title: Targeted Large-Scale Deletion of Bacterial Genomes Using CRISPR-Nickases.
abstract: Programmable CRISPR-Cas systems have augmented our ability to produce precise genome manipulations. Here we demonstrate and characterize the ability of CRISPR-Cas derived nickases to direct targeted recombination of both small and large genomic regions flanked by repetitive elements in Escherichia coli. While CRISPR directed double-stranded DNA breaks are highly lethal in many bacteria, we show that CRISPR-guided nickase systems can be programmed to make precise, nonlethal, single-stranded incisions in targeted genomic regions. This induces recombination events and leads to targeted deletion. We demonstrate that dual-targeted nicking enables deletion of 36 and 97 Kb of the genome. Furthermore, multiplex targeting enables deletion of 133 Kb, accounting for approximately 3% of the entire E. coli genome. This technology provides a framework for methods to manipulate bacterial genomes using CRISPR-nickase systems. We envision this system working synergistically with preexisting bacterial genome engineering methods.
authors.affiliation: School of Life Sciences, ‡School of Biological and Health Systems Engineering, Arizona State University , Tempe, Arizona 85287, United States.
authors.forename: Kylie
authors.initials: K
authors.lastname: Standage-Beier
authors.affiliation: School of Life Sciences, ‡School of Biological and Health Systems Engineering, Arizona State University , Tempe, Arizona 85287, United States.
authors.forename: Qi
authors.initials: Q
authors.lastname: Zhang
authors.affiliation: School of Life Sciences, ‡School of Biological and Health Systems Engineering, Arizona State University , Tempe, Arizona 85287, United States.
authors.forename: Xiao
authors.initials: X
authors.lastname: Wang
mesh_terms: D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D017353:Gene Deletion; D016680:Genome, Bacterial; D011995:Recombination, Genetic; D058615:Synthetic Biology
keywords: CRISPR; Cas9 nickase; chromosome deletion; direct repeats; genome engineering; recombination