Abstract:
:CRISPR-Cas9 technology has been at the forefront of the field of biology. The Streptococcus pyogenes (SpyCas9) protein forms a complex with guide RNA and can recognize and cleave double-stranded DNA through hybridization based on 20 base pairings. SpyCas9 has two nuclease domains, HNH and RuvC, each of which cuts each DNA strand, and both contain critical histidine residues. Although previously reported crystal structures provide useful geometric information, the extent to which these residues functionally contribute to catalysis is unknown. Here, we mutated histidine residues on HNH and RuvC domains to alanine or glycine and attempted to rescue the enzymatic activity by adding the imidazole molecule, using an in vitro DNA cleavage assay. H840A and H840G exhibited rescued enzymatic activity on the HNH domain following imidazole addition, suggesting that H840 acts as a general base. We also tested various chemicals and found that the pKa of imidazole derivatives, and not their molecular shape, correlated with the rescue effect. In contrast, both H983A and H983G on the RuvC domain did not exhibit a rescue effect following imidazole addition. Our chemical rescue approach will provide crucial insight into understanding Cas9 catalysis, complementing structural analyses.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Furuhata Y,Kato Ydoi
10.1021/acs.biochem.0c00766subject
Has Abstractpub_date
2021-01-26 00:00:00pages
194-200issue
3eissn
0006-2960issn
1520-4995journal_volume
60pub_type
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