Efficient engineering of a bacteriophage genome using the type I-E CRISPR-Cas system.
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.
Department of Clinical Microbiology and Immunology; Sackler School of Medicine; Tel Aviv University; Tel Aviv, Israel.
Ruth
R
Kiro
Department of Clinical Microbiology and Immunology; Sackler School of Medicine; Tel Aviv University; Tel Aviv, Israel.
Dror
D
Shitrit
Department of Clinical Microbiology and Immunology; Sackler School of Medicine; Tel Aviv University; Tel Aviv, Israel.
Udi
U
Qimron
D017123:Bacteriophage T7 / Q000235:genetics*; D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000502:physiology*; D044127:Epigenesis, Genetic; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D016679:Genome, Viral; D011995:Recombination, Genetic; D014764:Viral Proteins / Q000235:genetics
D029968:Escherichia coli Proteins; D014764:Viral Proteins
Bacteriophage T7; Escherichia coli; genetic engineering; homologous recombination; negative selection; positive selection; spacer targeting