which activated endogenous murine BAT. Future work will need to examine whether this cell-based strategy can activate BAT thermogenesis in humans.
Brown and brown-like beige/brite adipocytes dissipate energy and have been proposed as therapeutic targets to combat metabolic disorders. However, the therapeutic effects of cell-based therapy in humans remain unclear. Here, we created human brown-like (HUMBLE) cells by engineering human white preadipocytes using CRISPR-Cas9–SAM-gRNA to activate endogenous uncoupling protein 1 expression. Obese mice that received HUMBLE cell transplants showed a sustained improvement in glucose tolerance and insulin sensitivity, as well as increased energy expenditure. Mechanistically, increased arginine/nitric oxide (NO) metabolism in HUMBLE adipocytes promoted the production of NO that was carried by S-nitrosothiols and nitrite in red blood cells to activate endogenous brown fat and improved glucose homeostasis in recipient animals. Together, these data demonstrate the utility of using CRISPR-Cas9 technology to engineer human white adipocytes to display brown fat-like phenotypes and may open up cell-based therapeutic opportunities to combat obesity and diabetes.
We report the synthesis of a molecular machine, fabricated from nucleic acids, which is capable of digesting viral RNA and utilizing it to assemble additional copies of itself inside living cells. The machine’s body plan combines several parts that build upon the target RNA, assembling an immobile, DNA:RNA 4-way junction, which contains a single gene encoding a hammerhead ribozyme (HHR). Full assembly of the machine’s body from its parts enables the subsequent elongation of the gene and transcription of HHR molecules, followed by HHR-mediated digestion of the target molecule. This digestion converts the target to a building block suitable for participation in the assembly of more copies of the machine, mimicking biological heterotrophy. In this work we describe the general design of a prototypical machine, characterize its activity cycle and kinetics, and show that it can be efficiently and safely delivered into live cells. As a proof of principle, we constructed a machine that targets the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) GP64 gene, and show that it effectively suppresses viral propagation in a cell population, exhibiting predator/prey-like dynamics with the infecting virus. In addition, the machine significantly reduced viral infection, stress signaling, and innate immune activation inside virus-infected animals. This preliminary design could control the behavior of antisense therapies for a range of applications, particularly against dynamic targets such as viruses and cancer.
The neocortex has expanded during mammalian evolution. Overexpression studies in developing mouse and ferret neocortex have implicated the human-specific gene ARHGAP11B in neocortical expansion, but the relevance for primate evolution has been unclear.
Here, we provide functional evidence that ARHGAP11B causes expansion of the primate neocortex. ARHGAP11B expressed in fetal neocortex of the common marmoset under control of the gene’s own (human) promoter increased the numbers of basal radial glia progenitors in the marmoset outer subventricular zone, increased the numbers of upper-layer neurons, enlarged the neocortex, and induced its folding.
Thus, the human-specific ARHGAP11B drives changes in development in the nonhuman primate marmoset that reflect the changes in evolution that characterize human neocortical development.
Large deletions and genomic re-arrangements are increasingly recognized as common products of double-strand break repair at Clustered Regularly Interspaced, Short Palindromic Repeats—CRISPR associated protein 9 (CRISPR/Cas9) on-target sites. Together with well-known off-target editing products from Cas9 target misrecognition, these are important limitations, that need to be addressed. Rigorous assessment of Cas9-editing is necessary to ensure validity of observed phenotypes in Cas9-edited cell-lines and model organisms. Here the mechanisms of Cas9 specificity, and strategies to assess and mitigate unwanted effects of Cas9 editing are reviewed; covering guide-RNA design, RNA modifications, Cas9 modifications, control of Cas9 activity; computational prediction for off-targets, and experimental methods for detecting Cas9 cleavage. Although recognition of the prevalence of on-target and off-target effects of Cas9 editing has increased in recent years, broader uptake across the gene editing community will be important in determining the specificity of Cas9 across diverse applications and organisms.
Bacterial toxins represent a vast reservoir of biochemical diversity that can be repurposed for biomedical applications. Such proteins include a group of predicted interbacterial toxins of the deaminase superfamily, members of which have found application in gene-editing techniques1,2. Because previously described cytidine deaminases operate on single-stranded nucleic acids3, their use in base editing requires the unwinding of double-stranded DNA (dsDNA)—for example by a CRISPR-Cas9 system. Base editing within mitochondrial DNA (mtDNA), however, has thus far been hindered by challenges associated with the delivery of guide RNA into the mitochondria4. As a consequence, manipulation of mtDNA to date has been limited to the targeted destruction of the mitochondrial genome by designer nucleases9,10.Here we describe an interbacterial toxin, which we name DddA, that catalyses the deamination of cytidines within dsDNA. We engineered split-DddA halves that are non-toxic and inactive until brought together on target DNA by adjacently bound programmable DNA-binding proteins. Fusions of the split-DddA halves, transcription activator-like effector array proteins, and an uracil glycosylase inhibitor resulted in RNA-free DddA-derived cytosine base editors (DdCBEs) that catalyse C•G-to-T•A conversions in human mtDNA with high target specificity and product purity. We used DdCBEs to model a disease-associated mtDNA mutation in human cells, resulting in changes in respiration rates and oxidative phosphorylation. CRISPR-free DdCBEs enable the precise manipulation of mtDNA, rather than the elimination of mtDNA copies that results from its cleavage by targeted nucleases, with broad implications for the study and potential treatment of mitochondrial disorders.
…as the one-year anniversary of her landmark treatment approaches, Gray has just received good news: The billions of genetically modified cells doctors infused into her body clearly appear to be alleviating virtually all the complications of her disorder, sickle cell disease. “It’s wonderful. It’s the change I’ve been waiting on my whole life”, Gray told NPR, which has had exclusive access to chronicle her experience over the past year.
…The last time NPR spoke with Gray—in November—her doctors had just gotten the first hints the treatment might be working. Now, after nine months of careful testing, the treatment shows no signs of waning, making her doctors more confident than ever the experiment has been a success.
…The researchers conducting the study Gray started caution that it’s too soon to reach any firm conclusions about the long-term safety and effectiveness of the approach. Gray is just one patient who has been followed for what is still a relatively short period of time, they noted. But Gray’s experience so far, along with two other patients who received the same treatment for a similar disorder, indicate the therapy has been effective for her and may work for other patients as well, they said…At a meeting of the European Hematology Association on June 12, Frangoul and other researchers presented the latest results of their latest testing of Gray as well as two study subjects with a related condition, beta thalassemia. The latter also appear to be benefiting…The researchers also reported that the first patient to receive the same treatment for beta thalassemia in Germany has now been able to live without blood transfusions for 15 months. Previously, the researchers had reported data for that patient for nine months. In addition, four other beta thalassemia patients have been treated, including one who has been transfusion-free for five months, the researchers reported. While Gray and the beta thalassemia patients experienced some health complications following their procedures, none appears to have been due to the gene-edited cells and all recovered, according to the researchers.
“A huge change”: Perhaps most importantly, the changes appear to have translated into substantial health benefits for Gray. She hasn’t had any severe pain attacks since the treatment and hasn’t required any emergency room treatments, hospitalizations or blood transfusions. In each of the previous two years, Gray had required an average of seven hospitalizations and emergency room visits due to severe pain episodes as well as requiring regular blood transfusions. She has also been able to reduce substantially her need for powerful narcotics to alleviate her pain.
“It’s a very big deal for me”, Gray said. “It’s a huge change.”
Two people with beta thalassaemia and one with sickle cell disease no longer require blood transfusions, which are normally used to treat severe forms of these inherited diseases, after their bone marrow stem cells were gene-edited with CRISPR.
Result of this ongoing trial, which is the first to use CRISPR to treat inherited genetic disorders, were announced today at a virtual meeting of the European Hematology Association. “The preliminary results…demonstrate, in essence, a functional cure for patients with beta thalassaemia and sickle cell disease”, team member Haydar Frangoul at Sarah Cannon Research Institute in Nashville, Tennessee, said in a statement.
…In this trial, run by collaborating companies CRISPR Therapeutics and Vertex, bone marrow stem cells are removed from people and the gene that turns off fetal haemoglobin production is disabled with CRISPR. The remaining bone marrow cells are killed by chemotherapy, then replaced by edited cells. This is done to ensure that new blood cells are produced by the edited stem cells, but the chemotherapy can have serious side effects including infertility. The first two patients with beta thalassaemia no longer need blood transfusions since being treated 15 and five months ago. Nor does the patient with sickle cell disease, nine months after treatment. The results are excellent, says Marina Cavazzana at the Necker-Enfants Malades Hospital in Paris, France, whose team has treated a 13-year-old boy with sickle cell disease using a different approach.
Clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein (Cas) technology has been applied in plant breeding mainly on genes for improving single or multiple traits1,2,3,4. Here we show that this technology can also be used to restructure plant chromosomes. Using the Cas9 nuclease from Staphylococcus aureus5, we were able to induce reciprocal translocations in the Mbp range between heterologous chromosomes in Arabidopsis thaliana. Of note, translocation frequency was about 5× more efficient in the absence of the classical non-homologous end-joining pathway. Using egg-cell-specific expression of the Cas9 nuclease and consecutive bulk screening, we were able to isolate heritable events and establish lines homozygous for the translocation, reaching frequencies up to 2.5% for individual lines. Using molecular and cytological analysis, we confirmed that the chromosome-arm exchanges we obtained between chromosomes 1 and 2 and between chromosomes 1 and 5 of Arabidopsis were conservative and reciprocal. The induction of chromosomal translocations enables mimicking of genome evolution or modification of chromosomes in a directed manner, fixing or breaking genetic linkages between traits on different chromosomes. Controlled restructuring of plant genomes has the potential to transform plant breeding.
Aging is a degenerative process leading to tissue dysfunction and death. A proposed cause of aging is the accumulation of epigenetic noise, which disrupts youthful gene expression patterns that are required for cells to function optimally and recover from damage. Changes to DNA methylation patterns over time form the basis of ‘aging clocks’, but whether old individuals retain information to reset the clocks and, if so, whether it would improve tissue function is not known. Of all the tissues in the body, the central nervous system (CNS) is one of the first to lose regenerative capacity. Using the eye as a model tissue, we show that expression of Oct4, Sox2, and Klf4 genes (OSK) in mice resets youthful gene expression patterns and the DNA methylation age of retinal ganglion cells, promotes axon regeneration after optic nerve crush injury, and restores vision in a mouse model of glaucoma and in normal aged mice. This process, which we call the reversal of information loss via epigenetic reprogramming or REVIVER, requires non-global, active DNA demethylation by TET enzymes and the downstream enzyme TDG, indicating that alterations in DNA methylation patterns may not simply indicate age, but participate in aging. Thus, old tissues retain a faithful record of youthful epigenetic information that can be accessed for functional age reversal.
Psilocybin is a tryptamine-derived psychoactive alkaloid found mainly in the fungal genus Psilocybe, among others, and is the active ingredient in so-called “magic mushrooms”. Although its notoriety originates from its psychotropic properties and popular use as a recreational drug, clinical trials have recently recognized psilocybin as a promising candidate for the treatment of various psychological and neurological afflictions. In this work, we demonstrate the de novo biosynthetic production of psilocybin and related tryptamine derivatives in Saccharomyces cerevisiae by expression of a heterologous biosynthesis pathway sourced from Psilocybe cubensis. Additionally, we achieve improved product titers by supplementing the pathway with a novel cytochrome P450 reductase from P. cubensis. Further rational engineering resulted in a final production strain producing 627 ± 140 mg/L of psilocybin and 580 ± 276 mg/L of the dephosphorylated degradation product psilocin in triplicate controlled fed-batch fermentations in minimal synthetic media. Pathway intermediates baeocystin, nor norbaeocystin as well the dephosphorylated baeocystin degradation product norpsilocin were also detected in strains engineered for psilocybin production. We also demonstrate the biosynthetic production of natural tryptamine derivative aeruginascin as well as the production of a new-to-nature tryptamine derivative N-acetyl-4-hydroxytryptamine. These results lay the foundation for the biotechnological production of psilocybin in a controlled environment for pharmaceutical applications, and provide a starting point for the biosynthetic production of other tryptamine derivatives of therapeutic relevance.
A court in China on Monday sentenced He Jiankui, the researcher who shocked the global scientific community when he claimed that he had created the world’s first genetically edited babies, to three years in prison for carrying out “illegal medical practices.” In a surprise announcement from a trial that was closed to the public, the court in the southern city of Shenzhen found Dr. He guilty of forging approval documents from ethics review boards to recruit couples in which the man had H.I.V. and the woman did not, Xinhua, China’s official news agency, reported. Dr. He had said he was trying to prevent H.I.V. infections in newborns, but the state media on Monday said he deceived the subjects and the medical authorities alike.
Dr. He, 35, sent the scientific world into an uproar last year when he announced at a conference in Hong Kong that he had created the world’s first genetically edited babies—twin girls. On Monday, China’s state media said his work had resulted in a third genetically edited baby, who had been previously undisclosed.
Dr. He pleaded guilty and was also fined $430,000, according to Xinhua. In a brief trial, the court also handed down prison sentences to two other scientists who it said had “conspired” with him: Zhang Renli, who was sentenced to two years in prison, and Qin Jinzhou, who got a suspended sentence of one and a half years…The court said the trial had to be closed to the public to guard the privacy of the people involved.
If any swine is fit to be an organ donor for people, then the dozens of pigs snuffling around Qihan Bio’s facility in Hangzhou, China, may be the best candidates so far. The Chinese company and its U.S. collaborators reported today that they have used the genome editor CRISPR to create the most extensively genetically engineered pigs to date—animals whose tissues, the researchers say, finally combine all the features necessary for a safe and successful transplant into humans. “This is the first prototype”, says Luhan Yang, a geneticist at Qihan Bio. In a preprint published today on bioRxiv, Qihan researchers and collaborators, including Cambridge, Massachusetts-based eGenesis—which Yang co-founded with Harvard University geneticist George Church—described the new generation of animals and various tests on their cells; the researchers have already begun to transplant the pigs’ organs into nonhuman primates, a key step toward human trials.
…In the new study, the team for the first time combined these PERV “knockouts” with a suite of other changes to prevent immune rejection, for a record-setting 13 modified genes. In pig ear cells, they removed three genes coding for enzymes that help produce molecules on pig cells that provoke an immune response. They also inserted six genes that inhibit various aspects of the human immune response and three more that help regulate blood coagulation. The researchers then put the DNA-containing nuclei of these edited cells into eggs from pig ovaries collected at a slaughterhouse. These eggs developed into embryos that were implanted into surrogate mothers. Cells from the resulting piglets got another round of edits to remove the PERV sequences, after which their DNA went into another set of egg cells to create a new generation of pigs with all the desired edits. (In future, Yang says, the team will try to make all the modifications in a single generation.)
The resulting pigs appeared healthy and fertile with functioning organs, the team reports today. And initial tests of their cells in lab dishes suggest their organs will be much less prone to immune rejection than those of unmodified pigs: The tendency of the pig cells to bind to certain human antibodies was reduced by 90%, and the modified cells better survived interactions with human immune cells. But a key test is still to come: Yang says her team has begun to transplant organs from the highly edited pigs into monkeys to gauge their safety and longevity.
The combination of edits described in the new paper is “a technical feat”, says Marilia Cascalho, a transplant immunologist at the University of Michigan in Ann Arbor. “Whether it offers an advantage [over other engineered pig organs]… the jury is out on that”, she says…Yang says that Qihan plans to remain “laser-focused” on preclinical studies in 2020, but expects to be testing pig organs in humans within 5 years. Many in the field now feel an inevitable momentum around xenotransplantation: “There is so much need for organs”, Cascalho says. “I think it’s going to be a reality.”
Xenotransplantation, specifically the use of porcine organs for human transplantation, has long been sought after as an alternative for patients suffering from organ failure. However, clinical application of this approach has been impeded by two main hurdles: (1) risk of transmission of porcine endogenous retroviruses (PERVs) and (2) molecular incompatibilities between donor pigs and humans which culminate in rejection of the graft. We previously demonstrated that all 25 copies of the PERV elements in the pig genome could be inactivated and live pigs successfully generated. In this study, we improved the scale of porcine germline editing from targeting a single repetitive locus with CRISPR to engineering 18 different loci using multiple genome engineering methods. we engineered the pig genome at 42 alleles using CRISPR-Cas9 and transposon and produced PERVKO·3KO·9TG pigs which carry PERV inactivation, xeno-antigen KO and 9 effective human transgenes.. The engineered pigs exhibit normal physiology, fertility, and germline transmission of the edited alleles. In vitro assays demonstrated that these pigs gain significant resistance to human humoral and cell mediated damage, and coagulation dysregulations, similar to that of allotransplantation. Successful creation of PERVKO·3KO·9TG pigs represents a significant step forward towards safe and effective porcine xenotransplantation, which also represents a synthetic biology accomplishment of engineering novel functions in a living organism.
One Sentence Summary
Extensive genome engineering is applied to modify pigs for safe and immune compatible organs for human transplantation
Significance: Human and animal longevity is directly bound to their health span. While previous studies have provided evidence supporting this connection, therapeutic implementation of this knowledge has been limited. Traditionally, diseases are researched and treated individually, which ignores the interconnectedness of age-related conditions, necessitates multiple treatments with unrelated substances, and increases the accumulative risk of side effects. In this study, we address and overcome this deadlock by creating adeno-associated virus (AAV)-based antiaging gene therapies for simultaneous treatment of several age-related diseases. We demonstrate the modular and extensible nature of combination gene therapy by testing therapeutic AAV cocktails that confront multiple diseases in a single treatment. We observed that 1 treatment comprising 2 AAV gene therapies was efficacious against all 4 diseases.
Comorbidity is common as age increases, and currently prescribed treatments often ignore the interconnectedness of the involved age-related diseases. The presence of any one such disease usually increases the risk of having others, and new approaches will be more effective at increasing an individual’s health span by taking this systems-level view into account. In this study, we developed gene therapies based on 3 longevity associated genes (fibroblast growth factor 21 [FGF21], αKlotho, soluble form of mouse transforming growth factor-β receptor 2 [sTGFβR2]) delivered using adeno-associated viruses and explored their ability to mitigate 4 age-related diseases: obesity, type II diabetes, heart failure, and renal failure. Individually and combinatorially, we applied these therapies to disease-specific mouse models and found that this set of diverse pathologies could be effectively treated and in some cases, even reversed with a single dose. We observed a 58% increase in heart function in ascending aortic constriction ensuing heart failure, a 38% reduction in α-smooth muscle actin (αSMA) expression, and a 75% reduction in renal medullary atrophy in mice subjected to unilateral ureteral obstruction and a complete reversal of obesity and diabetes phenotypes in mice fed a constant high-fat diet. Crucially, we discovered that a single formulation combining 2 separate therapies into 1 was able to treat all 4 diseases. These results emphasize the promise of gene therapy for treating diverse age-related ailments and demonstrate the potential of combination gene therapy that may improve health span and longevity by addressing multiple diseases at once.
Crispr, for all its DNA-snipping precision, has always been best at breaking things. But if you want to replace a faulty gene with a healthy one, things get more complicated. In addition to programming a piece of guide RNA to tell Crispr where to cut, you have to provide a copy of the new DNA and then hope the cell’s repair machinery installs it correctly. Which, spoiler alert, it often doesn’t. Anzalone wondered if instead there was a way to combine those two pieces, so that one molecule told Crispr both where to make its changes and what edits to make. Inspired, he cinched his coat tighter and hurried home to his apartment in Chelsea, sketching and Googling late into the night to see how it might be done.
A few months later, his idea found a home in the lab of David Liu, the Broad Institute chemist who’d recently developed a host of more surgical Crispr systems, known as base editors. Anzalone joined Liu’s lab in 2018, and together they began to engineer the Crispr creation glimpsed in the young post-doc’s imagination. After much trial and error, they wound up with something even more powerful. The system, which Liu’s lab has dubbed “prime editing”, can for the first time make virtually any alteration—additions, deletions, swapping any single letter for any other—without severing the DNA double helix. “If Crispr-Cas9 is like scissors and base editors are like pencils, then you can think of prime editors to be like word processors”, Liu told reporters in a press briefing.
Why is that a big deal? Because with such fine-tuned command of the genetic code, prime editing could, according to Liu’s calculations, correct around 89% of the mutations that cause heritable human diseases. Working in human cell cultures, his lab has already used prime editors to fix the genetic glitches that cause sickle cell anemia, cystic fibrosis, and Tay-Sachs disease. Those are just three of more than 175 edits the group unveiled today in a scientific article published in the journal Nature.
The work “has a strong potential to change the way we edit cells and be transformative”, says Gaétan Burgio, a geneticist at the Australian National University who was not involved in the work, in an email. He was especially impressed at the range of changes prime editing makes possible, including adding up to 44 DNA letters and deleting up to 80. “Overall, the editing efficiency and the versatility shown in this paper are remarkable.”
…The bigger problem, according to folks like Burgio, is that prime editors are huge, in molecular terms. They’re so big that they won’t pack up neatly into the viruses researchers typically use to shuttle editing components into cells. These colossi might even clog a microinjection needle, making it difficult to deliver into mouse (or potentially human) embryos. That, says Burgio, could make prime editing a lot less practical than existing techniques.
CRISPR, an extraordinarily powerful genome-editing tool invented in 2012, can still be clumsy. It sometimes changes genes it shouldn’t, and it edits by hacking through both strands of DNA’s double helix, leaving the cell to clean up the mess—shortcomings that limit its use in basic research and agriculture and pose safety risks in medicine. But a new entrant in the race to refine CRISPR promises to steer around some of its biggest faults. “It’s a huge step in the right direction”, chemist George Church, a CRISPR pioneer at Harvard University, says about the work, which appears online today in Nature.
…Liu’s earlier handwork, base editing, does not cut the double-stranded DNA but instead uses the CRISPR targeting apparatus to shuttle an additional enzyme to a desired sequence, where it converts a single nucleotide into another. Many genetic traits and diseases are caused by a single nucleotide change, so base editing offers a powerful alternative for biotechnology and medicine. But the method has limitations, and it, too, often introduces off-target mutations.
Prime editing steers around shortcomings of both techniques by heavily modifying the Cas9 protein and the guide RNA. The altered Cas9 only “nicks” a single strand of the double helix, instead of cutting both. The new guide, called a pegRNA, contains an RNA template for a new DNA sequence, to be added to the genome at the target location. That requires a second protein, attached to Cas9: a reverse transcriptase enzyme, which can make a new DNA strand from the RNA template and insert it at the nicked site.
Liu, who has already formed a company around the new technology, Prime Medicine, stresses that to gain a place in the editing toolkit, it will have to prove robust and useful in many labs. Delivering the large construct of RNA and enzymes into living cells will also be difficult, and no one has yet shown it can work in an animal model.
Most genetic variants that contribute to disease1 are challenging to correct efficiently and without excess byproducts2,3,4,5. Here we describe prime editing, a versatile and precise genome editing method that directly writes new genetic information into a specified DNA site using a catalytically impaired Cas9 endonuclease fused to an engineered reverse transcriptase, programmed with a prime editing guide RNA (pegRNA) that both specifies the target site and encodes the desired edit. We performed more than 175 edits in human cells, including targeted insertions, deletions, and all 12 types of point mutation, without requiring double-strand breaks or donor DNA templates. We used prime editing in human cells to correct, efficiently and with few byproducts, the primary genetic causes of sickle cell disease (requiring a transversion in HBB) and Tay-Sachs disease (requiring a deletion in HEXA); to install a protective transversion in PRNP; and to insert various tags and epitopes precisely into target loci. Four human cell lines and primary post-mitotic mouse cortical neurons support prime editing with varying efficiencies. Prime editing shows higher or similar efficiency and fewer byproducts than homology-directed repair, has complementary strengths and weaknesses compared to base editing, and induces much lower off-target editing than Cas9 nuclease at known Cas9 off-target sites. Prime editing substantially expands the scope and capabilities of genome editing, and in principle could correct up to 89% of known genetic variants associated with human diseases.
Engineering biology with recombinant DNA, broadly called synthetic biology, has progressed tremendously in the last decade, owing to continued industrialization of DNA synthesis, discovery and development of molecular tools and organisms, and increasingly sophisticated modeling and analytic tools. However, we have yet to understand the full potential of engineering biology because of our inability to write and test whole genomes, which we call synthetic genomics. Substantial improvements are needed to reduce the cost and increase the speed and reliability of genetic tools. Here, we identify emerging technologies and improvements to existing methods that will be needed in four major areas to advance synthetic genomics within the next 10 years: genome design, DNA synthesis, genome editing, and chromosome construction (see table). Similar to other large-scale projects for responsible advancement of innovative technologies, such as the Human Genome Project, an international, cross-disciplinary effort consisting of public and private entities will likely yield maximal return on investment and open new avenues of research and biotechnology.
[Mukherjee traces the evolution of CAR T-cell therapy, a form of immunotherapy that uses engineered immune cells to eliminate cancer, beginning with the development of bone marrow transplantation by Fred Hutch’s Dr. E. Donnall Thomas. In his article, Mukherjee profiles recent T-cell therapy research by Dr. Carl June at the Perelman School of Medicine of the University of Pennsylvania and other leaders in the immunotherapy field including Drs. Steve Rosenberg and Michel Sadelain and the Hutch’s Drs. Stan Riddell and Phil Greenberg. In addition to the promising early successes with this new therapy, Mukherjee explores some of the challenges that remain to making these approaches more accessible and affordable. In particular, the staggering price of custom single-patient CAR-T immunotherapy is in the hundreds of thousands or millions of dollars, posing a challenge to health insurance and national healthcare systems.]
A Russian scientist says he is planning to produce gene-edited babies, an act that would make him only the second person known to have done this. It would also fly in the face of the scientific consensus that such experiments should be banned until an international ethical framework has agreed on the circumstances and safety measures that would justify them.
Molecular biologist Denis Rebrikov has told Nature he is considering implanting gene-edited embryos into women, possibly before the end of the year if he can get approval by then. Chinese scientist He Jiankui prompted an international outcry when he announced last November that he had made the world’s first gene-edited babies—twin girls.
…Rebrikov heads a genome-editing laboratory at Russia’s largest fertility clinic, the Kulakov National Medical Research Center for Obstetrics, Gynecology and Perinatology in Moscow and is a researcher at the Pirogov Russian National Research Medical University, also in Moscow. According to Rebrikov he already has an agreement with an HIV centre in the city to recruit women infected with HIV who want to take part in the experiment…[he] plans to implant embryos only into a subset of HIV-positive mothers who do not respond to standard anti-HIV drugs. Their risk of transmitting the infection to the child is higher. If editing successfully disables the CCR5 gene, that risk would be greatly reduced, Rebrikov says. ‘This is a clinical situation which calls for this type of therapy’, he says.
Five Russian couples who are deaf want to try the CRISPR gene-editing technique so they can have a biological child who can hear, biologist Denis Rebrikov has told New Scientist. He plans to apply to the relevant Russian authorities for permission in “a couple of weeks”…Both would-be parents in each couple have a recessive form of deafness, meaning that all their children would normally inherit the same condition. While the vast majority of genetic diseases can be prevented by screening IVF embryos before implantation, with no need for gene-editing, this is not an option for these couples. Several reports have suggested that—if it can be done safely—editing the genes of babies might be justified in this kind of situation…Now Rebrikov has told New Scientist that he also wants to prevent children inheriting a form of deafness caused by mutations in the GJB2 gene. In western Siberia, many people have a missing DNA letter in position 35 of the GJB2 gene. Having one copy has no effect, but those who inherit this mutation from both parents never develop the ability to hear. Rebrikov has found five couples in which both would-be parents are deaf because of this mutation and don’t want their children to be deaf too. So he plans to use CRISPR to correct this mutation in IVF embryos from these couples. All these embryos will have the mutation in both copies of the GJB2 gene—correcting one copy using a method known as homology-directed repair will prevent deafness. “Technically, it is achievable”, says Burgio.
Some 80% of active sniffer dogs deployed by South Korea’s quarantine agency are cloned, data showed Monday, as activists express their concerns over potential animal abuse. According to the Animal and Plant Quarantine Agency, 42 of its 51 sniffer dogs were cloned from parent animals as of April, indicating such cloned detection dogs are already making substantial contributions to the country’s quarantine activities. The number of cloned dogs first outpaced their naturally born counterparts in 2014, the agency said. Of the active cloned dogs, 39 are currently deployed at Incheon International Airport, the country’s main gateway.
Deploying cloned dogs can save time and money over training naturally born puppies as they maintain the outstanding traits of their parents, whose capabilities have already been verified in the field, according to experts. While the average cost of raising one detection dog is over 100 million won (US$85,600), it is less than half that when utilising cloned puppies, they said.
To extend the frontier of genome editing and enable the radical redesign of mammalian genomes, we developed a set of dead-Cas9 base editor (dBEs) variants that allow editing at tens of thousands of loci per cell by overcoming the cell death associated with DNA double-strand breaks (DSBs) and single-strand breaks (SSBs). We used a set of gRNAs targeting repetitive elements—ranging in target copy number from about 31 to 124,000 per cell. dBEs enabled survival after large-scale base editing, allowing targeted mutations at up to ~13,200 and ~2610 loci in 293T and human induced pluripotent stem cells (hiPSCs), respectively, three orders of magnitude greater than previously recorded. These dBEs can overcome current on-target mutation and toxicity barriers that prevent cell survival after large-scale genome engineering.
One Sentence Summary
Base editing with reduced DNA nicking allows for the simultaneous editing of >10,000 loci in human cells.
A gene drive biases the transmission of one of the two copies of a gene such that it is inherited more frequently than by random segregation. Highly efficient gene drive systems have recently been developed in insects, which leverage the sequence-targeted DNA cleavage activity of CRISPR-Cas9 and endogenous homology-directed repair mechanisms to convert heterozygous genotypes to homozygosity1,2,3,4. If implemented in laboratory rodents, similar systems would enable the rapid assembly of currently impractical genotypes that involve multiple homozygous genes (for example, to model multigenic human diseases). To our knowledge, however, such a system has not yet been demonstrated in mammals. Here we use an active genetic element that encodes a guide RNA, which is embedded in the mouse tyrosinase (Tyr) gene, to evaluate whether targeted gene conversion can occur when CRISPR-Cas9 is active in the early embryo or in the developing germline. Although Cas9 efficiently induces double-stranded DNA breaks in the early embryo and male germline, these breaks are not corrected by homology-directed repair. By contrast, Cas9 expression limited to the female germline induces double-stranded breaks that are corrected by homology-directed repair, which copies the active genetic element from the donor to the receiver chromosome and increases its rate of inheritance in the next generation. These results demonstrate the feasibility of CRISPR-Cas9-mediated systems that bias inheritance of desired alleles in mice and that have the potential to transform the use of rodent models in basic and biomedical research.
The under-representation of non-European samples in genome-wide association studies could ultimately restrict who benefits from medical advances through genomic science. Our aim was therefore to address the fundamental question whether causal variants for blood lipids are shared across populations.
A polygenic score based on established LDL-cholesterol-associated loci from European discovery samples had consistent effects on serum levels in samples from the UK, Uganda and Greek population isolates (correlation coefficient r = 0.23 to 0.28 per LDL standard deviation, p < 1.9×10−14). Trans-ethnic genetic correlations between European ancestry, Chinese and Japanese cohorts did not differ statistically-significantly from 1 for HDL, LDL and triglycerides. In each study, >60% of major lipid loci displayed evidence of replication with one exception. There was evidence for an effect on serum levels in the Ugandan samples for only 10% of major triglyceride loci. The PRS was only weakly associated in this group (r = 0.06, SE = 0.013). We establish trans-ethnic colocalization as a method to distinguish shared from population-specific trait loci.
Our results provide evidence for high levels of consistency of genetic associations for cholesterol biomarkers across populations. However, we also demonstrate that the degree of shared causal genetic architecture can be population-specific, trait-specific, and locus-specific. Efforts to implement genetic risk prediction in clinical settings should account for this.
Genome editing holds promise for correcting pathogenic mutations. However, it is difficult to determine off-target effects of editing due to single nucleotide polymorphism in individuals. Here, we developed a method named GOTI (Genome-wide Off-target analysis by Two-cell embryo Injection) to detect off-target mutations by editing one blastomere of two-cell mouse embryos using either CRISPR-Cas9 or base editors. Comparison of the whole genome sequences of progeny cells of edited vs. non-edited blastomeres at E14.5 showed that off-target single nucleotide variants (SNVs) were rare in embryos edited by CRISPR-Cas9 or adenine base editor, with a frequency close to the spontaneous mutation rate. In contrast, cytosine base editing induced SNVs with over 20× higher frequencies, requiring a solution to address its fidelity.
Photorespiration is required in C3 plants to metabolize toxic glycolate formed when ribulose-1,5-bisphosphate carboxylase-oxygenase oxygenates rather than carboxylates ribulose-1,5-bisphosphate. Depending on growing temperatures, photorespiration can reduce yields by 20 to 50% in C3 crops. Inspired by earlier work, we installed into tobacco chloroplasts synthetic glycolate metabolic pathways that are thought to be more efficient than the native pathway. Flux through the synthetic pathways was maximized by inhibiting glycolate export from the chloroplast. The synthetic pathways tested improved photosynthetic quantum yield by 20%. Numerous homozygous transgenic lines increased biomass productivity between 19 and 37% in replicated field trials. These results show that engineering alternative glycolate metabolic pathways into crop chloroplasts while inhibiting glycolate export into the native pathway can drive increases in C3 crop yield under agricultural field conditions.
Malaria control efforts require implementation of new technologies that manage insecticide resistance. Metarhizium pingshaense provides an effective, mosquito-specific delivery system for potent insect-selective toxins. A semifield trial in a MosquitoSphere (a contained, near-natural environment) in Soumousso, a region of Burkina Faso where malaria is endemic, confirmed that the expression of an insect-specific toxin (Hybrid) increased fungal lethality and the likelihood that insecticide-resistant mosquitoes would be eliminated from a site. Also, as Hybrid-expressing M. pingshaense is effective at very low spore doses, its efficacy lasted longer than that of the unmodified Metarhizium. Deployment of transgenic Metarhizium against mosquitoes could (subject to appropriate registration) be rapid, with products that could synergistically integrate with existing chemical control strategies to avert insecticide resistance.
Should we use human germline genome modification (HGGM) only when serious diseases are involved? This belief is the underlying factor in the article written by Kleiderman, Ravitsky and Knoppers to which I now respond. In my opinion, the answer to this question should be negative. In this paper, I attempt to show that there are no good reasons to think that this technology should be limited to serious diseases once it is sufficiently proven to be safe and efficient. In fact, opting otherwise would negatively harm human beings’ right to the highest standard of health that unmodified embryos could promote. Therefore, the issue should not be so much to define adequately what a serious disease is, but rather to elucidate whether this concept should play any role beyond the context of preimplantation genetic testing (PGT). This paper argues that we should not accept the similarity between technologies such as PGT and HGGM because they face different challenges and offer totaly different possibilities. Therefore, we are in urgent need to build a completely new ethical architecture that covers the application of germline editing in human embryos. As a part of that process, a much deeper debate on the necessity of distinguishing different disease types is required.
Studies of the relationship between genetic and phenotypic variation have historically been carried out on people of European ancestry. Efforts are underway to address this limitation, but until they succeed, the legacy of a Euro-centric bias in medical genetic studies will continue to hinder research, including the use of polygenic scores, which are individual-level metrics of genetic risk. Ongoing debate surrounds the generalizability of polygenic scores based on genome-wide association studies (GWAS) conducted in European ancestry samples, to non-European ancestry samples. We analyzed the first decade of polygenic scoring studies (2008-2017, inclusive), and found that 67% of studies included exclusively European ancestry participants and another 19% included only East Asian ancestry participants. Only 3.8% of studies were carried out on samples of African, Hispanic, or Indigenous peoples. We find that effect sizes for European ancestry-derived polygenic scores are only 36% as large in African ancestry samples, as in European ancestry samples (t=-10.056, df = 22, p = 5.5×10−10). Poorer performance was also observed in other non-European ancestry samples. Analysis of polygenic scores in the 1000Genomes samples revealed many strong correlations with global principal components, and relationships between height polygenic scores and height phenotypes that were highly variable depending on methodological choices in polygenic score construction. As polygenic score use increases in research, precision medicine, and direct-to-consumer testing, improved handling of linkage disequilibrium and variant frequencies (both of which currently reduce transferability of scores) across populations will improve polygenic score performance. These findings bolster the rationale for large-scale GWAS in diverse human populations.
The modern genetics revolution enabled rough calculations of individuals’ genetic liability for many phenotypes, including height, weight, and schizophrenia. Increasingly, polygenic scores, which are individual-level metrics of genetic liability, are available via direct-to-consumer testing, and they are already widely used in research. The performance of these scores depends on the availability of very large genetic studies, and consequently it is problematic that people of European ancestry are vastly over-represented in these studies. We quantify the magnitude of this problem on the performance of polygenic scores in global samples and also show ancestry-related properties of polygenic scores. These findings set benchmarks for future progress, and they demonstrate the need for large-scale genetic studies in diverse human populations.
Improved plant varieties are important in our attempts to face the challenges of a growing human population and limited planet resources. Plant breeding relies on meiotic crossovers to combine favourable alleles into elite varieties. However, meiotic crossovers are relatively rare, typically one to three per chromosome, limiting the efficiency of the breeding process and related activities such as genetic mapping.
Several genes that limit meiotic recombination were identified in the model species Arabidopsis thaliana. Mutation of these genes in Arabidopsis induces a large increase in crossover frequency. However, it remained to be demonstrated whether crossovers could also be increased in crop species hybrids.
We explored the effects of mutating the orthologues of FANCM, RECQ4 or FIGL1 on recombination in three distant crop species, rice (Oryza sativa), pea (Pisum sativum) and tomato (Solanum lycopersicum). We found that the single recq4 mutation increases crossovers about three-fold in these crops, suggesting that manipulating RECQ4 may be an universal tool for increasing recombination in plants.
Enhanced recombination could be used with other state-of-the-art technologies such as genomic selection, genome editing or speed breeding6 to enhance the pace and efficiency of plant improvement.
Mutations in the gene encoding dystrophin, a protein that maintains muscle integrity and function, cause Duchenne muscular dystrophy (DMD). The deltaE50-MD dog model of DMD harbors a mutation corresponding to a mutational “hotspot” in the human DMD gene. We used adeno-associated viruses to deliver CRISPR gene editing components to four dogs and examined dystrophin protein expression 6 weeks after intramuscular delivery (n = 2) or 8 weeks after systemic delivery (n = 2). After systemic delivery in skeletal muscle, dystrophin was restored to levels ranging from 3 to 90% of normal, depending on muscle type. In cardiac muscle, dystrophin levels in the dog receiving the highest dose reached 92% of normal. The treated dogs also showed improved muscle histology. These large-animal data support the concept that, with further development, gene editing approaches may prove clinically useful for the treatment of DMD.
The human population is growing, and as a result we need to produce more food whilst reducing the impact of farming on the environment. Selective breeding and genomic selection have had a transformational impact on livestock productivity, and now transgenic and genome-editing technologies offer exciting opportunities for the production of fitter, healthier and more-productive livestock. Here, we review recent progress in the application of genome editing to farmed animal species and discuss the potential impact on our ability to produce food.
Background: Identifying genetic relationships between complex traits in emerging adulthood can provide useful etiological insights into risk for psychopathology. College-age individuals are under-represented in genomic analyses thus far, and the majority of work has focused on the clinical disorder or cognitive abilities rather than normal-range behavioral outcomes.
Methods: This study examined a sample of emerging adults 18–22 years of age (n = 5947) to construct an atlas of polygenic risk for 33 traits predicting relevant phenotypic outcomes. 28 hypotheses were tested based on the previous literature on samples of European ancestry, and the availability of rich assessment data allowed for polygenic predictions across 55 psychological and medical phenotypes.
Results: Polygenic risk for schizophrenia (SZ) in emerging adults predicted anxiety, depression, nicotine use, trauma, and family history of psychological disorders. Polygenic risk for neuroticism predicted anxiety, depression, phobia, panic, neuroticism, and was correlated with polygenic risk for cardiovascular disease.
Conclusions: These results demonstrate the extensive impact of genetic risk for SZ, neuroticism, and major depression on a range of health outcomes in early adulthood. Minimal cross-ancestry replication of these phenomic patterns of polygenic influence underscores the need for more genome-wide association studies of non-European populations.
Understanding the role of rare variants is important in elucidating the genetic basis of human diseases and complex traits. It is widely believed that negative selection can cause rare variants to have larger per-allele effect sizes than common variants. Here, we develop a method to estimate the minor allele frequency (MAF) dependence of SNP effect sizes. We use a model in which per-allele effect sizes have variance proportional to [p(1−p)]α, where p is the MAF and negative values of α imply larger effect sizes for rare variants. We estimate α by maximizing its profile likelihood in a linear mixed model framework using imputed genotypes, including rare variants (MAF >0.07%). We applied this method to 25 UK Biobank diseases and complex traits (n = 113,851). All traits produced negative α estimates with 20 significantly negative, implying larger rare variant effect sizes. The inferred best-fit distribution of true α values across traits had mean −0.38 (s.e. 0.02) and standard deviation 0.08 (s.e. 0.03), with statistically-significant heterogeneity across traits (p = 0.0014). Despite larger rare variant effect sizes, we show that for most traits analyzed, rare variants (MAF <1%) explain less than 10% of total SNP-heritability. Using evolutionary modeling and forward simulations, we validated the α model of MAF-dependent trait effects and estimated the level of coupling between fitness effects and trait effects. Based on this analysis an average genome-wide negative selection coefficient on the order of 10−4 or stronger is necessary to explain the α values that we inferred.
Recent work has hinted at the linkage disequilibrium (LD)-dependent architecture of human complex traits, where SNPs with low levels of LD (LLD) have larger per-SNP heritability. Here we analyzed summary statistics from 56 complex traits (average n = 101,401) by extending stratified LD score regression to continuous annotations. We determined that SNPs with low LLD have statistically-significantly larger per-SNP heritability and that roughly half of this effect can be explained by functional annotations negatively correlated with LLD, such as DNase I hypersensitivity sites (DHSs). The remaining signal is largely driven by our finding that more recent common variants tend to have lower LLD and to explain more heritability (p = 2.38 × 10−104); the youngest 20% of common SNPs explain 3.9× more heritability than the oldest 20%, consistent with the action of negative selection. We also inferred jointly statistically-significant effects of other LD-related annotations and confirmed via forward simulations that they jointly predict deleterious effects.
Obesity is a risk factor for a wide variety of health problems. In a genome-wide association study (GWAS) of body mass index (BMI) in Japanese people (n = 173,430), we found 85 loci statistically-significantly associated with obesity (p < 5.0 × 10−8), of which 51 were previously unknown. We conducted trans-ancestral meta-analyses by integrating these results with the results from a GWAS of Europeans and identified 61 additional new loci. In total, this study identifies 112 novel loci, doubling the number of previously known BMI-associated loci. By annotating associated variants with cell-type-specific regulatory marks, we found enrichment of variants in CD19+ cells. We also found statistically-significant genetic correlations between BMI and lymphocyte count (p = 6.46 × 10−5, rg = 0.18) and between BMI and multiple complex diseases. These findings provide genetic evidence that lymphocytes are relevant to body weight regulation and offer insights into the pathogenesis of obesity.
Xenotransplantation is a promising strategy to alleviate the shortage of organs for human transplantation. In addition to the concern on pig-to-human immunological compatibility, the risk of cross-species transmission of porcine endogenous retroviruses (PERVs) has impeded the clinical application of this approach. Earlier, we demonstrated the feasibility of inactivating PERV activity in an immortalized pig cell line. Here, we confirmed that PERVs infect human cells, and observed the horizontal transfer of PERVs among human cells. Using CRISPR-Cas9, we inactivated all the PERVs in a porcine primary cell line and generated PERV-inactivated pigs via somatic cell nuclear transfer. Our study highlighted the value of PERV inactivation to prevent cross-species viral transmission and demonstrated the successful production of PERV-inactivated animals to address the safety concern in clinical xenotransplantation.
Genome editing has potential for the targeted correction of germline mutations. Here we describe the correction of the heterozygous MYBPC3 mutation in human preimplantation embryos with precise CRISPR-Cas9-based targeting accuracy and high homology-directed repair efficiency by activating an endogenous, germline-specific DNA repair response. Induced double-strand breaks (DSBs) at the mutant paternal allele were predominantly repaired using the homologous wild-type maternal gene instead of a synthetic DNA template. By modulating the cell cycle stage at which the DSB was induced, we were able to avoid mosaicism in cleaving embryos and achieve a high yield of homozygous embryos carrying the wild-type MYBPC3 gene without evidence of off-target mutations. The efficiency, accuracy and safety of the approach presented suggest that it has potential to be used for the correction of heritable mutations in human embryos by complementing preimplantation genetic diagnosis. However, much remains to be considered before clinical applications, including the reproducibility of the technique with other heterozygous mutations.
Polygenic risk scores are gaining more and more attention for estimating genetic risks for liabilities, especially for noncommunicable diseases. They are now calculated using thousands of DNA markers. In this paper, we compare the score distributions of two previously published very large risk score models within different populations. We show that the risk score model together with its risk stratification thresholds, built upon the data of one population, cannot be applied to another population without taking into account the target population’s structure. We also show that if an individual is classified to the wrong population, his/her disease risk can be systematically incorrectly estimated.
Photosynthesis is the basis of primary productivity on the planet. Crop breeding has sustained steady improvements in yield to keep pace with population growth increases. Yet these advances have not resulted from improving the photosynthetic process per se but rather of altering the way carbon is partitioned within the plant. Mounting evidence suggests that the rate at which crop yields can be boosted by traditional plant breeding approaches is wavering, and they may reach a “yield ceiling” in the foreseeable future. Further increases in yield will likely depend on the targeted manipulation of plant metabolism. Improving photosynthesis poses one such route, with simulations indicating it could have a significant transformative influence on enhancing crop productivity. Here, we summarize recent advances of alternative approaches for the manipulation and enhancement of photosynthesis and their possible application for crop improvement.
Reliable genome editing via Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 may provide a means to correct inherited diseases in patients. As proof of principle, we show that CRISPR/Cas9 can be used in vivo to selectively ablate the rhodopsin gene carrying the dominant S334ter mutation (Rho(S334)) in rats that model severe autosomal dominant retinitis pigmentosa. A single subretinal injection of guide RNA/Cas9 plasmid in combination with electroporation generated allele-specific disruption of Rho(S334), which prevented retinal degeneration and improved visual function.
We discuss a multiple-play multi-armed bandit (MAB) problem in which several arms are selected at each round. Recently, Thompson sampling (TS), a randomized algorithm with a Bayesian spirit, has attracted much attention for its empirically excellent performance, and it is revealed to have an optimal regret bound in the standard single-play MAB problem. In this paper, we propose the multiple-play Thompson sampling (MP-TS) algorithm, an extension of TS to the multiple-play MAB problem, and discuss its regret analysis. We prove that MP-TS for binary rewards has the optimal regret upper bound that matches the regret lower bound provided by Anantharam et al 1987. Therefore, MP-TS is the first computationally efficient algorithm with optimal regret. A set of computer simulations was also conducted, which compared MP-TS with state-of-the-art algorithms. We also propose a modification of MP-TS, which is shown to have better empirical performance.
Genome editing tools such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated system (Cas) have been widely used to modify genes in model systems including animal zygotes and human cells, and hold tremendous promise for both basic research and clinical applications. To date, a serious knowledge gap remains in our understanding of DNA repair mechanisms in human early embryos, and in the efficiency and potential off-target effects of using technologies such as CRISPR/Cas9 in human pre-implantation embryos. In this report, we used tripronuclear(3PN) zygotes to further investigate CRISPR/Cas9-mediated gene editing in human cells. We found that CRISPR/Cas9 could effectively cleave the endogenousβ-globin gene (HBB). However, the efficiency of homologous recombination directed repair (HDR) of HBB was low and the edited embryos were mosaic. Off-target cleavage was also apparent in these 3PN zygotes as revealed by the T7E1 assay and whole-exome sequencing. Furthermore, the endogenous delta-globin gene (HBD), which is homologous to HBB, competed with exogenous donor oligos to act as the repair template, leading to untoward mutations. Our data also indicated that repair of the HBB locus in these embryos occurred preferentially through the non-crossover HDR pathway. Taken together, our work highlights the pressing need to further improve the fidelity and specificity of the CRISPR/Cas9 platform, a prerequisite for any clinical applications of CRISPR/Cas9-mediated editing.
I was a college teacher when opportunity opened a path into academia. A fascination with totipotency channeled me into research on tissue culture. As I was more interested in contributions to food security than in scientific novelty, I turned my attention to the development of genetic modification technology for cereals. From my cell culture experience, I had reasons not to trust Agrobacterium for that purpose, and I developed direct gene transfer instead. In the early 1990s, I became aware of the problem of micronutrient deficiency, particularly vitamin A deficiency in rice-eating populations. Golden Rice, which contains increased amounts of provitamin A, was probably instrumental for the concept of biofortification to take off. I realized that this rice would remain an academic exercise if product development and product registration were not addressed, and this is what I focused on after my retirement. Although progress is slowly being made, had I known what this pursuit would entail, perhaps I would not have started. Hopefully Golden Rice will reach the needy during my lifetime.
[Keywords: Golden Rice, biofortification, genetic engineering, public good, GMO regulation, Autobiography]
Inheritance-biasing “gene drives” may be capable of spreading genomic alterations made in laboratory organisms through wild populations. We previously considered the potential for RNA-guided gene drives based on the versatile CRISPR/Cas9 genome editing system to serve as a general method of altering populations1. Here we report molecularly contained gene drive constructs in the yeast Saccharomyces cerevisiae that are typically copied at rates above 99% when mated to wild yeast. We successfully targeted both non-essential and essential genes, showed that the inheritance of an unrelated “cargo” gene could be biased by an adjacent drive, and constructed a drive capable of overwriting and reversing changes made by a previous drive. Our results demonstrate that RNA-guided gene drives are capable of efficiently biasing inheritance when mated to wild-type organisms over successive generations.
This document provides further details about materials, methods and additional analyses to accompany the research report “Proxy-Phenotype Method Identifies Common Genetic Variants Associated with Cognitive Performance.”
Genomic disorders resulting from large rearrangements of the genome remain an important unsolved issue in gene therapy. Chromosome transplantation, defined as the perfect replacement of an endogenous chromosome with a homologous one, has the potential of curing this kind of disorders. Here we report the first successful case of chromosome transplantation by replacement of an endogenous X chromosome carrying a mutation in the Hprt genewith a normal one in mouse embryonic stem cells (ESCs), correcting the genetic defect. The defect was also corrected by replacing the Y chromosome with an X chromosome. Chromosome transplanted clones maintained in vitro and in vivo features of stemness and contributed to chimera formation. Genome integrity was confirmed by cytogenetic and molecular genome analysis. The approach here proposed, with some modifications, might be used to cure various disorders due to other X chromosome aberrations in induced pluripotent stem (iPS) cells derived from affected patients.
An organism with a single recessive loss-of-function allele will typically have a wild-type phenotype, whereas individuals homozygous for two copies of the allele will display a mutant phenotype. We have developed a method called the mutagenic chain reaction (MCR), which is based on the CRISPR/Cas9 genome-editing system for generating autocatalytic mutations, to produce homozygous loss-of-function mutations. In Drosophila, we found that MCR mutations efficiently spread from their chromosome of origin to the homologous chromosome, thereby converting heterozygous mutations to homozygosity in the vast majority of somatic and germline cells. MCR technology should have broad applications in diverse organisms.
Many multi-step processes look like ‘leaky pipelines’, where a fractional loss/success happens at every step; such multiplicative processes can often be modeled as a log-normal distribution (or power law), with counterintuitive implications like skewed output distributions and large final differences from small differences in per-step success rates.
Statistical methodology has played a key role in scientific animal breeding. ~1hundred years of statistical developments in animal breeding are reviewed. Some of the scientific foundations of the field are discussed, and many milestones are examined from historical and critical perspectives. The review concludes with a discussion of some future challenges and opportunities arising from the massive amount of data generated by livestock, plant, and human genome projects.
The CRISPR/Cas9 system has been adapted as an efficient genome editing tool in laboratory animals such as mice, rats, zebrafish and pigs. Here, we report that CRISPR/Cas9 mediated approach can efficiently induce monoallelic and biallelic gene knockout in goat primary fibroblasts. Four genes were disrupted simultaneously in goat fibroblasts by CRISPR/Cas9-mediated genome editing. The single-gene knockout fibroblasts were successfully used for somatic cell nuclear transfer (SCNT) and resulted in live-born goats harboring biallelic mutations. The CRISPR/Cas9 system represents a highly effective and facile platform for targeted editing of large animal genomes, which can be broadly applied to both biomedical and agricultural applications.
Duchenne muscular dystrophy (DMD) is an inherited X-linked disease caused by mutations in the gene encoding dystrophin, a protein required for muscle fiber integrity. DMD is characterized by progressive muscle weakness and a shortened life span, and there is no effective treatment.
We used clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9)-mediated genome editing to correct the dystrophin gene (Dmd) mutation in the germ line of mdx mice, a model for DMD, and then monitored muscle structure and function. Genome editing produced genetically mosaic animals containing 2 to 100% correction of the Dmd gene. The degree of muscle phenotypic rescue in mosaic mice exceeded the efficiency of gene correction, likely reflecting an advantage of the corrected cells and their contribution to regenerating muscle.
With the anticipated technological advances that will facilitate genome editing of postnatal somatic cells, this strategy may one day allow correction of disease-causing mutations in the muscle tissue of patients with DMD.
Current antibiotics tend to be broad spectrum, leading to indiscriminate killing of commensal bacteria and accelerated evolution of drug resistance. Here, we use CRISPR-Cas technology to create antimicrobials whose spectrum of activity is chosen by design. RNA-guided nucleases (RGNs) targeting specific DNA sequences are delivered efficiently to microbial populations using bacteriophage or bacteria carrying plasmids transmissible by conjugation. The DNA targets of RGNs can be undesirable genes or polymorphisms, including antibiotic resistance and virulence determinants in carbapenem-resistant Enterobacteriaceae and enterohemorrhagic Escherichia coli. Delivery of RGNs significantly improves survival in a Galleria mellonella infection model. We also show that RGNs enable modulation of complex bacterial populations by selective knockdown of targeted strains based on genetic signatures. RGNs constitute a class of highly discriminatory, customizable antimicrobials that enact selective pressure at the DNA level to reduce the prevalence of undesired genes, minimize off-target effects and enable programmable remodeling of microbiota.
We demonstrate CRISPR-Cas9-mediated correction of a Fah mutation in hepatocytes in a mouse model of the human disease hereditary tyrosinemia. Delivery of components of the CRISPR-Cas9 system by hydrodynamic injection resulted in initial expression of the wild-type Fah protein in ~1⁄250 liver cells. Expansion of Fah-positive hepatocytes rescued the body weight loss phenotype. Our study indicates that CRISPR-Cas9-mediated genome editing is possible in adult animals and has potential for correction of human genetic diseases.
Schizophrenia is a highly heritable disorder. Genetic risk is conferred by a large number of alleles, including common alleles of small effect that might be detected by genome-wide association studies. Here we report a multi-stage schizophrenia genome-wide association study of up to 36,989 cases and 113,075 controls. We identify 128 independent associations spanning 108 conservatively defined loci that meet genome-wide statistical-significance, 83 of which have not been previously reported. Associations were enriched among genes expressed in brain, providing biological plausibility for the findings. Many findings have the potential to provide entirely new insights into aetiology, but associations at DRD2 and several genes involved in glutamatergic neurotransmission highlight molecules of known and potential therapeutic relevance to schizophrenia, and are consistent with leading pathophysiological hypotheses. Independent of genes expressed in brain, associations were enriched among genes expressed in tissues that have important roles in immunity, providing support for the speculated link between the immune system and schizophrenia.
Study Objectives: Earlier work described a mutation in DEC2 also known as BHLHE41 (basic helix-loophelix family member e41) as causal in a family of short sleepers, who needed just 6 h sleep per night. We evaluated whether there were other variants of this gene in two well-phenotyped cohorts.
Design: Sequencing of the BHLHE41 gene, electroencephalographic data, and delta power analysis and functional studies using cell-based luciferase.
Results: We identified new variants of the BHLHE41 gene in two cohorts who had either acute sleep deprivation (n = 200) or chronic partial sleep deprivation (n = 217). One variant, Y362H, at another location in the same exon occurred in one twin in a dizygotic twin pair and was associated with reduced sleep duration, less recovery sleep following sleep deprivation, and fewer performance lapses during sleep deprivation than the homozygous twin. Both twins had almost identical amounts of non rapid eye movement (NREM) sleep. This variant reduced the ability of BHLHE41 to suppress CLOCK/BMAL1 and NPAS2/BMAL1 transactivation in vitro. Another variant in the same exome had no effect on sleep or response to sleep deprivation and no effect on CLOCK/BMAL1 transactivation. Random mutagenesis identified a number of other variants of BHLHE41 that affect its function.
Conclusions: There are a number of mutations of BHLHE41. Mutations reduce total sleep while maintaining NREM sleep and provide resistance to the effects of sleep loss. Mutations that affect sleep also modify the normal inhibition of BHLHE41 of CLOCK/BMAL1 transactivation. Thus, clock mechanisms are likely involved in setting sleep length and the magnitude of sleep homeostasis.
Citation: Pellegrino R, Kavakli IH, Goel N, Cardinale CJ, Dinges DF, Kuna ST, Maislin G, Van Dongen HP, Tufik S, Hogenesch JB, Hakonarson H, Pack AI. A novel BHLHE41 variant is associated with short sleep and resistance to sleep deprivation in humans. SLEEP 2014;37(8):1327–1336.
It has long been recognized that generalized deficits in cognitive ability represent a core component of schizophrenia (SCZ), evident before full illness onset and independent of medication. The possibility of genetic overlap between risk for SCZ and cognitive phenotypes has been suggested by the presence of cognitive deficits in first-degree relatives of patients with SCZ; however, until recently, molecular genetic approaches to test this overlap have been lacking. Within the last few years, large-scale genome-wide association studies (GWAS) of SCZ have demonstrated that a substantial proportion of the heritability of the disorder is explained by a polygenic component consisting of many common single-nucleotide polymorphisms (SNPs) of extremely small effect. Similar results have been reported in GWAS of general cognitive ability. The primary aim of the present study is to provide the first molecular genetic test of the classic endophenotype hypothesis, which states that alleles associated with reduced cognitive ability should also serve to increase risk for SCZ. We tested the endophenotype hypothesis by applying polygenic SNP scores derived from a large-scale cognitive GWAS meta-analysis (~5000 individuals from nine nonclinical cohorts comprising the Cognitive Genomics consorTium (COGENT)) to four SCZ case-control cohorts. As predicted, cases had significantly lower cognitive polygenic scores compared to controls. In parallel, polygenic risk scores for SCZ were associated with lower general cognitive ability. In addition, using our large cognitive meta-analytic data set, we identified nominally statistically-significant cognitive associations for several SNPs that have previously been robustly associated with SCZ susceptibility. Results provide molecular confirmation of the genetic overlap between SCZ and general cognitive ability, and may provide additional insight into pathophysiology of the disorder.
The vast majority of genome-wide association study (GWAS) findings reported to date are from populations with European Ancestry (EA), and it is not yet clear how broadly the genetic associations described will generalize to populations of diverse ancestry. The Population Architecture Using Genomics and Epidemiology (PAGE) study is a consortium of multi-ancestry, population-based studies formed with the objective of refining our understanding of the genetic architecture of common traits emerging from GWAS.
In the present analysis of five common diseases and traits, including body mass index, type 2 diabetes, and lipid levels, we compare direction and magnitude of effects for GWAS-identified variants in multiple non-EA populations against EA findings.
We demonstrate that, in all populations analyzed, a statistically-significant majority of GWAS-identified variants have allelic associations in the same direction as in EA, with none showing a statistically-significant effect in the opposite direction, after adjustment for multiple testing. However, 25% of tagSNPs identified in EA GWAS have statistically-significantly different effect sizes in at least one non-EA population, and these differential effects were most frequent in African Americans where all differential effects were diluted toward the null. We demonstrate that differential LD between tagSNPs and functional variants within populations contributes statistically-significantly to dilute effect sizes in this population.
Although most variants identified from GWAS in EA populations generalize to all non-EA populations assessed, genetic models derived from GWAS findings in EA may generate spurious results in non-EA populations due to differential effect sizes. Regardless of the origin of the differential effects, caution should be exercised in applying any genetic risk prediction model based on tagSNPs outside of the ancestry group in which it was derived. Models based directly on functional variation may generalize more robustly, but the identification of functional variants remains challenging.
Author Summary: The number of known associations between human diseases and common genetic variants has grown dramatically in the past decade, most being identified in large-scale genetic studies of people of Western European origin. But because the frequencies of genetic variants can differ substantially between continental populations, it’s important to assess how well these associations can be extended to populations with different continental ancestry. Are the correlations between genetic variants, disease endpoints, and risk factors consistent enough for genetic risk models to be reliably applied across different ancestries? Here we describe a systematic analysis of disease outcome and risk-factor-associated variants (tagSNPs) identified in European populations, in which we test whether the effect size of a tagSNP is consistent across six populations with statistically-significant non-European ancestry. We demonstrate that although nearly all such tagSNPs have effects in the same direction across all ancestries (ie. variants associated with higher risk in Europeans will also be associated with higher risk in other populations), roughly a quarter of the variants tested have statistically-significantly different magnitude of effect (usually lower) in at least one non-European population. We therefore advise caution in the use of tagSNP-based genetic disease risk models in populations that have a different genetic ancestry from the population in which original associations were first made. We then show that this differential strength of association can be attributed to population-dependent variations in the correlation between tagSNPs and the variant that actually determines risk—the so-called functional variant. Risk models based on functional variants are therefore likely to be more robust than tagSNP-based models.
Bacteria and archaea have evolved adaptive immune defenses, termed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems, that use short RNA to direct degradation of foreign nucleic acids. Here, we engineer the type II bacterial CRISPR system to function with custom guide RNA (gRNA) in human cells. For the endogenous AAVS1 locus, we obtained targeting rates of 10 to 25% in 293T cells, 13 to 8% in K562 cells, and 2 to 4% in induced pluripotent stem cells. We show that this process relies on CRISPR components; is sequence-specific; and, upon simultaneous introduction of multiple gRNAs, can effect multiplex editing of target loci. We also compute a genome-wide resource of ~190 K unique gRNAs targeting ~40.5% of human exons. Our results establish an RNA-guided editing tool for facile, robust, and multiplexable human genome engineering.
In bacteria, foreign nucleic acids are silenced by clustered, regularly interspaced, short palindromic repeats (CRISPR)–CRISPR-associated (Cas) systems. Bacterial type II CRISPR systems have been adapted to create guide RNAs that direct site-specific DNA cleavage by the Cas9 endonuclease in cultured cells. Here we show that the CRISPR-Cas system functions in vivo to induce targeted genetic modifications in zebrafish embryos with efficiencies similar to those obtained using zinc finger nucleases and transcription activator-like effector nucleases.
Type II CRISPR immune systems in bacteria use a dual RNA-guided DNA endonuclease, Cas9, to cleave foreign DNA at specific sites. We show here that Cas9 assembles with hybrid guide RNAs in human cells and can induce the formation of double-strand DNA breaks (DSBs) at a site complementary to the guide RNA sequence in genomic DNA. This cleavage activity requires both Cas9 and the complementary binding of the guide RNA. Experiments using extracts from transfected cells show that RNA expression and/or assembly into Cas9 is the limiting factor for Cas9-mediated DNA cleavage. In addition, we find that extension of the RNA sequence at the 3’ end enhances DNA targeting activity in vivo. These results show that RNA-programmed genome editing is a facile strategy for introducing site-specific genetic changes in human cells.DOI:http://dx.doi.org/10.7554/eLife.00471.001.
Study Objectives: To determine if the large and highly reproducible interindividual differences in rates of performance deficit accumulation during sleep deprivation, as determined by the number of lapses on a sustained reaction time test, the Psychomotor Vigilance Task (PVT), arise from a heritable trait.
Design: Prospective, observational cohort study.
Setting: Academic medical center.
Participants: There were 59 monozygotic (mean age 29.2 ± 6.8 [SD] yr; 15 male and 44 female pairs) and 41 dizygotic (mean age 26.6 ± 7.6 yr; 15 male and 26 female pairs) same-sex twin pairs with a normal polysomnogram.
Interventions: Thirty-eight hr of monitored, continuous sleep deprivation.
Measurements and Results: Patients performed the 10-min PVT every 2 hr during the sleep deprivation protocol. The primary outcome was change from baseline in square root transformed total lapses (response time ≥ 500 ms) per trial. Patient-specific linear rates of performance deficit accumulation were separated from circadian effects using multiple linear regression. Using the classic approach to assess heritability, the intraclass correlation coefficients for accumulating deficits resulted in a broad sense heritability (h2) estimate of 0.834. The mean within-pair and among-pair heritability estimates determined by analysis of variance-based methods was 0.715. When variance components of mixed-effect multilevel models were estimated by maximum likelihood estimation and used to determine the proportions of phenotypic variance explained by genetic and nongenetic factors, 51.1% (standard error = 8.4%, p < 0.0001) of twin variance was attributed to combined additive and dominance genetic effects.
Conclusion: Genetic factors explain a large fraction of interindividual variance among rates of performance deficit accumulations on PVT during sleep deprivation.
Sleep deprivation can impair human health and performance. Habitual total sleep time and homeostatic sleep response to sleep deprivation are quantitative traits in humans. Genetic loci for these traits have been identified in model organisms, but none of these potential animal models have a corresponding human genotype and phenotype.
We have identified a mutation in a transcriptional repressor (hDEC2-P385R) that is associated with a human short sleep phenotype. Activity profiles and sleep recordings of transgenic mice carrying this mutation showed increased vigilance time and less sleep time than control mice in a zeitgeber time-dependent and sleep deprivation-dependent manner.
These mice represent a model of human sleep homeostasis that provides an opportunity to probe the effect of sleep on human physical and mental health.
Theories of justice traditionally have regarded people’s natural endowments as being fixed facts of the genetic lottery.1 Some theorists, such as Robert Nozick, believe that we own our traits, talents, abilities, and genes even though they were endowed to us by chance.2 Other theorists argue that the inequalities inherent in the natural distribution of talents and abilities place a moral obligation on us to compensate the less fortunate for their genetic disadvantages.3
The important point is that until now, theories of justice have regarded one’s genetic endowment as a fixed fact of nature rather than as a matter of justice. The ability to control the genetic endowment of future generations calls for a rethinking of the traditional theories of justice. This paper aims to investigate how one such theory—John Rawls’s—might be modified to help us respond to this new moral problem in ways that reflect more completely our considered convictions about fairness and justice.
I argue that Rawls’s theory as it stands does not give us satisfactory answers to questions about how to regulate genetic manipulation.4 Rawls’s failure to take natural primary goods into account in identifying the least advantaged leads him to counterintuitive conclusions about who in society is worst off. Similarly, worries about the inflexibility of social primary goods and the consequences these worries have for the instantiation of conditions of fair equality of opportunity are serious weaknesses in Rawls’s theory of justice.
I explain how we can modify Rawls’s theory into a framework that allows us to govern genetic manipulation in humans in ways that more fully accommodate the fixed points of our considered judgments about justice.51 go on to show how such a modified theory would instruct us to use technologies for genetic correction and enhancement. Assuming a safe, effective, and inexpensive means of genetic manipulation, the modified Rawlsian theory mandates certain kinds of genetic intervention while permitting or prohibiting others.