Research On The Structure And Functional Mechanism Of CRISPR/xCas9 3.7

CRISPR/Cas system is widely used as a gene editing tool in various fields of biotechnology and gene therapy.Sp Cas9(Streptococcus pyogenes Cas9,Sp Cas9)system is the most widely used gene editing tool.However,the compatibility of PAM(protospacer adjacent motif)recognition and off-target effects are two major obstacles in the application of this system.Interestingly,a variant of Sp Cas9,xCas9 3.7,which has 7 amino acid mutations(A262T,R324 L,S409I,E480 K,E543D,M694 I and E1219V),xCas9 3.7 can recognize multiple PAM sequences(5’-NG-3’,5’-GAA-3’ and 5’-GAT-3’)than wild type Sp Cas9 can only recognize the canonical 5’-NGG-3’ PAM sequence.Meanwhile,the off-target effects of xCas9 3.7 is much lower than that of wild type Sp Cas9.So far,the molecular mechanism of xCas9 3.7 with broad PAM compatibility and high DNA targeting specificity is not clear.In this project,the structure and functional mechanism of the xCas9 3.7-sg RNAds DNA ternary complex have been studied.X-ray diffraction technique was used to analyze the crystal structure of xCas9 3.7-sg RNA-ds DNA ternary complex,and combined with biochemical and molecular biological techniques to reveal the molecular mechanism of xCas9 3.7 with broad PAM compatibility and high DNA targeting specificity,which can provide clues and methods for rational engineering of more efficient Sp Cas9 variants or other Cas9 orthologs.In this study,xCas9 3.7-sg RNA-ds DNA ternary complex was obtained by expression and purification,and then the crystallization conditions of the ternary complex were preliminarily screened and optimized.Finally,the crystal structure of the xCas9 3.7-sg RNA-ds DNA ternary complex was analyzed by X-ray diffraction technique.Structural comparison between xCas9 3.7 complex and wild type Sp Cas9 complex revealed that,despite their overall similar architectures,the REC lobe of xCas9 3.7 undergoes significant conformational changes relative to the NUC lobe.Specifically,compared to that in Sp Cas9,the REC3 domain of xCas9 3.7 moves about 10 ? away from the RNA/DNA heteroduplex,leaving the 5’ end of the RNA/DNA heteroduplex solvent exposed.Meanwhile,the REC2 domain of xCas 3.7 also undergoes substantial conformational changes.In order to explore the molecular mechanism of xCas9 3.7 has broad PAM compatibility,it was found that a pair of salt bridges is established between E1219 and R1335 of wild type Sp Cas9 by structural comparison and in vitro cleavage assay,salt bridge-stabilized R1335 is critical for the stringent selection of PAM sequence.However,E1219 of xCas9 3.7 was replaced by V1219,which disrupts the salt bridges with R1335 and allows R1335 rotating without restriction,thus recognizing multiple PAM sequences.Therefore,xCas9 3.7 has a broad PAM compatibility.In order to explore the molecular mechanism of xCas9 3.7 with high DNA targeting specificity,the crystal structure of wild type Sp Cas9 complex and xCas9 3.7 complex were analyzed in depth and combined with biochemical experiments revealed that M694 of wild type Sp Cas9 forms the van der Waals contacts with the last two nucleobases of the DNA substrate.However,M694 of xCas9 3.7 was replaced by Ile and the interaction would be compromised.R324 and S409 of wild type Sp Cas9 are involved in intra-molecular interactions at the REC1-REC2 interface,mutations of these two residues(R324L and S409I)of xCas9 3.7 pertub the conformation of the REC3 domain that is interact with substrate DNA.Therefore,all the three mutations of xCas9 3.7(M694I,R324 L and S409I)cause significant conformational changes in the REC2-REC3 domain and make the REC2 and REC3 domains to far away from the RNA/DNA heteroduplex,leading to reduce contact with DNA substrate,thereby improving the specificity of targeted DNA.In this study,novel Sp Cas9 variants were designed based on the three-dimensional structure of xCas9 3.7 complex.Novel Sp Cas9 variants display high fidelity both in vitro biochemical assay and cellular assay.Taken together,we solved the crystal structure of xCas9 3.7-sg RNA-ds DNA ternary complex,our data revealed the molecular mechanisms of xCas9 3.7 with broad PAM compatibility and high DNA targeting specificity.Moreover,the novel Sp Cas9 variants designed based on the structural basis of xCas9 3.7 have high fidelity,which lays the foundation for design of more efficient Sp Cas9 variants or other Cas nucleases.It is of great significance to engineer novel high fidelity gene editing tools for gene therapy.