Program in Molecular Medicine
Amino Acids, Peptides, and Proteins | Bioinformatics | Enzymes and Coenzymes | Genomics | Molecular Biology | Nucleic Acids, Nucleotides, and Nucleosides | Systems Biology
CRISPR-Cas technologies have provided programmable gene editing tools that have revolutionized research. The leading CRISPR-Cas9 and Cas12a enzymes are ideal for programmed genetic manipulation, however, they are limited for genome-scale interventions. Here, we utilized a Cas3-based system featuring a processive nuclease, expressed endogenously or heterologously, for genome engineering purposes. Using an optimized and minimal CRISPR-Cas3 system (Type I-C) programmed with a single crRNA, large deletions ranging from 7 - 424 kb were generated in Pseudomonas aeruginosa with high efficiency and speed. By comparison, Cas9 yielded small deletions and point mutations. Cas3-generated deletion boundaries were variable in the absence of a homology-directed repair (HDR) template, and successfully and efficiently specified when present. The minimal Cas3 system is also portable; large deletions were induced with high efficiency in Pseudomonas syringae and Escherichia coli using an “all-in-one” vector. Notably, Cas3 generated bi-directional deletions originating from the programmed cut site, which was exploited to iteratively reduce a P. aeruginosa genome by 837 kb (13.5%) using 10 distinct crRNAs. We also demonstrate the utility of endogenous Cas3 systems (Type I-C and I-F) and develop an “anti-anti-CRISPR” strategy to circumvent endogenous CRISPR-Cas inhibitor proteins. CRISPR-Cas3 could facilitate rapid strain manipulation for synthetic biological and metabolic engineering purposes, genome minimization, and the analysis of large regions of unknown function.
CRISPR, Cas3, genome engineering, gene editing, Molecular Biology
Rights and Permissions
The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It is made available under a CC-BY-NC 4.0 International license.
DOI of Published Version
bioRxiv 860999; doi: https://doi.org/10.1101/860999. Link to preprint on bioRxiv service
Csörgő B, León LM, Chau-Ly IJ, Vasquez-Rifo A, Berry JD, Mahendra C, Crawford ED, Lewis JD, Bondy-Denomy J. (2019). A minimal CRISPR-Cas3 system for genome engineering [preprint]. University of Massachusetts Medical School Faculty Publications. https://doi.org/10.1101/860999. Retrieved from https://escholarship.umassmed.edu/faculty_pubs/1660
Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial 4.0 License
Amino Acids, Peptides, and Proteins Commons, Bioinformatics Commons, Enzymes and Coenzymes Commons, Genomics Commons, Molecular Biology Commons, Nucleic Acids, Nucleotides, and Nucleosides Commons, Systems Biology Commons