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BIMM 122 Lecture Notes #26 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 and plasmids 4. These systems are 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. These complexes facilitate target nucleic acid recognition by the CRISPR-Cas system. 16. This occurs because of sequence-specific hybridization between the CRISPRRNA and the target nucleic acid. 17. Target recognition is enthalpically driven (e.g., uses hydrophilic forces including ionic and polar bonds). 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 (High temperature protein G). 25. It has been suggested that these systems utilize a (poorly defined) Lamarckian mechanism. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPRassociated (Cas) systems protect prokaryotes from invading viruses and plasmids. These defense systems rely on small RNAs for sequence-specific detection and silencing of foreign nucleic acids. CRISPR/Cas systems are composed of cas genes organized in operon(s) and CRISPR array(s) consisting of genome-targeting sequences (called spacers) interspersed with identical repeats. CRISPR/Cas-mediated immunity occurs in three steps. In the adaptive phase, bacteria and archaea harboring one or more CRISPR loci respond to viral or plasmid challenge by integrating short fragments of foreign sequence (protospacers) into the host chromosome at the proximal end of the CRISPR array. In the expression and interference phases, transcription of the repeat-spacer element into precursor CRISPR RNA (pre-crRNA) molecules followed by enzymatic cleavage yields the short crRNAs that can pair with complementary protospacer sequences of invading viral or plasmid targets. Target recognition by crRNAs directs the silencing of the foreign sequences by means of Cas proteins that function in complex with the crRNAs. There are three types of CRISPR/Cas systems. The type I and III systems share some overarching features: specialized Cas endonucleases process the pre-crRNAs, and once mature, each crRNA assembles into a large multi-Cas protein complex capable of recognizing and cleaving nucleic acids complementary to the crRNA. In contrast, type II systems process pre-crRNAs by a different mechanism in which a trans-activating crRNA (tracrRNA) complementary to the repeat sequences in pre-crRNA triggers processing by the double-stranded (ds) RNA-specific ribonuclease RNase III in the presence of the Cas9 protein. Cas9 is thought to be the sole protein responsible for crRNA-guided silencing of foreign DNA. In type II systems, Cas9 proteins constitute a family of enzymes that require a base-paired structure formed between the activating tracrRNA and the targeting crRNA to cleave target dsDNA. Site-specific cleavage occurs at locations determined by both base-pairing complementarity between the crRNA and the target protospacer DNA and a short motif [referred to as the protospacer adjacent motif (PAM)] juxtaposed to the complementary region in the target DNA. The Cas9 endonuclease family proteins can be programmed with single RNA molecules to cleave specific DNA sites, thereby raising the possibility of developing a simple and versatile RNA-directed system to generate dsDNA breaks for genome targeting and editing (Jinek et al. 2012)). “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. Jinek, M., K. Chylinski, I. Fonfara, M. Hauer, J.A. Doudna, and E. Charpentier. (2012). A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337: 816-821. 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. (2012). Nature and intensity of selection pressure on CRISPR-associated genes. J Bacteriol. 194, 1216-25. 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.