Insights into the compact CRISPR–Cas9d system

Nature Communications, Journal Year: 2025, Volume and Issue: 16(1)

Published: March 12, 2025

Cas9d, the smallest known member of Cas9 family, employs a compact domain architecture for effective target cleavage. However, underlying mechanism remains unclear. Here, we present cryo-EM structures Cas9d–sgRNA complex in both target-free and target-bound states. Biochemical assays elucidated PAM recognition DNA cleavage mechanisms Cas9d. Structural comparisons revealed that at least 17 base pairs guide–target heteroduplex is required nuclease activity. Beyond its typical role as an adaptor between enzymes targets, sgRNA also provides structural support functional regulation A segment scaffold interacts with REC to form module. Upon binding, this module undergoes coordinated conformational rearrangement, enabling propagation facilitating This hybrid precisely monitors complementarity, resulting lower mismatch tolerance compared SpyCas9. Moreover, structure-guided engineering Cas9d protein led more system well-maintained Altogether, our findings provide insights into shed light on development high-fidelity mini-CRISPR tools. unique RNA-coordinated precise targeting. Structure-guided achieved smaller, CRISPR robust activity, advancing genome-editing applications.

Language: Английский

Analysis of metal-dependent DNA nicking activities by Cas endonucleases

Methods in enzymology on CD-ROM/Methods in enzymology, Journal Year: 2025, Volume and Issue: unknown, P. 117 - 142

Published: Jan. 1, 2025

Language: Английский