VATIS UPDATE Article

Investigators at the University of California, Berkeley, and Massachusetts General Hospital, the United States, have identified a key region within the Cas9 protein that governs how accurately CRISPR/Cas9 homes in on a target DNA sequence, and have tweaked it to produce a hyper-accurate gene editor with the lowest level of off-target cutting to date. The research team identified a protein domain, called REC3, that acts as a master controller of DNA cutting – an obvious target for re-engineering to improve accuracy even further.

This approach should help scientists customize variants of Cas9 – the protein that binds and cuts DNA – to minimize the chance that CRISPR/Cas9 will edit DNA at the wrong place, a key consideration when doing gene therapy in humans. In the current study, the researchers used a technique called single-molecule FRET (Förster resonance energy transfer) to precisely measure how the various protein domains in the Cas9-sgRNA protein complex – in particular, REC3, REC2, and HNH – move when the complex binds to DNA.

“Using single-molecule Förster resonance energy transfer (smFRET) experiments, we show that both SpCas9-HF1 and eSpCas9(1.1) are trapped in an inactive state when bound to mismatched targets,” said the authors. Specifically, the scientists determined that the specificity benefits conferred by eSpCas9(1.1) and SpCas9-HF1 could be explained by the fact that the threshold for the HNH conformational switch was much higher for these Cas9 variants. Findings from the new study have been published in the journal Nature.