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

Kylie Standage-Beier, Qi Zhang, Xiao Wang

Research output: Contribution to journalArticlepeer-review

74 Scopus citations


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
Issue number11
StatePublished - Nov 20 2015


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

ASJC Scopus subject areas

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


Dive into the research topics of 'Targeted Large-Scale Deletion of Bacterial Genomes Using CRISPR-Nickases'. Together they form a unique fingerprint.

Cite this