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

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

Author information

1
Department of Clinical Microbiology and Immunology; Sackler School of Medicine; Tel Aviv University; Tel Aviv, Israel.

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

PMID:
24457913
PMCID:
PMC3929423
DOI:
10.4161/rna.27766
[Indexed for MEDLINE]
Free PMC Article

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