Authors
Csörgő, BálintLeón, Lina M.
Chau-Ly, Ilea J.
Vasquez-Rifo, Alejandro
Berry, Joel D.
Mahendra, Caroline
Crawford, Emily D.
Lewis, Jennifer D.
Bondy-Denomy, Joseph
UMass Chan Affiliations
Program in Molecular MedicineDocument Type
PreprintPublication Date
2019-12-03Keywords
CRISPRCas3
genome engineering
gene editing
Molecular Biology
Amino Acids, Peptides, and Proteins
Bioinformatics
Enzymes and Coenzymes
Genomics
Molecular Biology
Nucleic Acids, Nucleotides, and Nucleosides
Systems Biology
Metadata
Show full item recordAbstract
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.Source
bioRxiv 860999; doi: https://doi.org/10.1101/860999. Link to preprint on bioRxiv service
DOI
10.1101/860999Permanent Link to this Item
http://hdl.handle.net/20.500.14038/29434Rights
The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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.Distribution License
http://creativecommons.org/licenses/by-nc/4.0/ae974a485f413a2113503eed53cd6c53
10.1101/860999
Scopus Count
Collections
Except where otherwise noted, this item's license is described as 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.