New tool for mass gene-edits
Researchers have created a new gene-editing technique that enables millions of genetic experiments to be performed simultaneously.
The new gene editing tool called Retron Library Recombineering (RLR) can generate millions of mutations simultaneously, and ‘barcodes’ mutant bacterial cells so that the entire pool can be screened at once.
While the CRISPR-Cas9 gene editing system has become the poster child for innovation in synthetic biology, it has some major limitations.
CRISPR-Cas9 can be programmed to find and cut specific pieces of DNA, but editing the DNA to create desired mutations requires tricking the cell into using a new piece of DNA to repair the break.
This bait-and-switch can be complicated to orchestrate, and can even be toxic to cells because Cas9 often cuts unintended, off-target sites as well.
RLR can be used in contexts where CRISPR is toxic or not feasible, and results in better editing rates.
Alternative gene editing techniques called ‘recombineering’ perform this bait-and-switch by introducing an alternate piece of DNA while a cell is replicating its genome, efficiently creating genetic mutations without breaking DNA.
These methods can be used in many cells at once to create complex pools of mutations for researchers to study.
Figuring out what the effects of those mutations are, however, requires that each mutant be isolated, sequenced, and characterised: a time-consuming and impractical task.
Retron Library Recombineering (RLR) makes this task easier by generating millions of mutations simultaneously, and can “barcode” mutant cells so that the entire pool can be screened at once.
“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 co-first author Dr Max Schubert, who works in the lab of the ‘godfather of genetic engineering’ Dr George Church.
“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.”