Continuous evolution of SpCas9 variants compatible with non-G PAMs

Here, we report the laboratory evolution and extensive characterization of three new SpCas9 variants capable of collectively recognizing NRNH PAMs (where R = A or G and H = A, C, or T) using three new phage-assisted continuous evolution (PACE) strategies for DNA binding, together with a secondary selection for DNA cleavage. Using a panel of 64 endogenous human genome target sites, we show that these variants recognize NAAH, NACH, NATH, and NAGH PAMs to mediate indel formation, cytosine base editing, and adenine base editing in human cells. Genome-wide off-target profiling reveals that these evolved variants display similar or improved DNA specificity compared to SpCas9 on its cognate NGG PAM. We exhaustively characterized the targeting capabilities of these evolved variants and the previously reported SpCas9-NG in human cells using a library of 11,776 genomically integrated protospacer-sgRNA pairs covering all NNNN PAMs. Finally, we used an evolved variant to enable A•T-to-G•C base editing of the founder sickle-cell anemia mutation of ß-globin to a non-pathogenic allele using an otherwise inaccessible CAC PAM. These evolved variants, together with previously reported SpCas9 mutants, in principle enable correction of ~95% of known pathogenic transition mutations by base editing, and greatly reduce the fraction of genomic sites that are inaccessible by Cas9-based methods.