Researchers at Harvard’s Wyss Institute for Biologically Inspired Engineering have developed a new gene editing tool that allows scientists to run millions of genetic experiments at once. They call it the Retron Library Recombineering (RLR) technique, and it uses segments of bacterial DNA called retrons that can produce fragments of single-stranded DNA.
When it comes to gene editing, CRISPR-Cas9 is probably the best known technique today. It has created waves in the scientific world in recent years, giving researchers the tool they need to easily change DNA sequences. It is more accurate than previously used techniques and it has a wide variety of potential applications, including life-saving treatments for a variety of diseases.
However, the tool has some significant limitations. It can be difficult to supply CRISPR-Cas9 materials in large numbers, which remains a problem for studies and experiments, among other things. Also, the way the technique works can be toxic to cells, because the Cas9 enzyme – the molecular “scissors” responsible for cutting DNA strands – often cuts non-target sites as well.
CRISPR-Cas9 physically cuts DNA to insert the mutant sequence into the genome during the repair process. Meanwhile, retrons can introduce the mutant DNA strand into a replicating cell, so that the strand can be incorporated into the daughter cells’ DNA. Furthermore, the sequences of retrons can serve as “bar codes” or “name labels”, allowing scientists to track individuals in a pool of bacteria. That means they can be used for genome editing without damaging the original DNA, and they can be used to run multiple experiments in one large mixture.
The Wyss Institute scientists tested RLR on E. coli bacteria and found that 90 percent of the population absorbed the retron sequence after making a few adjustments. They were also able to prove how useful it can be in large-scale genetic experiments. During their tests, they were able to find antibiotic resistance mutations in E. coli by sequencing the retrons’ barcodes instead of sequencing individual mutants, making the process a lot faster.
The study’s co-lead author, Max Schubert, explained:
“RLR enabled us to do something impossible with CRISPR: we randomly mined a bacterial genome, converted those genetic fragments into single-stranded DNA in situ and used them to screen millions of sequences at once. RLR is a simpler, more flexible tool. for editing genes that can be used for highly multiplexed experiments, eliminating the toxicity commonly seen with CRISPR and enhancing researchers’ ability to investigate mutations at the genome level.
For a long time, CRISPR was considered just a weird thing that bacteria did, and figuring out how to use it for genome engineering changed the world. Retrons are another bacterial innovation that could also make some significant advancements. “
There is still work to be done before RLR can be used widely, including improving and standardizing processing speed. However, the team believes it “can lead to new, exciting and unexpected innovations.”