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
'''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%
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). Asp10, Glu477, His701, and Asp704 ofSaCas9 are located at positions similar to those of the catalytic
identity, rmsd of 2.0 A˚ for 179 equivalent Ca atoms) and AnCas9
residues of SpCas9 (Asp10, Glu762, His983, and Asp986) and AnCas9 (Asp17, Glu505, His736, and Asp739). Indeed, the D10A, E477A, H701A, and D704A mutants of SaCas9 exhibited almost no DNA cleavage activity, suggesting that the SaCas9 RuvC domain
(PDB: 4OGE, 17% identity, rmsd of 3.0 A˚ for 169 equivalent Ca
cleaves the non-target DNA strand through a two-metal ion mechanism, as in other RNase H superfamily endonucleases. The HNH domain of SaCas9 has a ββα-metal fold, and shares structural similarity with those of SpCas9 (27% identity, rmsd of 1.8 A˚ for 93 equivalent Ca atoms) and AnCas9 (18% identity, rmsd of 2.6 A˚ for 98 equivalent Ca atoms). Asp556, His557, and Asn580 of SaCas9 are located at positions similar to those of the catalytic residues of SpCas9 (Asp839, His840, and Asn863) and AnCas9 (Asp581, His582, and Asn606). Indeed, the H557A and N580A mutants of SaCas9 almost completely lacked DNA cleavage activity, suggesting that the SaCas9 HNH domain cleaves the target DNA strand through a one-metal ion mechanism, as in other ββα-metal endonucleases. A structural comparison of SaCas9 with SpCas9 and AnCas9 revealed that the RuvC and HNH domains are connected by α-helical linkers, L1 and L2, and that notable differences exist in the relative arrangements between the two nuclease domains. A biochemical study suggested that PAM duplex binding to SpCas9 facilitates the cleavage of the target DNA strand by the HNH domain. However, in the PAM-containing quaternary complex structures of SaCas9 and SpCas9, the HNH domains are distant from the cleavage site of the target DNA strand. A structural comparison of SaCas9 with Thermus thermophilus RuvC in complex with a Holliday junction substrate indicated steric clashes between the L1 linker and the modeled non-target DNA strand, bound to the active site of the SaCas9 RuvC domain. These observations suggested that the binding of the non-target DNA strand to the RuvC domain may facilitate a conformational change of L1, thereby bringing the HNH domain to the scissile phosphate group in the target DNA strand.
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). The HNH domain of SaCas9 has a bba-metal fold, and shares
structural similarity with those of SpCas9 (27% identity, rmsd of
1.8 A˚ for 93 equivalent Ca atoms) and AnCas9 (18% identity,
rmsd of 2.6 A˚ for 98 equivalent Ca atoms) (Figure 6B). Asp556,
His557, and Asn580 of SaCas9 are located at positions similar
to those of the catalytic residues of SpCas9 (Asp839, His840,
and Asn863) and AnCas9 (Asp581, His582, and Asn606) (Figure
6B and Figure S3). Indeed, the H557A and N580A mutants
of SaCas9 almost completely lacked DNA cleavage activity
(Figures S7A and S7B), suggesting that the SaCas9 HNH
domain cleaves the target DNA strand through a one-metal ion mechanism, as in other bba-metal endonucleases (Biertu¨ mpfel
et al., 2007).
A structural comparison of SaCas9 with SpCas9 and AnCas9
revealed that the RuvC and HNH domains are connected by
a-helical linkers, L1 and L2, and that notable differences exist
in the relative arrangements between the two nuclease domains
(Figure 6C). A biochemical study suggested that PAM duplex
binding to SpCas9 facilitates the cleavage of the target DNA
strand by the HNH domain (Sternberg et al., 2014). However,
in the PAM-containing quaternary complex structures of SaCas9
and SpCas9, the HNH domains are distant from the cleavage site
of the target DNA strand (Figure 6C). A structural comparison of
SaCas9 with Thermus thermophilus RuvC in complex with a
Holliday junction substrate (Go´ recka et al., 2013) indicated steric
clashes between the L1 linker and the modeled non-target DNA
strand, bound to the active site of the SaCas9 RuvC domain
(Figures S7C and S7D). These observations suggested that the
binding of the non-target DNA strand to the RuvC domain may
facilitate a conformational change of L1, thereby bringing the
HNH domain to the scissile phosphate group in the target DNA
strand.


=See aslo=
=See aslo=

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Alexander Berchansky, Michal Harel
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