Retron-based genome editing has been inviting comparisons with CRISPR-based genome editing, especially since researchers have learned that retrons, like CRISPR systems, function as a sort of immune system in bacteria. However, the comparisons may be a little premature. Retron-based genome editing has yet to work in mammalian cells. Nonetheless, retrons have already been shown to offer unique advantages in genome editing applications.

The latest advance in retron-based genome editing comes from researchers at the Wyss Institute for Biologically Inspired Engineering at Harvard University and Harvard Medical School. They report that they have created a new tool for retron-based genome editing. The tool, which is called Retron Library Recombineering (RLR), may be used to perform high-throughput functional screens that surpass the scale and specificity of the screens enabled by CRISPR-Cas technology.

RLR generates up to millions of mutations simultaneously, and it inserts “barcodes” into mutant cells so that the entire pool can be screened at once, enabling massive amounts of data to be easily generated and analyzed. A description of how RLR performed in bacterial cells appeared April 29 in the Proceedings of the National Academy of Sciences, in a paper titled, “High-throughput functional variant screens via in vivo production of single-stranded DNA.”

“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,” the article’s authors wrote. “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.”

Retrons—complexes of DNA, RNA, and protein—are poorly understood bacterial retroelements that undergo targeted reverse-transcription, producing single-stranded multicopy satellite DNA (msDNA). Several research teams have shown that msDNA can function as a recombineering donor, creating specific edits in the genome. This capability suggests that retrons could function as part of a “donor only” genome editing method, unlike CRISPR, which is typically a “donor + guide” method.

Another attraction of retrons is that their sequences themselves can serve as “barcodes” that identify which individuals within a pool of bacteria have received each retron sequence, enabling dramatically faster, pooled screens of precisely created mutant strains.

Retron recombineering has been used in various studies, including synthetic biology studies, but editing rates were low—too low for retron-based genome editing to be practical for studying mutants of physiological interest.

To see if retron recombineering could be more efficient, the Wyss scientists first created circular plasmids of bacterial DNA that contained antibiotic resistance genes placed within retron sequences, as well as a single-stranded annealing protein (SSAP) gene to enable integration of the retron sequence into the bacterial genome. They inserted these retron plasmids into Escherichia 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.

Retron sequences (red) containing a mutation of interest (black notch) are introduced into a bacterial cell along with the enzyme reverse transcriptase (RT). The retron then produces ssDNA that is inserted into replicating DNA with the help of another enzyme called single-stranded annealing protein (SSAP). [Max Schubert / Wyss Institute at Harvard University]

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.

Now that they were confident that their retron ssDNA was incorporated into their bacteria’s genomes, the team tested whether they could use the retrons as a genetic sequencing “shortcut,” enabling many experiments to be performed in a mixture. Because each plasmid had its own unique retron sequence that can function as a “name tag,” they reasoned that they should be able to sequence the much shorter retron rather than the whole bacterial genome to determine which mutation the cells had received.

First, the team tested whether RLR could detect known antibiotic resistance mutations in E. coli. They found that it could – retron sequences containing these mutations were present in much greater proportions in their sequencing data compared with other mutations. The team also determined that RLR was sensitive and precise enough to measure small differences in resistance that result from very similar mutations. Crucially, gathering these data by sequencing barcodes from the entire pool of bacteria rather than isolating and sequencing individual mutants, dramatically speeds up the process.

Then, the researchers took RLR one step further to see if it could be used on randomly fragmented DNA, and find out how many retrons they could use at once. They chopped up the genome of a strain of E. coli highly resistant to another antibiotic, and used those fragments to build a library of tens of millions of genetic sequences contained within retron sequences in plasmids.

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

“The simplicity of RLR really shone in this experiment, because it allowed us to build a much bigger library than what we can currently use with CRISPR, in which we have to synthesize both a guide and a donor DNA sequence to induce each mutation,” said Max Schubert, PhD, the co-first author of the PNAS article and a postdoc in the lab of Wyss Core Faculty member George Church, PhD.

This library was then introduced into the RLR-optimized E. coli strain for analysis. Once again, the researchers found that retrons conferring antibiotic resistance could be easily identified by the fact that they were enriched relative to others when the pool of bacteria was sequenced.

“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.”

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