Efficient engineering of a bacteriophage genome using the type I-E CRISPR-Cas system

RNA Biol. 2014;11(1):42-4. doi: 10.4161/rna.27766. Epub 2014 Jan 22.

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) system has recently been used to engineer genomes of various organisms, but surprisingly, not those of bacteriophages (phages). Here we present a method to genetically engineer the Escherichia coli phage T7 using the type I-E CRISPR-Cas system. T7 phage genome is edited by homologous recombination with a DNA sequence flanked by sequences homologous to the desired location. Non-edited genomes are targeted by the CRISPR-Cas system, thus enabling isolation of the desired recombinant phages. This method broadens CRISPR Cas-based editing to phages and uses a CRISPR-Cas type other than type II. The method may be adjusted to genetically engineer any bacteriophage genome.

Keywords: Bacteriophage T7; Escherichia coli; genetic engineering; homologous recombination; negative selection; positive selection; spacer targeting.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacteriophage T7 / genetics*
  • CRISPR-Cas Systems*
  • Clustered Regularly Interspaced Short Palindromic Repeats / physiology*
  • Epigenesis, Genetic
  • Escherichia coli / metabolism*
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism
  • Genome, Viral
  • Recombination, Genetic
  • Viral Proteins / genetics

Substances

  • Escherichia coli Proteins
  • Viral Proteins