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

Weizhong Chen; Hongyuan Zhang; Yifei Zhang; Yu Wang; Jianhua Gan; Quanjiang Ji

doi:10.1371/journal.pbio.3000496

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

PLoS Biol. 2019 Oct 11;17(10):e3000496. doi: 10.1371/journal.pbio.3000496. eCollection 2019 Oct.

Authors

Weizhong Chen¹, Hongyuan Zhang¹, Yifei Zhang¹, Yu Wang¹, Jianhua Gan², Quanjiang Ji¹

Affiliations

¹ School of Physical Science and Technology, ShanghaiTech University, Shanghai, China.
² State Key Laboratory of Genetic Engineering, Collaborative Innovation Center of Genetics and Development, Shanghai Public Health Clinical Center, School of Life Sciences, Fudan University, Shanghai, China.

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Amino Acid Substitution
CRISPR-Associated Protein 9 / chemistry
CRISPR-Associated Protein 9 / genetics*
CRISPR-Associated Protein 9 / metabolism
CRISPR-Cas Systems*
Cloning, Molecular
Clustered Regularly Interspaced Short Palindromic Repeats*
Crystallography, X-Ray
DNA / chemistry
DNA / genetics*
DNA / metabolism
Escherichia coli / genetics
Escherichia coli / metabolism
Gene Editing
Gene Expression
Genetic Vectors / chemistry
Genetic Vectors / metabolism
Isoenzymes / chemistry
Isoenzymes / genetics
Isoenzymes / metabolism
Klebsiella pneumoniae / genetics
Klebsiella pneumoniae / metabolism
Models, Molecular
Mutagenesis, Site-Directed / methods
Mutation
Nucleotide Motifs
Protein Binding
Protein Engineering / methods
RNA, Guide, CRISPR-Cas Systems / chemistry
RNA, Guide, CRISPR-Cas Systems / genetics*
RNA, Guide, CRISPR-Cas Systems / metabolism
Recombinant Proteins / chemistry
Recombinant Proteins / genetics
Recombinant Proteins / metabolism
Streptococcus pyogenes / genetics
Streptococcus pyogenes / metabolism

Substances

Isoenzymes
RNA, Guide, CRISPR-Cas Systems
Recombinant Proteins
DNA
CRISPR-Associated Protein 9
Cas9 endonuclease Streptococcus pyogenes

Grants and funding

This work was financially supported by the National Natural Science Foundation of China (21922705, 91753127, 31700123 to Q. Ji and 21907066 to W. Chen), the Shanghai Committee of Science and Technology, China (19QA1406000, 17ZR1449200 to Q. Ji), and the Shanghai Sailing Program (18YF1416500 to W. Chen). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.