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ARS Home » Pacific West Area » Albany, California » Plant Gene Expression Center » Research » Publications at this Location » Publication #378888

Research Project: Mining Collections of Wild Germplasm and Novel Defense Regulators for Enhanced Plant Defenses

Location: Plant Gene Expression Center

Title: A compact Cascade-Cas3 system for targeted genome engineering

item CSORGO, BALINT - University Of San Francisco
item LEON, LINA - University Of San Francisco
item CHAU-LY, ILEA - University Of California
item VASQUEZ-RIFO, ALEJANDRO - University Of San Francisco
item BERRY, JOEL - University Of San Francisco
item MAHENDRA, CAROLINE - University Of San Francisco
item CRAWFORD, EMILY - University Of San Francisco
item Lewis, Jennifer
item BONDY-DENOMY, JOSEPH - University Of San Francisco

Submitted to: Nature Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/15/2020
Publication Date: 10/19/2020
Citation: Csorgo, B., Leon, L., Chau-Ly, I., Vasquez-Rifo, A., Berry, J., Mahendra, C., Crawford, E., Lewis, J.D., Bondy-Denomy, J. 2020. A compact Cascade-Cas3 system for targeted genome engineering. Nature Methods. 17:1183-1190.

Interpretive Summary: Many bacteria, such as Pseudomonas syringae, cause disease in crop plants by injecting a suite of effector proteins into the plant. Effectors have dual roles - they contribute to bacterial virulence and disease symptoms in susceptible hosts or are recognized by the immune system in resistant hosts. However, it is difficult to show how each effector protein contributes to bacterial virulence and disease symptoms or to effector recognition because effectors can have overlapping functions. We developed a gene editing technique in P. syringae which allows the removal of entire clusters of effector genes. This tool will help identify the function of effector proteins and determine which ones contribute most to disease or defense responses. This work will help identify bacterial genes which could be modified by biotechnology and enhance food security.

Technical Abstract: 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. coli, P. 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.