CRISPR-Cas9: Difference between revisions
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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 | 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 | ||
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. | 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. | ||
Structure-Guided Engineering of SaCas9 Transcription Activators and Inducible Nucleases | |||
Using the crystal structure of SaCas9, we sought to conduct | |||
structure-guided engineering to further expand the CRISPRCas9 | |||
toolbox, as we have done previously using the SpCas9 | |||
crystal structure. Given the similarities in the overall domain | |||
organizations of SaCas9 and SpCas9, we initially explored the | |||
feasibility of engineering the SaCas9 sgRNA, to develop robust | |||
transcription activators. In the SpCas9 structure, the tetraloop | |||
and stem loop 2 of the sgRNA are exposed to the solvent (Nishimasu | |||
et al., 2014; Anders et al., 2014) (Figure S4D), and | |||
permitted the insertion of RNA aptamers into the sgRNA to | |||
create robust RNA-guided transcription activators (Konermann | |||
et al., 2015). To generate the SaCas9-based activator system, | |||
we created a catalytically inactive version of SaCas9 (dSaCas9) | |||
by introducing the D10A and N580A mutations to inactivate the | |||
RuvC and HNH domains, respectively, and attached VP64 to | |||
the C terminus of dSaCas9 (Figures 7A and 7B). The sgRNA scaffold | |||
was modified by the insertion of the MS2 aptamer stem loop | |||
(MS2-SL), to allow the recruitment of MS2-p65-HSF1 transcriptional | |||
activation modules (Figure 7A). To evaluate the dSaCas9- | |||
based activator design, we constructed a transcriptional | |||
activation reporter system, consisting of tandem sgRNA target | |||
=See aslo= | =See aslo= | ||