Researchers at the Wyss Institute for Biological Inspired Engineering at Harvard have developed a new gene editing tool that allows scientists to conduct millions of genetic experiments simultaneously. 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 editing genes, CRISPR-Cas9 is probably the most popular technique these days. It has made waves in the scientific world in recent years, giving researchers the tools they need to be able to easily manipulate DNA sequences. It is more accurate than previously used techniques and has a wide variety of potential uses, including life-saving treatments for various diseases.
However, the tool has some major limitations. It could be difficult to supply CRISPR-Cas9 materials in large numbers, which on the one hand remains a problem for studies and experiments. The way the technology works can also be toxic to cells, as the Cas9 enzyme – the molecular “scissors” responsible for cutting strands of DNA – often cuts non-target sites as well.
CRISPR-Cas9 physically cuts DNA to incorporate the mutated sequence into its genome during the repair process. In the meantime, retrons can introduce the mutated strand of DNA into a replicating cell so that the strand can be built into the DNA of daughter cells. In addition, the sequences of retrons can serve as “barcodes” or “name tags” so that scientists can track people in a pool of bacteria. That is, they can be used to manipulate the genome without damaging the native DNA, and they can be used to run multiple experiments in a large mix.
The scientists at the Wyss Institute tested RLR on E. coli bacteria and found that 90 percent of the population incorporated the Retron sequence after some optimization. They were also able to prove how useful it can be in massive genetic experiments. During their tests, they were able to find antibiotic resistance mutations in E. coli by sequencing the retron’s barcodes instead of sequencing individual mutants, which sped up the process significantly.
The study’s co-first author, Max Schubert, stated:
“With RLR we were able to do something that is impossible with CRISPR: We accidentally dismantled a bacterial genome, converted these genetic fragments in situ into single-stranded DNA and thus screened millions of sequences at the same time. RLR is a simpler, more flexible gene editing tool, which can be used for highly multiplexed experiments, it eliminates the toxicity often observed with CRISPR and improves the ability of researchers to examine mutations at the genome level …
CRISPR was long considered just a strange thing bacteria did, and how to use it for genomic engineering changed the world. Retrons are another bacterial innovation that could bring some important advances as well. “
Much work remains to be done before RLR can be widely deployed, including improving and standardizing the processing rate. However, the team believes it “can lead to new, exciting, and unexpected innovations”.
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