New high-throughput analysis reveals parameters affecting guideRNA efficiency

A recent report in Nature Methods by one of the leading geneticist, George Church, unveils factors that affect genome editing efficiency with CRISPR. The group from the Wyss Institute and Harvard Medical School employed a library-on-library approach to figure out why certain gRNAs are more effective than others. They carried out two separate experiments; first they transfected cells with a target lentiviral library as well as a gRNA plasmid library, which enabled the scientists to analyze how a certain sequence affect genome editing rates. Not quite surprising, there was a massive variation among different gRNAs at different targets, but the most important base was found to be the PAM adjacent base, with G being the most efficient and T least preferred. Next, they transfected the gRNA plasmid library into 293T cells and looked at endogenous genome editing. This experiment allowed to analyze the effect of epigenetic parameters on genome editing efficiency. Indeed, there was a robust correlation between DN-ase I hypersensitive sites (i.e. more open chromatin, transcriptionally active regions) and editing efficiency. Finally, it is interesting to note that there was no tangible relationship between gRNA effectiveness and off-target activity, moreover some of the most active gRNAs had the least off-target effects.

The group made a prediction algorithm available
online: https://crispr.med.harvard.edu/sgRNAScorer/