Expanded-PAM Streptococcus pyogenes Cas9 (xCas9)

Characteristics

Cleavage chemistry is unchanged from SpCas9 - a 20-nucleotide spacer, RuvC and HNH catalysis, a blunt double-strand break - while PAM readout is relaxed. All evolved clones share the substitutions E480K, E543D and E1219V; the xCas9 3.6-3.13 lineage containing xCas9 3.7 adds A262T, S409I and M694I. In HEK293T cells xCas9 3.7 gives 41% indels at NGG sites, matching SpCas9 at 41%, and higher indel frequencies at NGT (38% versus 8.6%), NGA (32% versus 20%) and NGC (13% versus 6.3%), averaging 1.6- to 6.4-fold greater apparent cleavage than SpCas9 across the non-NGG sites tested. GUIDE-seq at EMX1 detects zero off-target reads for xCas9 3.7 against 1,132 for SpCas9. xCas9 has the lowest mismatch tolerance of SpCas9, xCas9 and SpCas9-NG.

Applications: Nuclease-mediated gene disruption at NGG and non-NGG target sites in HEK293T and U2OS cells. Catalytically dead dxCas9(3.7) fused to VPR drives targeted transcriptional activation. Substituting xCas9(3.7) for SpCas9 in the cytosine base editor BE3 and the adenine base editor ABE extends both editors to NG, GAA and GAT target sites.

Limitations: Independent head-to-head comparisons find the broadened PAM range weaker than first reported. In pooled screens across thousands of loci SpCas9-NG outperforms xCas9 at every PAM and genome-engineering modality tested, and at NGH PAMs (H = A, C or T) xCas9 is often indistinguishable from wild-type SpCas9. Across 26,478 lentivirally integrated and 78 endogenous human target sites SpCas9-NG shows the broader PAM compatibility.

References

1. Hu et al. (2018). Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature - Hu 2018 xCas9