Targeted Large-Scale Deletion of Bacterial Genomes Using CRISPR-Nickases

Kylie Standage-Beier, Qi Zhang, Xiao Wang

Research output: Contribution to journalArticle

37 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)1217-1225
Number of pages9
JournalACS Synthetic Biology
Volume4
Issue number11
DOIs
StatePublished - Nov 20 2015

Keywords

  • Cas9 nickase
  • chromosome deletion
  • CRISPR
  • direct repeats
  • genome engineering
  • recombination

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology (miscellaneous)
  • Biomedical Engineering

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