CRISPR-Cas9: Difference between revisions
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In SpCas9, Glu1108 and Ser1109, in the phosphate lock loop, hydrogen bond with the phosphate group between dA(1) and dT1 in the target DNA strand (referred to as the +1 phosphate), thereby contributing to the target DNA unwinding. The present structure revealed that SaCas9 also has the phosphate lock loop, although it shares limited sequence similarity to that of SpCas9. In SaCas9, the +1 phosphate between dA(1) and dG1, in the target DNA strand, hydrogen bonds with the main-chain amide groups of Asp786 and Thr787 and the side chain of Thr787 in the phosphate lock loop. These interactions result in the rotation of the +1 phosphate, thereby facilitating base-pairing between dG1 in the target DNA strand and C20 in the sgRNA. Indeed, the SaCas9 T787A mutant showed reduced DNA cleavage activity (Figure 5C), confirming the functional significance of Thr787 in the phosphate lock loop. These observations indicated the conserved molecular mechanism of target DNA unwinding in SaCas9 and SpCas9. | In SpCas9, Glu1108 and Ser1109, in the phosphate lock loop, hydrogen bond with the phosphate group between dA(1) and dT1 in the target DNA strand (referred to as the +1 phosphate), thereby contributing to the target DNA unwinding. The present structure revealed that SaCas9 also has the phosphate lock loop, although it shares limited sequence similarity to that of SpCas9. In SaCas9, the +1 phosphate between dA(1) and dG1, in the target DNA strand, hydrogen bonds with the main-chain amide groups of Asp786 and Thr787 and the side chain of Thr787 in the phosphate lock loop. These interactions result in the rotation of the +1 phosphate, thereby facilitating base-pairing between dG1 in the target DNA strand and C20 in the sgRNA. Indeed, the SaCas9 T787A mutant showed reduced DNA cleavage activity (Figure 5C), confirming the functional significance of Thr787 in the phosphate lock loop. These observations indicated the conserved molecular mechanism of target DNA unwinding in SaCas9 and SpCas9. | ||
RuvC and HNH Nuclease Domains | |||
The RuvC domain of SaCas9 has an RNase H fold, and shares | |||
structural similarity with those of SpCas9 (PDB: 4UN3, 26% | |||
identity, rmsd of 2.0 A˚ for 179 equivalent Ca atoms) and AnCas9 | |||
(PDB: 4OGE, 17% identity, rmsd of 3.0 A˚ for 169 equivalent Ca | |||
atoms) (Figure 6A). Asp10, Glu477, His701, and Asp704 of | |||
SaCas9 are located at positions similar to those of the catalytic | |||
residues of SpCas9 (Asp10, Glu762, His983, and Asp986) and | |||
AnCas9 (Asp17, Glu505, His736, and Asp739) (Figure 6A and | |||
Figure S3). Indeed, the D10A, E477A, H701A, and D704A mutants | |||
of SaCas9 exhibited almost no DNA cleavage activity (Figures | |||
S7A and S7B), suggesting that the SaCas9 RuvC domain | |||
cleaves the non-target DNA strand through a two-metal ion | |||
mechanism, as in other RNase H superfamily endonucleases | |||
(Go´ recka et al., 2013). | |||
=See aslo= | =See aslo= | ||