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Precision Engineering of the Genome, Epigenome and Transcriptome | EK24


A compact Cascade-Cas3 system for targeted genome engineering and gene discovery


Mar 8, 2021 12:00am ‐ Mar 8, 2021 12:00am

Description

A compact Cascade-Cas3 system for targeted genome engineering and gene discovery Balint Csorgo, Lina M. Leon, Alex Wirganowicz, Joseph Bondy-Denomy University of California, San Francisco CRISPR-Cas technologies are programmable gene editing tools that have revolutionized research. The leading Cas9 and Cas12a enzymes have been optimized for smaller scale modifications and are limited for larger, genome-scale interventions. To expand the current genome-scale editing toolset, we utilize 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) generates large deletions (7 – 424 kb) in Pseudomonas aeruginosa with near-100% efficiency, while Cas9 yields small deletions and point mutations. Cas3 generated bi-directional deletions originating from the programmed site, which was utilized to reduce the P. aeruginosa genome by 1.05 Mb (~19.5 %). 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 a variety of bacteria, including E. coli, P. syringae, and Klebsiella pneumoniae. We are employing Cas3 as an unbiased discovery tool in a range of clinical and environmental isolates of P. aeruginosa to identify previously uncharacterized bacterial immune systems. Overall, Cascade-Cas3 facilitates rapid strain manipulation for synthetic biology, genome minimization, screening, and the analysis/removal of large genomic regions.

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