title: Using the CRISPR-Cas System to Positively Select Mutants in Genes Essential for Its Function.
abstract: The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated proteins (Cas) comprise a prokaryotic adaptive defense system against foreign nucleic acids. This defense is mediated by Cas proteins, which are guided by sequences flanked by the repeats, called spacers, to target nucleic acids. Spacers designed against the prokaryotic self chromosome are lethal to the prokaryotic cell. This self-killing of the bacterium by its own CRISPR-Cas system can be used to positively select genes that participate in this killing, as their absence will result in viable cells. Here we describe a positive selection assay that uses this feature to identify E. coli mutants encoding an inactive CRISPR-Cas system. The procedure includes establishment of an assay that detects this self-killing, generation of transposon insertion mutants in random genes, and selection of viable mutants, suspected as required for this lethal activity. This procedure enabled us to identify a novel gene, htpG, that is required for the activity of the CRISPR-Cas system. The procedures described here can be adjusted to various organisms to identify genes required for their CRISPR-Cas activity.
authors.affiliation: Department of Clinical Microbiology and Immunology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, 69978, Israel.
authors.forename: Ido
authors.initials: I
authors.lastname: Yosef
authors.forename: Moran G
authors.initials: MG
authors.lastname: Goren
authors.forename: Rotem
authors.initials: R
authors.lastname: Edgar
authors.forename: Udi
authors.initials: U
authors.lastname: Qimron
mesh_terms: D064113:CRISPR-Cas Systems / Q000235:genetics*; D004251:DNA Transposable Elements / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D016296:Mutagenesis; D009154:Mutation; D010641:Phenotype; D016133:Polymerase Chain Reaction; D014169:Transformation, Bacterial
chemical_list: D004251:DNA Transposable Elements