To generate Cas9s that recognize PAM sequences beyond their canonical NGG site (where N represents any DNA base), the authors established a PACE system in Escherichia coli in which a selection phage carries a library of mutated, catalytically inactive SpCas9 variants in place of a gene (gene III) that the phage requires to propagate. Gene III is instead positioned in an accessory plasmid downstream of a PAM library and protospacer and expressed in E. coli along with a sgRNA. When these E. coli are infected with the selection phage, only phage carrying SpCas9 that can bind to the PAM and protospacer in the accessory plasmid can activate gene III and be propagated. Thus, only Cas9 variants with broadened PAM compatibilities are propagated in subsequent E. coli host cells. By using accessory plasmid libraries expressing all possible three-letter PAMs (NNN) in their PACE system, the team isolated 14 evolved Cas9 variants, named xCas9 3.0–xCas9 3.13.
After restoring the catalytic activity of the xCas9 variants the authors showed that several of these cleave DNA sites with NG, NNG, GAA, GAT or CAA PAMs in bacterial cells. They then demonstrated that xCas9 variants are active at a range of PAMs in human cells using three different approaches. First, catalytically inactive xCas9 3.6 and xCas9 3.7 fused to a transcriptional activator could activate a GFP reporter downstream of a target protospacer containing NGT, NGA, NGC, NNG, GAA or GAT PAMs. xCas9 3.7 also activated the transcription of six endogenous loci in human cells. Second, in HEK293T cells xCas9 3.7 cleaved endogenous genomic sites at NG, GAA and GAT PAMs more efficiently than SpCas9. Third, xCas9 3.6 and xCas9 3.7 forms of cytidine and adenine base editors, which can directly convert one base pair to another, were more efficient than spCas9 at introducing point mutations at endogenous genomic sites containing NGG, NG, GAA or GAT PAMs. Thus, xCas9 proteins target a broad range of PAMs in mammalian cells.
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