Using the CRISPR-Cas System to Positively Select Mutants in Genes Essential for Its Function
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.
Key wordsDefense mechanism Phage-host interaction Non-cas genes Autoimmunity Positive selection
This research was supported by the Israel Science Foundation grant 611/10 to U.Q., and the Marie Curie International Reintegration Grant PIRG-2010-266717 to R.E.
- 24.Yosef I, Goren MG, Kiro R, Edgar R, Qimron U (2011) High-temperature protein G is essential for activity of the Escherichia coli clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system. Proc Natl Acad Sci U S A 108(50):20136–20141. doi: 10.1073/pnas.1113519108 CrossRefPubMedCentralPubMedGoogle Scholar
- 25.Vercoe RB, Chang JT, Dy RL, Taylor C, Gristwood T, Clulow JS, Richter C, Przybilski R, Pitman AR, Fineran PC (2013) Cytotoxic chromosomal targeting by CRISPR/Cas systems can reshape bacterial genomes and expel or remodel pathogenicity islands. PLoS Genet 9(4):e1003454. doi: 10.1371/journal.pgen.1003454 CrossRefPubMedCentralPubMedGoogle Scholar
- 26.Kitagawa M, Ara T, Arifuzzaman M, Ioka-Nakamichi T, Inamoto E, Toyonaga H, Mori H (2005) Complete set of ORF clones of Escherichia coli ASKA library (a complete set of E. coli K-12 ORF archive): unique resources for biological research. DNA Res 12(5):291–299. doi: 10.1093/dnares/dsi012 CrossRefPubMedGoogle Scholar
- 27.Wilson K (1994) Preparation of genomic DNA from bacteria, p. 2.4. 1-2.4. 5. InIn FA Ausubel, R. Brent, RE Kingston, DD Moore, JG Seidman, JA Smith, and K. Struhl. Current protocols in molecular biology John Wiley & Sons, Inc, New York, NYGoogle Scholar
- 28.Larsen RA, Wilson MM, Guss AM, Metcalf WW (2002) Genetic analysis of pigment biosynthesis in Xanthobacter autotrophicus Py2 using a new, highly efficient transposon mutagenesis system that is functional in a wide variety of bacteria. Arch Microbiol 178(3):193–201. doi: 10.1007/s00203-002-0442-2 CrossRefPubMedGoogle Scholar
- 29.Walker CB, Stolyar S, Chivian D, Pinel N, Gabster JA, Dehal PS, He Z, Yang ZK, Yen HC, Zhou J, Wall JD, Hazen TC, Arkin AP, Stahl DA (2009) Contribution of mobile genetic elements to Desulfovibrio vulgaris genome plasticity. Environ Microbiol 11(9):2244–2252. doi: 10.1111/j.1462-2920.2009.01946.x CrossRefPubMedGoogle Scholar