CRISPR RNA or crRNA is a RNA transcript from the CRISPR locus.[1] CRISPR-Cas (clustered, regularly interspaced short palindromic repeats - CRISPR associated systems) is an adaptive immune system found in bacteria and archaea to protect against mobile genetic elements, like viruses, plasmids, and transposons.[2] The CRISPR locus contains a series of repeats interspaced with unique spacers. These unique spacers can be acquired from MGEs.[2]

Transcripts of the CRISPR Genetic Locus and Maturation of pre-crRNA.

Pre-crRNA is formed after the transcription of the CRISPR locus and before being processed by Cas proteins. Mature crRNA transcripts contain a partial conserved section of repeat and a sequence of spacer that is complementary to the target DNA.[3] crRNA forms an effector complex with a single nuclease or multiple Cas proteins called a Cascade (CRISPR-associated complex for antiviral defense).[3][1] Once the effector complex is formed a Cas nuclease or single effector protein will cause interference guided by the crRNA match.[4]

Function

Type-I

Type-I CRISPR systems are characterized by Cas3, a nuclease-helicase protein, and the multi-subunit Cascade (CRISPR-associated complex for antiviral defense). The crRNA can form a complex with the Cas proteins in the Cascade and guide the complex to the target DNA sequence. Cas3 is recruited for the nuclease-helicase activity.[5]

Typically in the Cascade, Cas6 generates the mature crRNAs while Cas5 and Cas7 process and stabilize the crRNA.[6]

Type-II

Type-II CRISPR systems[7] are characterized by the single signature nuclease Cas9.[8] In type-II CRISPR systems crRNA and tracrRNA (trans-activating CRISPR RNA) can form a complex known as the guide RNA or gRNA.[9] The crRNA within the gRNA is what matches up with the target sequence or protospacer after the PAM is found. Once the match is made Cas9 will make a double-stranded break.

Stages of CRISPR immunity for type-I, type-II, and type-III.

Type-III

Type-III CRISPR systems are characterized by Cas10, an RNA cleaving protein.[10] Similar to type-I, a large subunit effector complex is formed and crRNA guides the complex to the target sequence. Cas6 helps to generate the mature crRNA.[10]

Type-IV

Type-IV CRISPR systems do not have an effector nuclease and are associated with plasmids and prophages. A Cas6-like enzyme is associated with the maturation of the crRNA. Not all type-IV systems have a CRISPR locus and therefore do not have crRNA.[11]

Type-V

Type-V CRISPR systems are characterized by Cas12, a nuclease that can cleave ssDNA, dsDNA, and RNA.[7] Like Cas9, Cas12 is the single effector nuclease. Type-V systems process pre-crRNA without tracrRNA. The mature crRNA in complex with Cas12 target the DNA sequence of interest and cleave the DNA.[12]

Type-VI

Type-VI CRISPR systems are characterized by Cas13, a single effector protein that targets RNA. Like the type-V system, Cas13 can process the pre-crRNA without tracrRNA. The mature crRNA in complex with Cas13 guides the complex to the target RNA and degrades it.[13]

References

  1. 1 2 Gasiunas, Giedrius; Barrangou, Rodolphe; Horvath, Philippe; Siksnys, Virginijus (2012-09-25). "Cas9–crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria". Proceedings of the National Academy of Sciences. 109 (39): E2579-86. doi:10.1073/pnas.1208507109. ISSN 0027-8424. PMC 3465414. PMID 22949671.
  2. 1 2 Faure, Guilhem; Shmakov, Sergey A.; Yan, Winston X.; Cheng, David R.; Scott, David A.; Peters, Joseph E.; Makarova, Kira S.; Koonin, Eugene V. (August 2019). "CRISPR–Cas in mobile genetic elements: counter-defence and beyond". Nature Reviews Microbiology. 17 (8): 513–525. doi:10.1038/s41579-019-0204-7. ISSN 1740-1534. S2CID 174809341.
  3. 1 2 Karvelis, Tautvydas; Gasiunas, Giedrius; Miksys, Algirdas; Barrangou, Rodolphe; Horvath, Philippe; Siksnys, Virginijus (2013-05-01). "crRNA and tracrRNA guide Cas9-mediated DNA interference in Streptococcus thermophilus". RNA Biology. 10 (5): 841–851. doi:10.4161/rna.24203. ISSN 1547-6286. PMC 3737341. PMID 23535272.
  4. ↑ Jinek, Martin; Chylinski, Krzysztof; Fonfara, Ines; Hauer, Michael; Doudna, Jennifer A.; Charpentier, Emmanuelle (2012-08-17). "A programmable dual RNA-guided DNA endonuclease in adaptive bacterial immunity". Science. 337 (6096): 816–821. Bibcode:2012Sci...337..816J. doi:10.1126/science.1225829. ISSN 0036-8075. PMC 6286148. PMID 22745249.
  5. ↑ Sinkunas, Tomas; Gasiunas, Giedrius; Fremaux, Christophe; Barrangou, Rodolphe; Horvath, Philippe; Siksnys, Virginijus (2011-04-06). "Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system". The EMBO Journal. 30 (7): 1335–1342. doi:10.1038/emboj.2011.41. ISSN 0261-4189. PMC 3094125. PMID 21343909.
  6. ↑ Brendel, Jutta; Stoll, Britta; Lange, Sita J.; Sharma, Kundan; Lenz, Christof; Stachler, Aris-Edda; Maier, Lisa-Katharina; Richter, Hagen; Nickel, Lisa; Schmitz, Ruth A.; Randau, Lennart; Allers, Thorsten; Urlaub, Henning; Backofen, Rolf; Marchfelder, Anita (2014-03-07). "A Complex of Cas Proteins 5, 6, and 7 Is Required for the Biogenesis and Stability of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-derived RNAs (crRNAs) in Haloferax volcanii". The Journal of Biological Chemistry. 289 (10): 7164–7177. doi:10.1074/jbc.M113.508184. ISSN 0021-9258. PMC 3945376. PMID 24459147.
  7. 1 2 Makarova, Kira S.; Wolf, Yuri I.; Iranzo, Jaime; Shmakov, Sergey A.; Alkhnbashi, Omer S.; Brouns, Stan J. J.; Charpentier, Emmanuelle; Cheng, David; Haft, Daniel H.; Horvath, Philippe; Moineau, Sylvain; Mojica, Francisco J. M.; Scott, David; Shah, Shiraz A.; Siksnys, Virginijus (February 2020). "Evolutionary classification of CRISPR–Cas systems: a burst of class 2 and derived variants". Nature Reviews Microbiology. 18 (2): 67–83. doi:10.1038/s41579-019-0299-x. ISSN 1740-1534. PMC 8905525. PMID 31857715.
  8. ↑ Heler, Robert; Samai, Poulami; Modell, Joshua W.; Weiner, Catherine; Goldberg, Gregory W.; Bikard, David; Marraffini, Luciano A. (2015-03-12). "Cas9 specifies functional viral targets during CRISPR-Cas adaptation". Nature. 519 (7542): 199–202. Bibcode:2015Natur.519..199H. doi:10.1038/nature14245. ISSN 0028-0836. PMC 4385744. PMID 25707807.
  9. ↑ Charpentier, Emmanuelle; Richter, Hagen; van der Oost, John; White, Malcolm F. (2015-05-01). "Biogenesis pathways of RNA guides in archaeal and bacterial CRISPR-Cas adaptive immunity". FEMS Microbiology Reviews. 39 (3): 428–441. doi:10.1093/femsre/fuv023. ISSN 0168-6445. PMC 5965381. PMID 25994611.
  10. 1 2 Kolesnik, Matvey V.; Fedorova, Iana; Karneyeva, Karyna A.; Artamonova, Daria N.; Severinov, Konstantin V. (2021-10-01). "Type III CRISPR-Cas Systems: Deciphering the Most Complex Prokaryotic Immune System". Biochemistry (Moscow). 86 (10): 1301–1314. doi:10.1134/S0006297921100114. ISSN 1608-3040. PMC 8527444. PMID 34903162.
  11. ↑ Pinilla-Redondo, Rafael; Mayo-Muñoz, David; Russel, Jakob; Garrett, Roger A.; Randau, Lennart; Sørensen, Søren J.; Shah, Shiraz A. (2020-02-28). "Type IV CRISPR-Cas systems are highly diverse and involved in competition between plasmids". Nucleic Acids Research. 48 (4): 2000–2012. doi:10.1093/nar/gkz1197. ISSN 1362-4962. PMC 7038947. PMID 31879772.
  12. ↑ Paul, Bijoya; Montoya, Guillermo (February 2020). "CRISPR-Cas12a: Functional overview and applications". Biomedical Journal. 43 (1): 8–17. doi:10.1016/j.bj.2019.10.005. ISSN 2319-4170. PMC 7090318. PMID 32200959.
  13. ↑ O'Connell, Mitchell R. (2019-01-04). "Molecular Mechanisms of RNA Targeting by Cas13-containing Type VI CRISPR-Cas Systems". Journal of Molecular Biology. 431 (1): 66–87. doi:10.1016/j.jmb.2018.06.029. ISSN 1089-8638. PMID 29940185. S2CID 49414939.
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