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BIMM 122 The CRISPR-Cas System of Bacterial Immunity Dr. Milton Saier “Man's mind, once stretched by a new idea, never regains its original dimensions.” – Oliver Wendell Holmes 1. Clustered regulatory interspaced short palindromic repeats (CRISPRs) target invading phage and plasmids. 2. They are found in many bacteria and most archaea. 3. They deal with environmental stressors including invading viruses. 4. It is described as the “bacterial immune system.” 5. The CRISPR repeats incorporate “spacers” homologous to fragments of the alien viral or plasmid genomes. 6. Possibly, the host incorporates short sequences from invading genetic elements (DNA) (virus or plasmid) into the CRISPR region. 7. The mechanism might comprise a constantly changing, co-evolutionary “arms race” between the host and invading agents. 8. The CRISPR and cas genes appear to evolve rapidly, accounting for their tremendous sequence divergence. 9. The process is sequence-specific, involving a variable cassette of CRISPRassociated (cas) genes. 10. There are at least four cas genes that are unique to and may be conserved in microorganisms having the CRISPR system. 11. The 4 Cas proteins of Yersinia (Csy 1-4) assemble into a 350 kDa ribonucleoprotein complex; some other bacteria/archaea have more cas genes. 12. Cas proteins from various organisms may be homologous, having common origins and structures. 13. Sequence similarity between these systems is meager, but they have conserved structural features. 14. There appear to be three phylogenetically distinct groups of these systems, all possibly related. 15. This complex facilitates target nucleic acid recognition by the CRISPR-Cas system. 16. This occurs because of sequence-specific hybridization between the CRISPRRNA 17. Target recognition is enthalpically driven. 18. Target recognition is localized to a “seed sequence” at the 5’ end of the CRISPR RNA spacer. 19. The Cas complex is a crescent shaped particle in many bacteria including E. coli. 20. When these sequences are transcribed and processed with small (s)RNAs, they guide the Cas system to recognize, bind to and cleave the invading genetic material. 21. This “adaptive immunity system” (at the nucleic acid rather than the protein levels) uses a library of small non-coding RNAs as a potential weapon against fast-evolving viruses and plasmids. 22. The system reveals the increasingly dynamic nature of prokaryotic genomes. 23. There are CRISPR polymerases, and “Repeat-associated Mysterious Proteins” (RAMPs), containing RNA recognition motif (RRM) domains. 24. CRISPR-Cas promotors are regulated, among other regulators, by H-NS, which is involved in induction, and by a chaperone protein, HtpG. 25. It has been suggested that these systems utilizes a (poorly defined) Lamarckian mechanism. “Evolution, like the tinker, does not produce innovations from scratch. It works on what already exists, transforming a system to give a new function, or combining several systems to produce a more complex one.” - F. Jacob from “The Possible and the Actual” References Bhaya, D., Davison, M., and Barrangou, R. (2011). CRISPR-Cas systems in bacteria and archaea: versatile small RNAs for adaptive defense and regulation. Annu Rev Genet 45, 273-297. Makarova, K.S., Aravind, L., Wolf, Y.I., and Koonin, E.V. (2011). Unification of Cas protein families and a simple scenario for the origin and evolution of CRISPR-Cas systems. Biol Direct 6, 38. Makarova, K.S., Haft, D.H., Barrangou, R., Brouns, S.J., Charpentier, E., Horvath, P., Moineau, S., Mojica, F.J., Wolf, Y.I., Yakunin, A.F., et al. (2011). Evolution and classification of the CRISPR-Cas systems. Nat Rev Microbiol 9, 467-477. Makarova, K.S., Wolf, Y.I., Snir, S., and Koonin, E.V. (2011). Defense islands in bacterial and archaeal genomes and prediction of novel defense systems. J Bacteriol 193, 6039-6056. Pul, U., Wurm, R., Arslan, Z., Geissen, R., Hofmann, N., and Wagner, R. (2010). Identification and characterization of E. coli CRISPR-cas promoters and their silencing by H-NS. Mol Microbiol 75, 1495-1512. Takeuchi, N., Wolf, Y.I., Makarova, K.S., and Koonin, E.V. (2011). Nature and Intensity of Selection Pressure on CRISPR-Associated Genes. J Bacteriol. Wiedenheft, B., van Duijn, E., Bultema, J.B., Waghmare, S.P., Zhou, K., Barendregt, A., Westphal, W., Heck, A.J., Boekema, E.J., Dickman, M.J., et al. (2011). RNAguided complex from a bacterial immune system enhances target recognition through seed sequence interactions. Proc Natl Acad Sci U S A 108, 10092-10097. Yosef, I., Goren, M.G., Kiro, R., Edgar, R., and 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, 20136-20141.