New Gene Editing Tool for Millions of Edits

There is a new gene-editing tool called Retron Library Recombineering (RLR) that makes this task easier that has been developed by George Church and his team. RLR generates up to millions of mutations simultaneously, and “barcodes” mutant cells so that the entire pool can be screened at once, enabling massive amounts of data to be easily generated and analyzed. The achievement, which has been accomplished in bacterial cells.

RLR enabled us to do something that’s impossible to do with CRISPR: we randomly chopped up a bacterial genome, turned those genetic fragments into single-stranded DNA in situ, and used them to screen millions of sequences simultaneously,” said co-first author Max Schubert, Ph.D., a postdoc in the lab of Wyss Core Faculty member George Church, Ph.D. “RLR is a simpler, more flexible gene editing tool that can be used for highly multiplexed experiments, which eliminates the toxicity often observed with CRISPR and improves researchers’ ability to explore mutations at the genome level.

Retrons’ existence has been known for decades, but the function of the ssDNA they produce flummoxed scientists from the 1980s until June 2020, when a team finally figured out that retron ssDNA detects whether a virus has infected the cell, forming part of the bacterial immune system.

RTR has multiple components: the genetic snippet that contains a mutation and two proteins. RT and SSAP (reverse transcriptase and single-stranded annealing proteins) transform the retron into ssDNA and let it insert itself into a dividing cell. Eetrons carry the genetic code we want to insert. RT makes it into a more compatible form that’s called ssDNA. SSAP sticks it into DNA as it’s dividing.

Retrons enable the rapid production and screening of millions of trackable DNA variations and their effects on bacteria simultaneously. Credit: Max Schubert / Wyss Institute at Harvard University

Retrons: from enigma to engineering tool

Retrons are segments of bacterial DNA that undergo reverse transcription to produce fragments of single-stranded DNA (ssDNA). Retrons’ existence has been known for decades, but the function of the ssDNA they produce flummoxed scientists from the 1980s until June 2020, when a team finally figured out that retron ssDNA detects whether a virus has infected the cell, forming part of the bacterial immune system.

While retrons were originally seen as simply a mysterious quirk of bacteria, researchers have become more interested in them over the last few years because they, like CRISPR, could be used for precise and flexible gene editing in bacteria, yeast, and even human cells.

“For a long time, CRISPR was just considered a weird thing that bacteria did, and figuring out how to harness it for genome engineering changed the world. Retrons are another bacterial innovation that might also provide some important advances,” said Schubert. His interest in retrons was piqued several years ago because of their ability to produce ssDNA in bacteria – an attractive feature for use in a gene editing process called oligonucleotide recombineering.

To see if they could actually use retrons to achieve efficient recombineering with retrons, Schubert and his colleagues first created circular plasmids of bacterial DNA that contained antibiotic resistance genes placed within retron sequences, as well as an SSAP gene to enable integration of the retron sequence into the bacterial genome. They inserted these retron plasmids into E. coli bacteria to see if the genes were successfully integrated into their genomes after 20 generations of cell replication. Initially, less than 0.1% of E. coli bearing the retron recombineering system incorporated the desired mutation.

To improve this disappointing initial performance, the team made several genetic tweaks to the bacteria. First, they inactivated the cells’ natural mismatch repair machinery, which corrects DNA replication errors and could therefore be “fixing” the desired mutations before they were able to be passed on to the next generation. They also inactivated two bacterial genes that code for exonucleases – enzymes that destroy free-floating ssDNA. These changes dramatically increased the proportion of bacteria that incorporated the retron sequence, to more than 90% of the population.

“Being able to analyze pooled, barcoded mutant libraries with RLR enables millions of experiments to be performed simultaneously, allowing us to observe the effects of mutations across the genome, as well as how those mutations might interact with each other,” said senior author George Church, who leads the Wyss Institute’s Synthetic Biology Focus Area and is also a Professor of Genetics at HMS. “This work helps establish a road map toward using RLR in other genetic systems, which opens up many exciting possibilities for future genetic research.”

Another feature that distinguishes RLR from CRISPR is that the proportion of bacteria that successfully integrate a desired mutation into their genome increases over time as the bacteria replicate, whereas CRISPR’s “one shot” method tends to either succeed or fail on the first try. RLR could potentially be combined with CRISPR to improve its editing performance, or could be used as an alternative in the many systems in which CRISPR is toxic.

More work remains to be done on RLR to improve and standardize editing rate, but excitement is growing about this new tool. RLR’s simple, streamlined nature could enable the study of how multiple mutations interact with each other, and the generation of a large number of data points that could enable the use of machine learning to predict further mutational effects.

PNAS – High-throughput functional variant screens via in vivo production of single-stranded DNA

Significance

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.

Abstract

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.

SOURCES – PNAS, Wyss Institute at Harvard
Written by Brian Wang, Nextbigfuture.com

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