New Collaborative Paper from the Lewis Lab

A minimal Cascade-Cas3 system for targeted genome engineering, by Bálint Csörgő, Lina M. León, Ilea J. Chau-Ly, Alejandro Vasquez-Rifo, Joel D. Berry, Caroline Mahendra, Emily D. Crawford, Jennifer D. Lewis and Joseph Bondy-Denomy. Nature Methods (in press). 


CRISPR-Cas technologies are programmable gene editing tools that have revolutionized research. However, the leading Cas9 and Cas12a enzymes are limited for genome-scale interventions. Here, we utilized the processive nuclease Cas3, together with a minimal Type I-C Cascade-based system for targeted genome engineering. DNA cleavage guided by a single CRISPR RNA (crRNA) generated large deletions (7 – 424 kb) in Pseudomonas aeruginosa with near-100% efficiency, while Cas9 yielded small deletions and point mutations. Cas3 generated bi-directional deletions originating from the programmed site, which was exploited to reduce the P. aeruginosa genome by 837 kb (13.5 %). Large deletion boundaries were efficiently specified by a homology-directed repair (HDR) template during Cascade-Cas3, but not Cas9, targeting. A transferable “all-in-one” vector was functional in E. coliP. syringae, and Klebsiella pneumoniae, and endogenous CRISPR-Cas utility was enhanced with an “anti-anti-CRISPR” strategy. Cascade-Cas3 facilitates rapid strain manipulation for synthetic biology, genome minimization, screening, and the analysis/removal of large genomic regions.