CRISPR

CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) are loci containing multiple short direct repeats that are found in the genomes of approximately 40% of bacteria and 90% of archaea.[2][3] CRISPR functions as a prokaryotic immune system, in that it confers resistance to exogenous genetic elements such as plasmids and phages.[4][5] The CRISPR system provides a form of acquired immunity. Short segments of foreign DNA, called spacers, are incorporated into the genome between CRISPR repeats, and serve as a 'memory' of past exposures.[6] CRISPR spacers are then used to recognize and silence exogenous genetic elements in a manner analogous to RNAi in eukaryotic organisms.[6]

Contents

Discovery of CRISPR

The clustered genomic repeats that are today known as CRISPR were first described in 1987 for the bacterium Escherichia coli.[7] In 2000, similar clustered repeats were identified in the genomes of additional bacteria and archaea, and were termed Short Regularly Spaced Repeats (SRSR).[8] SRSR were renamed CRISPR in 2002.[9] A set of genes, some encoding putative nuclease or helicase proteins, were found to be associated with CRISPR repeats (the cas, or CRISPR-associated, genes).[9]

CRISPR locus structure

CRISPR repeats and spacers

CRISPR repeats range in size from 24 to 48 base pairs.[10] They usually show some dyad symmetry, implying the formation of a secondary structure such as a hairpin, but are not truly palindromic.[11] CRISPR repeats are separated by spacers of similar length.[10] Some CRISPR spacer sequences have identity to sequences from plasmids and phage[12][13][14], although some spacers have identity to the prokaryote's own genome (self-targeting spacers).[15] New spacers can be added rapidly in response to phage infection.[16]

cas genes and CRISPR subtypes

The CRISPR-associated (cas) genes are often associated with CRISPR repeat-spacer arrays. More than forty different Cas protein families have been described.[10] Of these protein families, Cas1 appears to be ubiquitous among different CRISPR/Cas systems. Particular combinations of cas genes and repeat structures have been used to define 8 CRISPR subtypes (Ecoli, Ypest, Nmeni, Dvulg, Tneap, Hmari, Apern, and Mtube), some of which are associated with an additional gene module encoding repeat-associated mysterious proteins (RAMPs).[10] More than one CRISPR subtype may occur in a single genome. The sporadic distribution of the CRISPR/Cas subtypes suggests that the system is subject to horizontal gene transfer during microbial evolution.

CRISPR associated protein
crystal structure of a crispr-associated protein from thermus thermophilus
Identifiers
Symbol CRISPR_assoc
Pfam PF08798
Pfam clan CL0362
InterPro IPR010179
CRISPR associated protein Cas2
crystal structure of a hypothetical protein tt1823 from thermus thermophilus
Identifiers
Symbol CRISPR_Cas2
Pfam PF09827
InterPro IPR019199
CRISPR-associated protein Cse1
Identifiers
Symbol CRISPR_Cse1
Pfam PF09481
InterPro IPR013381
CRISPR-associated protein Cse2
Identifiers
Symbol CRISPR_Cse2
Pfam PF09485
InterPro IPR013382

CRISPR mechanism

Exogenous DNA is apparently processed by proteins encoded by some of the CRISPR-associated (cas) genes into small elements (of ~30bp in length), which are then somehow inserted into the CRISPR locus near the leader sequence. RNAs from the CRISPR loci are constituitively expressed and are processed by Cas proteins to small RNAs composed of individual exogenously-derived sequence elements with some flanking repeat sequence. The RNAs guide other Cas proteins to silence exogenous genetic elements at the RNA or DNA level.[1][17] There is evidence for functional diversity among the different CRISPR subtypes. The Cse (Cas subtype Ecoli) proteins (called CasA-E in E. coli) form a functional complex, Cascade, that processes CRISPR RNA transcripts into spacer-repeat units that are retained by Cascade.[18] In other prokaryotes, Cas6 processes the CRISPR transcripts. Interestingly, CRISPR-based phage inactivation in E. coli requires Cascade and Cas3, but not Cas1 and Cas2. The Cmr (Cas RAMP module) proteins found in Pyrococcus furiosus and other prokaryotes form a functional complex with small CRISPR RNAs that recognizes and cleaves complementary target RNAs.

Evolutionary significance and possible applications

A bioinformatic study has shown that the CRISPRs are evolutionarily conserved and cluster into related types. Many show signs of a conserved secondary structure.[11]

Through the CRISPR-Cas mechanism bacteria can acquire immunity against certain phages and thus halt further transmission of targeted phages. For this reason, some researchers have proposed that the CRISPR-Cas system is a Lamarckian inheritance mechanism.[19] Others investigated the coevolution of host and viral genomes by analysis of CRISPR sequences.[20]

There are several proposals for CRISPR-derived biotechnology:[21]

References

  1. ^ a b c Horvath P, Barrangou R (January 2010). "CRISPR/Cas, the immune system of bacteria and archaea". Science 327 (5962): 167–70. doi:10.1126/science.1179555. PMID 20056882. 
  2. ^ 71/79 Archaea, 463/1008 Bacteria CRISPRdb, Date: 19.6.2010
  3. ^ Grissa I, Vergnaud G, Pourcel C (2007). "The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats". BMC Bioinformatics 8: 172. doi:10.1186/1471-2105-8-172. PMC 1892036. PMID 17521438. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=1892036. 
  4. ^ Barrangou R, Fremaux C, Deveau H, et al. (March 2007). "CRISPR provides acquired resistance against viruses in prokaryotes". Science 315 (5819): 1709–12. doi:10.1126/science.1138140. PMID 17379808. 
  5. ^ Marraffini LA, Sontheimer EJ (December 2008). "CRISPR Interference Limits Horizontal Gene Transfer in Staphylococci by Targeting DNA". Science 322 (5909): 1843–5. doi:10.1126/science.1165771. PMC 2695655. PMID 19095942. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=2695655. 
  6. ^ a b c Marraffini LA, Sontheimer EJ (February 2010). "CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea". Nat Rev Genet 11 (3): 181–190. doi:10.1038/nrg2749. PMC 2928866. PMID 20125085. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=2928866. 
  7. ^ Ishino Y, Shinagawa H, Makino K, Amemura M, Nakata A (1987). "Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product". J Bacteriol 169 (12): 5429–33. PMC 213968. PMID 3316184. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=213968. 
  8. ^ Mojica FJM, Díez-Villaseñor C, Soria E, Juez G (2000). "Biological significance of a family of regularly spaced repeats in the genomes of Archaea, Bacteria and mitochondria". Mol Microbiol 36 (1): 244–6. doi:10.1046/j.1365-2958.2000.01838.x. PMID 10760181. 
  9. ^ a b Jansen R, Embden JD, Gaastra W, Schouls LM (2002). "Identification of genes that are associated with DNA repeats in prokaryotes". Mol Microbiol 43 (6): 1565–75. doi:10.1046/j.1365-2958.2002.02839.x. PMID 11952905. 
  10. ^ a b c d Haft DH, Selengut J, Mongodin EF, Nelson KE (2005). "A Guild of 45 CRISPR-Associated (Cas) Protein Families and Multiple CRISPR/Cas Subtypes Exist in Prokaryotic Genomes". PLoS Comput Biol. 1 (6): e60. doi:10.1371/journal.pcbi.0010060. PMC 1282333. PMID 16292354. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=1282333. 
  11. ^ a b Kunin V, Sorek R, Hugenholtz P (2007). "Evolutionary conservation of sequence and secondary structures in CRISPR repeats". Genome Biol 8 (4): R61. doi:10.1186/gb-2007-8-4-r61. PMC 1896005. PMID 17442114. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=1896005. 
  12. ^ Mojica FJ, Díez-Villaseñor C, García-Martínez J, Soria E (February 2005). "Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements". J. Mol. Evol. 60 (2): 174–82. doi:10.1007/s00239-004-0046-3. PMID 15791728. 
  13. ^ Bolotin A, Quinquis B, Sorokin A, Ehrlich SD (August 2005). "Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin". Microbiology (Reading, Engl.) 151 (Pt 8): 2551–61. doi:10.1099/mic.0.28048-0. PMID 16079334. 
  14. ^ Pourcel C, Salvignol G, Vergnaud G (2005). "CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies". Microbiology 151 (Pt 3): 653–63. doi:10.1099/mic.0.27437-0. PMID 15758212. 
  15. ^ Stern A, Keren L, Wurtzel O, Amitai G, Sorek R (August 2010). "Self-targeting by CRISPR: gene regulation or autoimmunity?". Trends Genet. 26 (8): 335–40. doi:10.1016/j.tig.2010.05.008. PMC 2910793. PMID 20598393. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=2910793. 
  16. ^ Tyson GW, Banfield JF (January 2008). "Rapidly evolving CRISPRs implicated in acquired resistance of microorganisms to viruses". Environ. Microbiol. 10 (1): 200–7. doi:10.1111/j.1462-2920.2007.01444.x. PMID 17894817. 
  17. ^ Makarova KS, Grishin NV, Shabalina SA, Wolf YI, Koonin EV (2006). "A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action". Biol Direct 1: 7. doi:10.1186/1745-6150-1-7. PMC 1462988. PMID 16545108. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=1462988. 
  18. ^ Brouns SJ, Jore MM, Lundgren M, et al. (August 2008). "Small CRISPR RNAs guide antiviral defense in prokaryotes". Science 321 (5891): 960–4. doi:10.1126/science.1159689. PMID 18703739. 
  19. ^ Koonin EV, Wolf YI (2009). "Is evolution Darwinian or/and Lamarckian?". Biol Direct 4: 42. doi:10.1186/1745-6150-4-42. PMC 2781790. PMID 19906303. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=2781790. 
  20. ^ Heidelberg JF, Nelson WC, Schoenfeld T, Bhaya D (2009). Ahmed, Niyaz. ed. "Germ Warfare in a Microbial Mat Community: CRISPRs Provide Insights into the Co-Evolution of Host and Viral Genomes". PLoS ONE 4 (1): e4169. doi:10.1371/journal.pone.0004169. PMC 2612747. PMID 19132092. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=2612747. 
  21. ^ Sorek R, Kunin V, Hugenholtz P (2008). "CRISPR--a widespread system that provides acquired resistance against phages in bacteria and archaea". Nat Rev Microbiol 6 (3): 181–6. doi:10.1038/nrmicro1793. PMID 18157154. 

Further reading

External links

Gallery of secondary structure images
CRISPR-DR2: Secondary structure taken from the Rfam database. Family RF01315.  
CRISPR-DR5: Secondary structure taken from the Rfam database. Family RF011318.  
CRISPR-DR6: Secondary structure taken from the Rfam database. Family RF01319.  
CRISPR-DR8: Secondary structure taken from the Rfam database. Family RF01321.  
CRISPR-DR9: Secondary structure taken from the Rfam database. Family RF01322.  
CRISPR-DR19: Secondary structure taken from the Rfam database. Family RF01332.  
CRISPR-DR41: Secondary structure taken from the Rfam database. Family RF01350.  
CRISPR-DR52: Secondary structure taken from the Rfam database. Family RF01365.  
CRISPR-DR57: Secondary structure taken from the Rfam database. Family RF01370.  
CRISPR-DR65: Secondary structure taken from the Rfam database. Family RF01378.