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genetics/​genome-synthesis directory

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“Continuous Cell-Free Replication and Evolution of Artificial Genomic DNA in a Compartmentalized Gene Expression System”, Okauchi & Ichihashi 2021

2021-okauchi.pdf: “Continuous Cell-Free Replication and Evolution of Artificial Genomic DNA in a Compartmentalized Gene Expression System”⁠, Hiroki Okauchi, Norikazu Ichihashi (2021-11-15; ⁠, ; similar):

In all living organisms, genomic DNA continuously replicates by the proteins encoded in itself and undergoes evolution through many generations of replication. This continuous replication coupled with gene expression and the resultant evolution are fundamental functions of living things, but they have not previously been reconstituted in cell-free systems.

In this study, we combined an artificial DNA replication scheme with a reconstituted gene expression system and microcompartmentalization to realize these functions. Circular DNA replicated through rolling-circle replication followed by homologous recombination catalyzed by the proteins, phi29 DNA polymerase, and Cre recombinase expressed from the DNA. We encapsulated the system in microscale water-in-oil droplets and performed serial dilution cycles. Isolated circular DNAs at Round 30 accumulated several common mutations, and the isolated DNA clones exhibited higher replication abilities than the original DNA due to its improved ability as a replication template, increased polymerase activity, and a reduced inhibitory effect of polymerization by the recombinase.

The artificial genomic DNA, which continuously replicates using self-encoded proteins and autonomously improves its sequence, provides an useful starting point for the development of more complex artificial cells.

[Keywords: DNA replication, cell-free synthetic biology, artificial cell, Darwinian evolution]

“Molecular Recording of Sequential Cellular Events into DNA”, Loveless et al 2021

“Molecular recording of sequential cellular events into DNA”⁠, Theresa B. Loveless, Courtney K. Carlson, Vincent J. Hu, Catalina A. Dentzel Helmy, Guohao Liang, Michelle Ficht et al (2021-11-07; ⁠, ; similar):

Genetically encoded DNA recorders noninvasively convert transient biological events into durable mutations in a cell’’s genome, allowing for the later reconstruction of cellular experiences using high-throughput DNA sequencing. Existing DNA recorders have achieved high-information recording, durable recording, prolonged recording over multiple timescales, multiplexed recording of several user-selected signals, and temporally resolved signal recording, but not all at the same time. We present a DNA recorder called peCHYRON (prime editing Cell HistorY Recording by Ordered iNsertion) that does. In peCHYRON, prime editor guide RNAs (pegRNAs) insert a variable triplet DNA sequence alongside a constant propagation sequence that deactivates the previous and activates the next step of insertion. This process results in the sequential accumulation of regularly spaced insertion mutations at a synthetic locus. Accumulated insertions are permanent throughout editing because peCHYRON uses a prime editor that avoids cutting both DNA strands, which risks deletions. Editing continues indefinitely because each insertion adds the complete sequence needed to initiate the next step. Constitutively expressed pegRNAs generate insertion patterns that support straightforward reconstruction of cell lineage relationships. Pulsed expression of different pegRNAs enables the reconstruction of pulse sequences, which may be coupled to biological stimuli for temporally-resolved multiplexed event recording.

“Sense Codon Reassignment Enables Viral Resistance and Encoded Polymer Synthesis”, Robertson et al 2021

2021-robertson.pdf: “Sense codon reassignment enables viral resistance and encoded polymer synthesis”⁠, Wesley E. Robertson, Louise F. H. Funke, Daniel de la Torre, Julius Fredens, Thomas S. Elliott, Martin Spinck et al (2021-06-04; similar):

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.

“The Second Decade of Synthetic Biology: 2010–2020”, Meng & Ellis 2020

“The second decade of synthetic biology: 2010–2020”⁠, Fankang Meng, Tom Ellis (2020-10-14; similar):

Synthetic biology is among the most hyped research topics this century, and in 2010 it entered its teenage years. But rather than these being a problematic time, we’ve seen synthetic biology blossom and deliver many new technologies and landmark achievements.

  1. …Looking back at 2010, the biggest synthetic biology story of the year was the complete synthesis of a working bacterial genome by a team at the J. Craig Venter Institute (JCVI)
  2. …Could hard biological problems such as context, noise, burden and cross-reactivity really be solved to allow us to engineer cells like we wire-up electronic circuits? Well, thanks to a lot of challenging technical biology and biological engineering work undertaken by many in the field, but especially MIT’s Chris Voigt, the answer to this was yes.
  3. …It’s no surprise therefore, that synthetic biology groups were the first to pounce on gene editing technologies like CRISPR as they appeared in 2011 and 2012.
  4. …While there’s no doubt that CRISPR was the breakthrough of the decade in biosciences, it’s perhaps its forerunner TALENs (TAL-Effector Nucleases) that deserve more credit in revolutionizing how synthetic biology changed in the past 10 years.
  5. …The drop in cost for gene synthesis can mostly be attributed to new methods for printing thousands of oligonucleotides in parallel on chips to make ‘oligo pools’ and teaming this with next generation sequencing (NGS) as a much more cost-effective method for validating assembled DNA.
  6. …High-power computation also opened up new frontiers in what can be modelled and predicted in the last 10 years…This helped inform JCVI’s project towards a minimal genome, which delivered a further landmark in 2016 with the impressive engineering of a bacteria with a minimized synthetic genome
  7. …Synthetic genomics also moved into eukaryotes with the international Sc2.0 consortium constructing highly-modified, yet fully-functional synthetic versions of Baker’s yeast chromosomes
  8. …DNA also became a way to store data, initially just in vitro via chemical synthesis, but then also in cells via ‘molecular recorder’ genetic systems that use recombinases or CRISPR to modify DNA as cells grow, divide and change their gene expression
  9. …Academic achievements include engineering cells to fix CO2 and nitrogen, and getting yeast to make opioids and cannabinoids.

…A multibillion dollar industry now exists that makes chemicals, drugs, proteins, probiotics, sensors, fertilisers, textiles, food and many other things from engineered cells.

“Technological Challenges and Milestones for Writing Genomes: Synthetic Genomics Requires Improved Technologies”, Ostrov et al 2019

2019-ostrov.pdf: “Technological challenges and milestones for writing genomes: Synthetic genomics requires improved technologies”⁠, Nili Ostrov, Jacob Beal, Tom Ellis, D. Benjamin Gordon, Bogumil J. Karas, Henry H. Lee, Scott C. Lenaghan et al (2019-10-18; ; similar):

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.

“Programmed Chromosome Fission and Fusion Enable Precise Large-scale Genome Rearrangement and Assembly”, Wang et al 2019

2019-wang.pdf: “Programmed chromosome fission and fusion enable precise large-scale genome rearrangement and assembly”⁠, Kaihang Wang, Daniel de la Torre, Wesley E. Robertson, Jason W. Chin (2019-08-30; similar):

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.

“Total Synthesis of Escherichia Coli With a Recoded Genome”, Fredens et al 2019

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 et al (2019-05-15; similar):

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.

“Beyond Editing to Writing Large Genomes”, Chari & Church 2017

2017-chari.pdf: “Beyond editing to writing large genomes”⁠, Raj Chari, George M. Church (2017-01-01)

“Capacity-approaching DNA Storage”, Erlich & Zielinski 2016

“Capacity-approaching DNA storage”⁠, Yaniv Erlich, Dina Zielinski (2016-09-09; similar):

Humanity produces data at exponential rates, creating a growing demand for better storage devices. DNA molecules are an attractive medium to store digital information due to their durability and high information density. Recent studies have made large strides in developing DNA storage schemes by exploiting the advent of massive parallel synthesis of DNA oligos and the high throughput of sequencing platforms. However, most of these experiments reported small gaps and errors in the retrieved information. Here, we report a strategy to store and retrieve DNA information that is robust and approaches the theoretical maximum of information that can be stored per nucleotide. The success of our strategy lies in careful adaption of recent developments in coding theory to the domain specific constrains of DNA storage. To test our strategy, we stored an entire computer operating system, a movie, a gift card, and other computer files with a total of 2.14×106 bytes in DNA oligos. We were able to fully retrieve the information without a single error even with a sequencing throughput on the scale of a single tile of an Illumina sequencing flow cell. To further stress our strategy, we created a deep copy of the data by PCR amplifying the oligo pool in a total of nine successive reactions, reflecting one complete path of an exponential process to copy the file 218×1012 times. We perfectly retrieved the original data with only five million reads. Taken together, our approach opens the possibility of highly reliable DNA-based storage that approaches the information capacity of DNA molecules and enables virtually unlimited data retrieval.

“Design, Synthesis, and Testing toward a 57–codon Genome”, Ostrov et al 2016

2016-ostrov.pdf: “Design, synthesis, and testing toward a 57–codon genome”⁠, Nili Ostrov, Matthieu Landon, Marc Guell, Gleb Kuznetsov, Jun Teramoto, Natalie Cervantes, Minerva Zhou et al (2016-08-19; similar):

Recoding—the repurposing of genetic codons—is a powerful strategy for enhancing genomes with functions not commonly found in nature. Here, we report computational design, synthesis, and progress toward assembly of a 3.97-megabase, 57-codon Escherichia coli genome in which all 62,214 instances of 7 codons were replaced with synonymous alternatives across all protein-coding genes. We have validated 63% of recoded genes by individually testing 55 segments of 50 kilobases each. We observed that 91% of tested essential genes retained functionality with limited fitness effect. We demonstrate identification and correction of lethal design exceptions, only 13 of which were found in 2,229 genes. This work underscores the feasibility of rewriting genomes and establishes a framework for large-scale design, assembly, troubleshooting, and phenotypic analysis of synthetic organisms.


Recoding and repurposing genetic codons: By recoding bacterial genomes, it is possible to create organisms that can potentially synthesize products not commonly found in nature. By systematic replacement of 7 codons with synonymous alternatives for all protein-coding genes, Ostrov et al 2016 recoded the Escherichia coli genome. The number of codons in the E. coli genetic code was reduced from 64 to 57 by removing instances of the UAG stop codon and excising 2 arginine codons, 2 leucine codons, and 2 serine codons. Over 90% functionality was successfully retained. In 10 cases, reconstructed bacteria were not viable, but these few failures offered interesting insights into genome-design challenges and what is needed for a viable genome.

“Large-scale de Novo DNA Synthesis: Technologies and Applications”, Kosuri & Church 2014

“Large-scale de novo DNA synthesis: technologies and applications”⁠, Sriram Kosuri, George M. Church (2014; similar):

For over 60 years, the synthetic production of new DNA sequences has helped researchers understand and engineer biology.

Here we summarize methods and caveats for the de novo synthesis of DNA, with particular emphasis on recent technologies that allow for large-scale and low-cost production.

In addition, we discuss emerging applications enabled by large-scale de novo DNA constructs, as well as the challenges and opportunities that lie ahead.

Synthetic biology

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Phosphoramidite

Wikipedia

Oligonucleotide synthesis

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Mycoplasma laboratorium

Wikipedia

Human genome

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Human Genome Project - Write

Wikipedia

Base pair

Wikipedia

Artificial gene synthesis

Wikipedia

Miscellaneous