Molecular basis for the PAM expansion and fidelity enhancement of an evolved Cas9 nuclease.

Hongyuan Zhang,Jianhua Gan,Yu Wang,Quanjiang Ji,Weizhong Chen,Yifei Zhang,Bon-Kyoung Koo

doi:10.1371/journal.pbio.3000496

Hongyuan Zhang, Jianhua Gan + Show 5 more

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https://doi.org/10.1371/journal.pbio.3000496

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Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems have been harnessed as powerful genome editing tools in diverse organisms. However, the off-target effects and the protospacer adjacent motif (PAM) compatibility restrict the therapeutic applications of these systems. Recently, a Streptococcus pyogenes Cas9 (SpCas9) variant, xCas9, was evolved to possess both broad PAM compatibility and high DNA fidelity. Through determination of multiple xCas9 structures, which are all in complex with single-guide RNA (sgRNA) and double-stranded DNA containing different PAM sequences (TGG, CGG, TGA, and TGC), we decipher the molecular mechanisms of the PAM expansion and fidelity enhancement of xCas9. xCas9 follows a unique two-mode PAM recognition mechanism. For non-NGG PAM recognition, xCas9 triggers a notable structural rearrangement in the DNA recognition domains and a rotation in the key PAM-interacting residue R1335; such mechanism has not been observed in the wild-type (WT) SpCas9. For NGG PAM recognition, xCas9 applies a strategy similar to WT SpCas9. Moreover, biochemical and cell-based genome editing experiments pinpointed the critical roles of the E1219V mutation for PAM expansion and the R324L, S409I, and M694I mutations for fidelity enhancement. The molecular-level characterizations of the xCas9 nuclease provide critical insights into the mechanisms of the PAM expansion and fidelity enhancement of xCas9 and could further facilitate the engineering of SpCas9 and other Cas9 orthologs.

Highlights

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 systems, originally discovered in prokaryotic immune systems, have been harnessed and engineered for robust genome editing in diverse organisms [1,2,3,4,5,6,7,8]
To examine the DNA-targeting activity of xCas9 in vivo, we substituted the WT Streptococcus pyogenes Cas9 (SpCas9) gene with the xCas9 gene in the bacterial cytosine base-editing system pBECKP [49], which was constructed by fusing a cytosine deaminase with a Cas9 nickase (Cas9D10A)
For the CGT and GGT sites, pBECKP-xCas9 showed a much higher editing efficiency (71.5% ± 1.7% and 42.4% ± 1.1% for CGT protospacer adjacent motif (PAM); 47.9% ± 3.6% for GGT PAM) than WT SpCas9 (2.77% ± 0.17% and 0.57% ± 0.51% for CGT PAM; 6.32% ± 0.46% for GGT PAM), which was consistent with the results in vitro

Summary

Introduction

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems, originally discovered in prokaryotic immune systems, have been harnessed and engineered for robust genome editing in diverse organisms [1,2,3,4,5,6,7,8]. Mechanistic study of an evolved Cas nuclease xCas. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

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