Impacts of DNA Supercoiling on the Sequence-Dependent Nuclease Activity of CRISPR-Cas9 with Truncated Guides.

ABSTRACT: CRISPR-Cas9 is natively present in the adaptive immune systems of a multitude of bacteria and has been adapted as an effective genome engineering tool. The Cas9 effector enzyme, which is composed of a single Cas9 protein and a single-guide RNA (sgRNA), identifies and cleaves double-stranded DNA targets through a series of conformational changes that require DNA distortion and unwinding. While most studies of Cas9 specificity have focused on the DNA sequence, the role of intrinsic DNA physical properties ("DNA shape") in modulating Cas9 activity remains insufficiently defined. We previously showed that with a 16-nucleotide (-nt) truncated guide, the intrinsic DNA duplex dissociation energy at the PAM+(17-20) segment beyond the RNA-DNA hybrid tunes Cas9 cleavage rates of linear substrates. H

SUBMITTER: Schuster I 

PROVIDER: S-EPMC12893335 | biostudies-literature | 2026 Feb

REPOSITORIES: biostudies-literature