CRISPR-Cas9: Difference between revisions
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A structural comparison of SaCas9, SpCas9, and AnCas9 revealed that, despite the lack of sequence homology, their PI domains share a similar protein fold. The PI domains consist of the TOPO domain, comprising a three-stranded anti-parallel β-sheet (β1–β3) flanked by several α-helices, and the C-terminal domain, comprising a twisted six-stranded anti-parallel β-sheet (β4–β9) (β7 in SpCas9 adopts a loop conformation). In both SaCas9 and SpCas9, the major groove of the PAM duplex is read by the β5–β7 region in their PI domains. The 3rd G in the 5'-NNGRRT-3' PAM is recognized by Arg1015 in SaCas9 , whereas the 3rd G in the 5'-NGG-3' PAM is recognized by Arg1335 in SpCas9 in a similar manner. However, there are notable differences in the PI domains of SaCas9 and SpCas9, consistent with their distinct PAM specificities. Arg1333 of SpCas9, which recognizes the 2nd G in the 5'-NGG-3' PAM, is replaced with Pro1013 in SaCas9. In addition, SpCas9 lacks the amino acid residues equivalent to Asn985/Asn986 (β5) and Arg991 (β6) of SaCas9, because the b5–b6 region of SpCas9 is shorter than that of SaCas9. Moreover, Asn985, Asn986, Arg991, and Arg1015 in SaCas9 are replaced with Asp1030, Thr1031, Lys1034, and Lys1061 in AnCas9, respectively, suggesting that the PAM of AnCas9 is different from those of SaCas9 and SpCas9 (although the sequence remains unknown). Together, these structural findings demonstrate that the distinct PAM specificities of the Cas9 orthologs are primarily defined by the specific differences in the PAM-interacting residues in the PI domains. | A structural comparison of SaCas9, SpCas9, and AnCas9 revealed that, despite the lack of sequence homology, their PI domains share a similar protein fold. The PI domains consist of the TOPO domain, comprising a three-stranded anti-parallel β-sheet (β1–β3) flanked by several α-helices, and the C-terminal domain, comprising a twisted six-stranded anti-parallel β-sheet (β4–β9) (β7 in SpCas9 adopts a loop conformation). In both SaCas9 and SpCas9, the major groove of the PAM duplex is read by the β5–β7 region in their PI domains. The 3rd G in the 5'-NNGRRT-3' PAM is recognized by Arg1015 in SaCas9 , whereas the 3rd G in the 5'-NGG-3' PAM is recognized by Arg1335 in SpCas9 in a similar manner. However, there are notable differences in the PI domains of SaCas9 and SpCas9, consistent with their distinct PAM specificities. Arg1333 of SpCas9, which recognizes the 2nd G in the 5'-NGG-3' PAM, is replaced with Pro1013 in SaCas9. In addition, SpCas9 lacks the amino acid residues equivalent to Asn985/Asn986 (β5) and Arg991 (β6) of SaCas9, because the b5–b6 region of SpCas9 is shorter than that of SaCas9. Moreover, Asn985, Asn986, Arg991, and Arg1015 in SaCas9 are replaced with Asp1030, Thr1031, Lys1034, and Lys1061 in AnCas9, respectively, suggesting that the PAM of AnCas9 is different from those of SaCas9 and SpCas9 (although the sequence remains unknown). Together, these structural findings demonstrate that the distinct PAM specificities of the Cas9 orthologs are primarily defined by the specific differences in the PAM-interacting residues in the PI domains. | ||
Mechanism of Target DNA Unwinding | |||
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 (Anders | |||
et al., 2014) (Figure 5F). The present structure revealed that | |||
SaCas9 also has the phosphate lock loop, although it shares | |||
limited sequence similarity to that of SpCas9 (Figure 5G and Figure | |||
S3). 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 (Figure 5G). 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. | |||
=See aslo= | =See aslo= | ||