Now Pruitt, working with clinical fellow Alejandro Chavez in the laboratory of George Church at Harvard Medical School, has devised a potential solution to this problem, which is published on the biology preprint server, bioRxiv.
Cas9 can tolerate a single mismatch between the guide RNA and its target sequence, but it likely cannot tolerate much more. With this in mind, the team devised a screen for guide RNAs that would cut at a specific mutation conferring antibiotic resistance but not if it also cuts the wild-type allele, using guide RNAs that each contained two mutations relative to the wild-type sequence. Once the target mutated into the undesired sequence, the guide would be off by only a single mismatch, allowing Cas9 to cut and disable the undesired copy.
The team first applied this strategy to a specific mutation in the beta-lactamase gene, which encodes resistance to ampicillin, in a highly mutagenic background. Cells containing a control guide RNA grew rapidly on ampicillin, while cells containing a dual-mutant “tuned guide RNA” (tgRNA) did not. In those cells, appearance of the unwanted mutation induced rapid cleavage, removing that gene from the population.
The researchers next applied the same approach to streptomycin and rifampicin resistance, showing that the strategy not only works on targets in the E. coli chromosome itself but that it also can be multiplexed. They also showed that it can work in the complex environment of the mouse gastrointestinal tract.
According to Chavez, this method—should it translate to higher organisms—has potential applications for gene therapy, synthetic biology, and evolutionary biology, among other fields. It could be used, for instance, to ensure that genetically modified bacteria don’t acquire specific unwanted traits or to study organismal responses to stress when common escape pathways are cut off. All that’s required is a pool of candidate guide RNAs—in this case, 27. “That’s not very expensive,” Chavez said. “It’s laborious, but not very expensive.”
Chavez, A., et al., “Precise Cas9 targeting enables genomic mutation prevention,” BioRxiv, June 14, 2016 (doi: 10.1101/058974).