After being identified in the 1980s, it was thought that retrons were just an odd feature of some bacterial cells. But eventually, scientists showed that retrons are a complex of an enzyme, DNA, and RNA, and it enables a bacterial cell to generate single-stranded DNA molecules (ssDNA). It took decades, but scientists showed that the ssDNA produced by retrons can help bacteria defend against invading viruses. Researchers knew they had potential as gene editors, just like CRISPR.
"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 co-first study author Max Schubert, Ph.D., a postdoc in the lab of Wyss Core Faculty member George Church, Ph.D.
Scientists have now been able to use retrons to engineer a new gene-editing system. The tool is called Retron Library Recombineering (RLR). Millions of genetic mutations can be made at the same time, and mutant cells get tagged with a barcode that allows for a pool of cells to be screened at the same time. With computational tools, tons of data can easily be created and analyzed. The work has been reported in the Proceedings of the National Academy of Sciences (PNAS).
The gene-editing mechanism that utilizes retrons is called oligonucleotide recombineering. Instead of cutting the DNA with an enzyme and initiating a natural repair process that integrates new DNA, like some CRISPR techniques, this tool incorporates new DNA into the cells' genomes with a protein called a single-stranded annealing protein (SSAP). The SSAP adds the new, mutant DNA into the genome as a cell divides, and the daughter cells carry the new sequence.
"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 Max Schubert. "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."
The sequences of the retrons that get integrated into the genomes of daughter cells serve as identifiers, or barcodes, so cells can be grouped and analyzed together. Sequencing data sorts out the individual cells afterward.
"We figured that retrons should give us the ability to produce ssDNA within the cells we want to edit rather than trying to force them into the cell from the outside, and without damaging the native DNA, which were both very compelling qualities," said co-first author Daniel Goodman, Ph.D., a former Graduate Research Fellow at the Wyss Institute who is now at UCSF.
First, the researchers used plasmids, circular pieces of DNA, which contained antibiotic resistance genes inside of retron sequences along with an SSAP gene, and attempted to integrate it into bacterial cells. The process worked but it was incredibly inefficient. Next, they deactivated genes that would destroy ssDNA and tamp down DNA repair mechanisms in the bacteria, and these changes dramatically improved the process. The team found that 90 percent of the bacterial population integrated the retron sequence into their genome. The researchers also ensured that very small sequence differences could be detected by RLR.
"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 study author George Church. "This work helps establish a road map toward using RLR in other genetic systems, which opens up many exciting possibilities for future genetic research."
This process is also different from CRISPR because the bacteria can continue to incorporate a desired mutation into their genome over time as they replicate. It may also be possible to combine the two methods.