[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 forRNA-guided cleavage of double-stranded DNA and can be harnessed forgenome 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 mRNAcargo 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 thatSEND is a modular platform suited for development as an efficient therapeutic delivery modality.
Here, we show that transgenic expression of the human RNAdemethylaseFTO 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 rootmeristem 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) inplant 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.
Oocytes mature in a specialized fluid-filled sac, the ovarian follicle, which provides signals needed for meiosis and germ cell growth. Methods have been developed to generate functional oocytes from pluripotent stem cell–derived primordial germ cell–like cells (PGCLCs) when placed in culture with embryonic ovarian somatic cells. In this study, we developed culture conditions to recreate the stepwise differentiation process from pluripotent cells to fetal ovarian somatic cell–like cells (FOSLCs). When FOSLCs were aggregatedwith PGCLCs derived from mouse embryonic stem cells, the PGCLCs entered meiosis to generate functional oocytes capable of fertilization and development to live offspring. Generating functional mouse oocytes in a reconstituted ovarian environment provides a method for in vitro oocyte production and follicle generation for a better understanding of mammalian reproduction.
2021-gillmore.pdf: “CRISPR–Cas9 In Vivo Gene Editing for Transthyretin Amyloidosis”, Julian D. Gillmore, Ed Gane, Jorg Taubel, Justin Kao, Marianna Fontana, Michael L. Maitland, Jessica Seitzer, Daniel O’Connell, Kathryn R. Walsh, Kristy Wood, Jonathan Phillips, Yuanxin Xu, Adam Amaral, Adam P. Boyd, Jeffrey E. Cehelsky, Mark D. McKee, Andrew Schiermeier, Olivier Harari, Andrew Murphy, Christos A. Kyratsous, Brian Zambrowicz, Randy Soltys, David E. Gutstein, John Leonard, Laura Sepp-Lorenzino, David Lebwohl (2021-06-26; backlinks):
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 asingle 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 ATTRamyloidosis 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.)
It is widely hypothesized that removing cellular transfer RNAs (tRNAs)—making their cognate codons unreadable—might create a genetic firewall to viral infection and enable sense codon reassignment. However, it has been impossible to test these hypotheses.
In this work, following synonymous codon compression and laboratory evolution in Escherichia coli, we deleted the tRNAs and release factor 1, which normally decode 2 sense codons and a stop codon; the resulting cells could not read the canonical genetic code and were completely resistant to a cocktail of viruses. We reassigned these codons to enable the efficient synthesis of proteins containing 3 distinct noncanonical amino acids. Notably, we demonstrate the facile reprogramming of our cells for the encoded translation of diverse noncanonical heteropolymers and macrocycles.
2021-newby.pdf: “Base editing of haematopoietic stem cells rescues sickle cell disease in mice”, Gregory A. Newby, Jonathan S. Yen, Kaitly J. Woodard, Thiyagaraj Mayuranathan, Cicera R. Lazzarotto, Yichao Li, Heather Sheppard-Tillman, Shaina N. Porter, Yu Yao, Kalin Mayberry, Kelcee A. Everette, Yoonjeong Jang, Christopher J. Podracky, Elizabeth Thaman, Christophe Lechauve, Akshay Sharma, Jordana M. Henderson, Michelle F. Richter, Kevin T. Zhao, Shannon M. Miller, Tina Wang, Luke W. Koblan, Anton P. McCaffrey, John F. Tisdale, Theodosia A. Kalfa, Shondra M. Pruett-Miller, Shengdar Q. Tsai, Mitchell J. Weiss, David R. Liu (2021-06-02):
Sickle cell disease (SCD) is caused by a mutation in the β-globin geneHBB.
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 a one-time autologous treatment for SCD that eliminates pathogenic HBBS, generates benign HBBG, and minimizes the undesired consequences of double-strand DNA breaks.
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.
2021-musunuru.pdf: “In vivo CRISPR base editing of PCSK9 durably lowers cholesterol in primates”, Kiran Musunuru, Alexandra C. Chadwick, Taiji Mizoguchi, Sara P. Garcia, Jamie E. DeNizio, Caroline W. Reiss, Kui Wang, Sowmya Iyer, Chaitali Dutta, Victoria Clendaniel, Michael Amaonye, Aaron Beach, Kathleen Berth, Souvik Biswas, Maurine C. Braun, Huei-Mei Chen, Thomas V. Colace, John D. Ganey, Soumyashree A. Gangopadhyay, Ryan Garrity, Lisa N. Kasiewicz, Jennifer Lavoie, James A. Madsen, Yuri Matsumoto, Anne Marie Mazzola, Yusuf S. Nasrullah, Joseph Nneji, Huilan Ren, Athul Sanjeev, Madeleine Shay, Mary R. Stahley, Steven H. Y. Fan, Ying K. Tam, Nicole M. Gaudelli, Giuseppe Ciaramella, Leslie E. Stolz, Padma Malyala, Christopher J. Cheng, Kallanthottathil G. Rajeev, Ellen Rohde, Andrew M. Bellinger, Sekar Kathiresan (2021-05-19; backlinks):
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.
2021-nunez.pdf: “Genome–wide programmable transcriptional memory by CRISPR–based epigenome editing”, James K. Nuñez, Jin Chen, Greg C. Pommier, J. Zachery Cogan, Joseph M. Replogle, Carmen Adriaens, Gokul N. Ramadoss, Quanming Shi, King L. Hung, Avi J. Samelson, Angela N. Pogson, James Y. S. Kim, Amanda Chung, Manuel D. Leonetti, Howard Y. Chang, Martin Kampmann, Bradley E. Bernstein, Volker Hovestadt, Luke A. Gilbert, Jonathan S. Weissman (2021-04-29):
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 ofiPSCs 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 genesincluding those lacking canonical CpG islands (CGIs) and reveal a widetargeting window extending beyond annotated CGIs.
The broad ability of CRISPRoff to initiate heritable gene silencingeven 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]
2021-trujillo.pdf: “Reintroduction of the archaic variant of NOVA1 in cortical organoids alters neurodevelopment”, Cleber A. Trujillo, Edward S. Rice, Nathan K. Schaefer, Isaac A. Chaim, Emily C. Wheeler, Assael A. Madrigal, Justin Buchanan, Sebastian Preissl, Allen Wang, Priscilla D. Negraes, Ryan A. Szeto, Roberto H. Herai, Alik Huseynov, Mariana S. A. Ferraz, Fernando S. Borges, Alexandre H. Kihara, Ashley Byrne, Maximillian Marin, Christopher Vollmers, Angela N. Brooks, Jonathan D. Lautz, Katerina Semendeferi, Beth Shapiro, Gene W. Yeo, Stephen E. P. Smith, Richard E. Green, Alysson R. Muotri (2021-02-12):
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 Oct4is required for the expansion of CNCC developmental potential to formfacial 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 a 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 uniformtranscriptional 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 ofOct4+ 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.
2021-koblan.pdf: “In vivo base editing rescues Hutchinson-Gilford progeria syndrome in mice”, Luke W. Koblan, Michael R. Erdos, Christopher Wilson, Wayne A. Cabral, Jonathan M. Levy, Zheng-Mei Xiong, Urraca L. Tavarez, Lindsay M. Davison, Yantenew G. Gete, Xiaojing Mao, Gregory A. Newby, Sean P. Doherty, Narisu Narisu, Quanhu Sheng, Chad Krilow, Charles Y. Lin, Leslie B. Gordon, Kan Cao, Francis S. Collins, Jonathan D. Brown, David R. Liu (2021-01-06; backlinks):
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 approximately 14 years. Adenine base editors (ABEs) convert targeted A•T base pairs to G•C base pairs with minimalby-products and without requiring double-strand DNA breaks or donorDNA templates. Here we describe the use of an ABE to directly correctthe pathogenic HGPS mutation in cultured fibroblasts derived fromchildren with progeria and in a mouse model of HGPS. Lentiviraldelivery of the ABE to fibroblasts from children with HGPS resulted in87–91% 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 a unique material for reproductive biology and medicine.
2020-lu.pdf: “Reprogramming to recover youthful epigenetic information and restore vision”, Yuancheng Lu, Benedikt Brommer, Xiao Tian, Anitha Krishnan, Margarita Meer, Chen Wang, Daniel L. Vera, Qiurui Zeng, Doudou Yu, Michael S. Bonkowski, Jae-Hyun Yang, Songlin Zhou, Emma M. Hoffmann, Margarete M. Karg, Michael B. Schultz, Alice E. Kane, Noah Davidsohn, Ekaterina Korobkina, Karolina Chwalek, Luis A. Rajman, George M. Church, Konrad Hochedlinger, Vadim N. Gladyshev, Steve Horvath, Morgan E. Levine, Meredith S. Gregory-Ksander, Bruce R. Ksander, Zhigang He, David A. Sinclair (2020-12-02; backlinks):
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 andregenerative 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 restoresyouthful 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-inducedreprogramming 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.
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.
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-basedstrategy 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.
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 programmableDNA-binding proteins. Fusions of the split-DddA halves, transcription activator-like effector array proteins, and a 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 hightarget specificity and product purity. We used DdCBEs to model adisease-associated mtDNA mutation in human cells, resulting in changes in respiration rates and oxidative phosphorylation. CRISPR-free DdCBEsenable the precise manipulation of mtDNA, rather than the eliminationof mtDNA copies that results from its cleavage by targeted nucleases, with broad implications for the study and potential treatment of mitochondrial disorders.
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 five times 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 patternsand 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 TETenzymes and the downstream enzyme TDG, indicating that alterations inDNA 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.
In honeybees, the ability of workers to produce daughters asexually, ie., thelytokous parthenogenesis, is restricted to a single subspecies inhabiting the Cape region of South Africa, Apis mellifera capensis. Thelytoky has unleashed new selective pressures and the evolution of traits such as social parasitism, invasiveness, and social cancer. Thelytoky arises from an abnormal meiosis that results in the fusion of two maternal pronuclei, restoring diploidy in newly laid eggs. The genetic basis underlying thelytoky is disputed. To resolve this controversy, we generated a backcross between thelytokous A. m. capensis and non-thelytokous A. m. scutellata from the neighboring population and looked for evidence of genetic markers that co-segregated with thelytokous reproduction in 49 backcross females. We found that markers associated with the gene GB45239 on chromosome 11, including non-synonymous variants, showed consistent co-segregation with thelytoky, whereas no other region did so. Alleles associated with thelytoky were present in all A. m. capensis genomes examined but were absent from all other honeybees worldwide including A. m. scutellata. GB45239 is derived in A. m. capensis and has a putative role in chromosome segregation. It is expressed in ovaries and is downregulated in thelytokous bees, likely because of polymorphisms in the promoter region. Our study reveals how mutations affecting the sequence and/or expression of a single gene can change the reproductive mode of a population.
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-Sachsdisease (requiring a deletion in HEXA); to install a protectivetransversion 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.
Early human embryonic development involves extensive lineage diversification, cell-fate specification and tissue patterning1. Despite its basic and clinical importance, early human embryonic development remains relatively unexplained owing to interspecies divergence2,3 and limited accessibility to human embryo samples. Here we report that human pluripotent stem cells (hPSCs) in a microfluidic device recapitulate, in a highly controllable and scalable fashion, landmarks of the development of the epiblast and amniotic ectoderm parts of the conceptus, including lumenogenesis of the epiblast and the resultant pro-amniotic cavity, formation of a bipolar embryonic sac, and specification of primordial germ cells and primitive streak cells. We further show that amniotic ectoderm-like cells function as a signalling centre to trigger the onset of gastrulation-like events in hPSCs. Given its controllability and scalability, the microfluidic model provides a powerful experimental system to advance knowledge of human embryology and reproduction. This model could assist in the rational design of differentiation protocols of hPSCs for disease modelling and cell therapy, and in high-throughput drug and toxicity screens to prevent pregnancy failure and birth defects.
The design and creation of synthetic genomes provide a powerful approach to understanding and engineering biology. However, it is often limited by the paucity of methods for precise genome manipulation. Here, we demonstrate the programmed fission of the Escherichia coli genome into diverse pairs of synthetic chromosomes and the programmed fusion of synthetic chromosomes to generate genomes with user-defined inversions and translocations. We further combine genome fission, chromosome transplant, and chromosome fusion to assemble genomic regions from different strains into a single genome. Thus, we program the scarless assembly of new genomes with nucleotide precision, a key step in the convergent synthesis of genomes from diverse progenitors. This work provides a set of precise, rapid, large-scale (megabase) genome-engineering operations for creating diverse synthetic genomes.
2019-fredens.pdf: “Total synthesis of Escherichia coli with a recoded genome”, Julius Fredens, Kaihang Wang, Daniel de la Torre, Louise F. H. Funke, Wesley E. Robertson, Yonka Christova, Tiongsun Chia, Wolfgang H. Schmied, Daniel L. Dunkelmann, Václav Beránek, Chayasith Uttamapinant, Andres Gonzalez Llamazares, Thomas S. Elliott, Jason W. Chin (2019-05-15; backlinks):
Nature uses 64 codons to encode the synthesis of proteins from the genome, and chooses 1 sense codon—out of up to 6 synonyms—to encode each amino acid. Synonymous codon choice has diverse and important roles, and many synonymous substitutions are detrimental. Here we demonstrate that the number of codons used to encode the canonical amino acids can be reduced, through the genome-wide substitution of target codons by defined synonyms. We create a variant of Escherichia coli with a four-megabase synthetic genome through a high-fidelity convergent total synthesis. Our synthetic genome implements a defined recoding and refactoring scheme—with simple corrections at just seven positions—to replace every known occurrence of two sense codons and a stop codon in the genome. Thus, we recode 18,214 codons to create an organism with a 61-codon genome; this organism uses 59 codons to encode the 20 amino acids, and enables the deletion of a previously essential transfer RNA.
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.
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.
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 thesimilarity between technologies such as PGT and HGGM because they face different challenges and offer totally 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.
2018-roth.pdf: “Reprogramming human T cell function and specificity with non-viral genome targeting”, Theodore L. Roth, Cristina Puig-Saus, Ruby Yu, Eric Shifrut, Julia Carnevale, P. Jonathan Li, Joseph Hiatt, Justin Saco, Paige Krystofinski, Han Li, Victoria Tobin, David N. Nguyen, Michael R. Lee, Amy L. Putnam, Andrea L. Ferris, Jeff W. Chen, Jean-Nicolas Schickel, Laurence Pellerin, David Carmody, Gorka Alkorta-Aranburu, Daniela del Gaudio, Hiroyuki Matsumoto, Montse Morell, Ying Mao, Min Cho, Rolen M. Quadros, Channabasavaiah B. Gurumurthy, Baz Smith, Michael Haugwitz, Stephen H. Hughes, Jonathan S. Weissman, Kathrin Schumann, Jonathan H. Esensten, Andrew P. May, Alan Ashworth, Gary M. Kupfer, Siri Atma W. Greeley, Rosa Bacchetta, Eric Meffre, Maria Grazia Roncarolo, Neil Romberg, Kevan C. Herold, Antoni Ribas, Manuel D. Leonetti, Alexander Marson
Dog cloning as a concept is no longer infeasible. Starting with Snuppy, the first cloned dog in the world, somatic cell nuclear transfer (SCNT) has been continuously developed and used for diversepurposes. In this article we summarise the current method for SCNT, the normality of cloned dogs and the application of dog cloning not only for personal reasons, but also for public purposes.
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.
2018-akcakaya.pdf: “In vivo CRISPR editing with no detectable genome-wide off-target mutations”, Pinar Akcakaya, Maggie L. Bobbin, Jimmy A. Guo, Jose Malagon-Lopez, Kendell Clement, Sara P. Garcia, Mick D. Fellows, Michelle J. Porritt, Mike A. Firth, Alba Carreras, Tania Baccega, Frank Seeliger, Mikael Bjursell, Shengdar Q. Tsai, Nhu T. Nguyen, Roberto Nitsch, Lorenz M. Mayr, Luca Pinello, Mohammad Bohlooly-Y, Martin J. Aryee, Marcello Maresca, J. Keith Joung
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 pigcell line. Here, we confirmed that PERVs infect human cells, andobserved the horizontal transfer of PERVs among human cells. UsingCRISPR-Cas9, we inactivated all the PERVs in a porcine primary cellline and generated PERV-inactivated pigs via somatic cell nucleartransfer. 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.
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]