Probing the stability of the SpCas9-DNA complex after cleavage.

ABSTRACT: CRISPR-Cas9 is a ribonucleoprotein complex that sequence-specifically binds and cleaves double-stranded DNA. Wildtype Cas9 and its nickase and cleavage-incompetent mutants have been used in various biological techniques due to their versatility and programmable specificity. Cas9 has been shown to bind very stably to DNA even after cleavage of the individual DNA strands, inhibiting further turnovers and considerably slowing down in-vivo repair processes. This poses an obstacle in genome editing applications. Here, we employed single-molecule magnetic tweezers to investigate the binding stability of different Streptococcus pyogenes Cas9 variants after cleavage by challenging them with supercoiling. We find that different release mechanisms occur depending on which DNA strand is cleaved. Afte

SUBMITTER: Aldag P 

PROVIDER: S-EPMC8643700 | biostudies-literature | 2021 Dec

REPOSITORIES: biostudies-literature