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CRISPR associated protein
PDB 1wj9 EBI.jpg
crystal structure of a crispr-associated protein from thermus thermophilus
Pfam clanCL0362
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Diagram of the possible mechanism for CRISPR.[1]



CRISPRs are often associated with cas genes that code for proteins related to CRISPRs. The CRISPR/Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and phages[5][6] and provides a form of acquired immunity. CRISPR spacers recognize and silence these exogenous genetic elements like RNAi in eukaryotic organisms.[2]

Since 2012, the CRISPR/Cas system has been used for gene editing (silencing, enhancing or changing specific genes) that even works in eukaryotes like mice and primates.[7] By inserting a plasmid containing cas genes and specifically designed CRISPRs, an organism's genome can be cut at any desired location.


Bacteria may incorporate foreign DNA in other circumstances and even to scavenge damaged DNA from their environment.[8]

Repeats were first described in 1987 for the bacterium Escherichia coli.[9] In 2000, similar clustered repeats were identified in additional bacteria and archaea and were termed Short Regularly Spaced Repeats (SRSR).[10] SRSR were renamed CRISPR in 2002.[11] 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).[11]



In 2007 Barrangou, Horvath (food industry scientists at Danisco) and others showed that they could alter the resistance of Streptococcus thermophilus to phage attack with spacer DNA.[12]



CRISPR was first shown to work as a genome engineering/editing tool in human cell culture by 2012[13][14] It has since been used in a wide range of organisms including bakers yeast (S. cerevisiae),[15] zebra fish,[16] nematodes (C. elegans),[17] plants,[18] mice,[19] and several other organisms.


Libraries of tens of thousands of guide RNAs are now available.[12]


Gene-editing predecessors[edit]


Locus structure[edit]

Repeats and spacers[edit]

CRISPR repeats range in size from 24 to 48 base pairs.[23] They usually show some dyad symmetry, implying the formation of a secondary structure such as a hairpin, but are not truly palindromic.[24] Repeats are separated by spacers of similar length.[23] Some CRISPR spacer sequences exactly match sequences from plasmids and phages,[25][26][27] although some spacers match the prokaryote's genome (self-targeting spacers).[28] New spacers can be added rapidly in response to phage infection.[29]

Cas genes and CRISPR subtypes[edit]

CRISPR-associated (cas) genes are often associated with CRISPR repeat-spacer arrays. As of 2013, more than forty different Cas protein families had been described.[23] 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).[23] 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
PDB 1wj9 EBI.jpg
crystal structure of a crispr-associated protein from thermus thermophilus
Pfam clanCL0362
CRISPR associated protein Cas2
PDB 1zpw EBI.jpg
crystal structure of a hypothetical protein tt1823 from thermus thermophilus
CRISPR-associated protein Cse1
CRISPR-associated protein Cse2


Exogenous DNA is apparently processed by proteins encoded by Cas genes into small elements (~30 base pairs in length), which are then somehow inserted into the CRISPR locus near the leader sequence. RNAs from the CRISPR loci are constitutively expressed and are processed by Cas proteins to small RNAs composed of individual, exogenously-derived sequence elements with a flanking repeat sequence. The RNAs guide other Cas proteins to silence exogenous genetic elements at the RNA or DNA level.[1][30] Evidence suggests functional diversity among 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 Cascade retains.[31] 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. RNA-guided CRISPR enzymes are classified as type V restriction enzymes.

Evolutionary significance[edit]


Through the CRISPR/Cas mechanism, bacteria can acquire immunity to certain phages and thus halt further transmission of targeted phages. For this reason, some researchers have proposed that CRISPR/Cas is a Lamarckian inheritance mechanism.[32] Others investigated the coevolution of host and viral genomes by analysis of CRISPR sequences.[33]



The proof-of-principle demonstration of selective engineered redirection of the CRISPR/Cas system in 2012[35] provided a first step toward realization of proposals for CRISPR-derived biotechnology:[36]


Editas Medicine, a $43 million startup, aims to develop treatments that employ CRISPR/Cas to make edits to single base pairs and larger stretches of DNA. Inherited diseases such as cystic fibrosis, sickle-cell anemia and Huntington's disease are caused by single base pair mutations; CRISPR/Cas technology has the potential to correct these errors. The "corrected" gene remains in its normal location on its chromosome, which preserves the way the cell normally activate inhibits its expression.[40]





Mouse models[edit]





Reversible knockdown[edit]





Use by phages[edit]


Automation and library support[edit]



A provisional US patent application on the use of the CRISPR system for editing genes and regulating gene expression was filed by the inventors on May 12, 2012. Subsequent applications were combined and on March 6, 2014 the resulting patent application was published by the USPTO.[43] The patent rights have been assigned by the inventors to the Regents of the University of California and to the University of Vienna.


  7. ^ Wiedenheft, B; Sternberg, SH; Doudna, JA (Feb 15, 2012). "RNA-guided genetic silencing systems in bacteria and archaea.". Nature 482 (7385): 331–8. PMID 22337052. 
  30. ^ Makarova, K. S.; Grishin, N. V.; Shabalina, S. A.; Wolf, Y. I.; Koonin, E. V. (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". Biology Direct 1: 7. doi:10.1186/1745-6150-1-7. PMC 1462988. PMID 16545108.  edit
  31. ^ Brouns, S. J. J.; Jore, M. M.; Lundgren, M.; Westra, E. R.; Slijkhuis, R. J. H.; Snijders, A. P. L.; Dickman, M. J.; Makarova, K. S.; Koonin, E. V.; Van Der Oost, J. (2008). "Small CRISPR RNAs Guide Antiviral Defense in Prokaryotes". Science 321 (5891): 960–964. doi:10.1126/science.1159689. PMID 18703739.  edit

Further reading[edit]

External links[edit]