Fast DNA Editing Tool to Boost Genetic Research

Scientists led by Wyss Institute for Biologically Inspired Engineering at Harvard University have developed a new gene editing tool that could significantly boost genetic research on mutations. The new tool offers advantages over CRISPR, including better editing rates and the possibility to be used in contexts where CRISPR is toxic or not feasible.

CRISPR pioneers Emmanuelle Charpentier and Jennifer Doudna were awarded the 2020 Nobel Prize in Chemistry for initiating a wave of powerful gene editing technologies that seem poised to change the world. See our review of the book, A Crack in Creation: Gene Editing and the Unthinkable Power to Control Evolution (2017), co-authored by Doudna.

The new gene editing tool is called “Retron Library Recombineering” (RLR). It can generate up to millions of mutations simultaneously. RLR can also “barcode” mutant bacterial cells so that an entire pool of bacteria can be screened at once, enabling massive amounts of data to be easily generated and analyzed.

"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," says research leader George Church in a press release issued by Wyss Institute. "This work helps establish a road map toward using RLR in other genetic systems, which opens up many exciting possibilities for future genetic research."

"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," says researcher 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."

Retrons are segments of bacterial DNA that, like CRISPR, can be used for precise and flexible gene editing in bacterial and even human cells. Recombineering is a gene editing technique that works by introducing an alternate piece of DNA into a cell while the cell is replicating its genome, creating genetic mutations without breaking DNA.

"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,” explains Schubert. “Retrons are another bacterial innovation that might also provide some important advances."

Retrons can "barcode" the individuals within a pool of bacteria that have received each retron sequence, enabling dramatically faster, pooled screens of precisely-created mutant strains.

A paper is published in PNAS. It shows that RLR could detect known antibiotic resistance mutations in bacteria, and that RLR is sensitive and precise enough to measure small differences in resistance that result from very similar mutations. Future research is expected to shed light on how multiple mutations interact with each other, and generate massive amounts of data that could enable the use of machine learning to predict further mutational effects.

Direct applications of RLR to future medicine haven’t been suggested so far. But the same was true of CRISPR only a few years ago. It seems likely to me that RLR could find direct and very powerful applications to DNA editing.