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20  structures 167  species 1  interaction 180  sequences 1  architecture

Family: CRISPR_assoc (PF08798)

Summary: CRISPR associated protein

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This is the Wikipedia entry entitled "CRISPR". More...

CRISPR Edit Wikipedia article

Diagram of the possible mechanism for CRISPR.[1]

CRISPRs (clustered regularly interspaced short palindromic repeats) are DNA loci containing short repetitions of base sequences. Each repetition is followed by short segments of "spacer DNA" from previous exposures to a virus.[2]

CRISPRs are found in approximately 40% of sequenced eubacteria genomes and 90% of sequenced archaea.[3][4]

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 cut these exogenous genetic elements in a manner analogous to RNAi in eukaryotic organisms.[2]

Since 2013, the CRISPR/Cas system has been used for gene editing (adding, disrupting or changing the sequence of specific genes) and gene regulation in species throughout the tree of life.[7] By delivering the Cas9 protein and appropriate guide RNAs into a cell, the organism's genome can be cut at any desired location.

It may be possible to use CRISPR to build RNA-guided gene drives capable of altering the genomes of entire populations.[8]


Bacteria may incorporate foreign DNA in other circumstances and even scavenge damaged DNA from their environment.[9]

Repeats were first described in 1987 for the bacterium Escherichia coli.[10] In 2000, similar clustered repeats were identified in additional bacteria and archaea and were termed Short Regularly Spaced Repeats (SRSR).[11] SRSR were renamed CRISPR in 2002.[12] 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).[12]

Simplified diagram of a CRISPR locus. The three major components of a CRISPR locus are shown: cas genes, a leader sequence, and a repeat-spacer array. Repeats are shown as grey boxes and spacers are colored bars. While most CRISPR loci contain each of the three components, the arrangement is not always as shown.[1][2]

In 2005, three independent researchers showed that CRISPR spacers showed homology to several phage DNA and extrachromosomal DNA such as plasmids. This was an indication that the CRISPR/cas system could have a role in adaptive immunity in bacteria.[1] Koonin and colleagues proposed that spacers serve as a template for RNA molecules, analogously to eukaryotic cells that use a system called RNA interference.[13]

In 2007 Barrangou, Horvath (food industry scientists at Danisco) and Moineau's group at Université Laval (Canada) showed that they could alter the resistance of Streptococcus thermophilus to phage attack with spacer DNA.[13]

Doudna and Charpentier had independently been exploring CRISPR-associated proteins to learn how bacteria deploy spacers in their immune defenses. They jointly studied a simpler CRISPR system that relies on a protein called Cas9. They found that bacteria respond to an invading phage by transcribing spacers and palindromic DNA into a long RNA molecule that the cell then uses tracrRNA and Cas9 to cut it into pieces called crRNAs.[13]

Cas9 is a nuclease, an enzyme specialized for cutting DNA, with two active cutting sites, one for each strand of the double helix. The team demonstrated that they could disable one or both sites while preserving Cas9's ability to home in on its target DNA. Jinek combined tracrRNA and spacer RNA into a "single-guide RNA" molecule that, mixed with Cas9, could find and cut the correct DNA targets. Jinek et al proposed that such synthetic guide RNAs might be able to be used for gene editing.[13]

CRISPR was first shown to work as a genome engineering/editing tool in human cell culture by 2012[14][15] It has since been used in a wide range of organisms including baker's yeast (S. cerevisiae),[16] zebra fish (D. rerio),[17] flies (D. melanogaster),[18] nematodes (C. elegans),[19] plants,[20] mice,[21] and several other organisms.

Additionally CRISPR has been modified to make programmable transcription factors that allow scientists to target and activate or silence specific genes.[22]

Libraries of tens of thousands of guide RNAs are now available.[13]

The first evidence that CRISPR can reverse disease symptoms in living animals was demonstrated in March 2014, when MIT researchers cured mice of a rare liver disorder.[23]

Gene-editing predecessors

In the early 2000s, researchers developed zinc finger nucleases, synthetic proteins whose DNA-binding domains enable them to cut DNA at specific spots. Later, synthetic nucleases called TALENs provided an easier way to target specific DNA and were predicted to surpass zinc fingers. They both depend on making custom proteins for each DNA target, a more cumbersome procedure than guide RNAs. CRISPRs are more efficient and can target more genes than these earlier techniques.[24]

Locus structure

Repeats and spacers

CRISPR loci range in size from 24 to 48 base pairs.[25] They usually show some dyad symmetry, implying the formation of a secondary structure such as a hairpin, but are not truly palindromic.[26] Repeats are separated by spacers of similar length.[25] Some CRISPR spacer sequences exactly match sequences from plasmids and phages,[27][28][29] although some spacers match the prokaryote's genome (self-targeting spacers).[30] New spacers can be added rapidly in response to phage infection.[31]

Cas genes and CRISPR subtypes

CRISPR-associated (cas) genes are often associated with CRISPR repeat-spacer arrays. Extensive comparative genomics have identified many different cas genes; an initial analysis of 40 bacterial and archaeal genomes suggested that there may be 45 cas gene families, with only two genes, cas1 and cas2, universally present.[25] The current CRISPR classification groups cas operons into three major divisions, each with multiple subdivisions based on cas1 phylogeny and cas operon gene complement.[32] Aside from cas1 and cas2, the three major divisions have vastly different sets of constituent genes, with each of the subdivisions characterised by a ‘signature gene’ found exclusively in that subdivision. Many organisms contain multiple CRISPR-Cas systems suggesting that they are compatible and may even share components.[33][34] The sporadic distribution of the CRISPR/Cas subtypes suggests that the system is subject to horizontal gene transfer during microbial evolution.

Signature genes and their putative functions for the major and minor CRISPR-cas types.
Cas type Signature gene Function Reference
I Cas3 Single-stranded DNA nuclease (HD domain) and ATP-dependent helicase [35]
IA Cas8a Subunit of the interference module [36]
IB Cas8b
IC Cas8c
ID Cas10d contains a domain homologous the palm domain of nucleic acid polymerases and nucleotide cyclases [32][37]
IE Cse1
IF Csy1 Not Determined
II Cas9 RuvC and HNH domain containing nuclease [38]
IIA Csn2 Not Determined
IIB Cas4 Not Determined
IIC Characterized by the absence of either Csn2 or Cas4 [39]
III Cas10 Homolog of Cas10d and Cse1 [37]
IIIA Csm2 Not Determined
IIIB Cmr5 Not Determined
CRISPR associated protein
PDB 1wj9 EBI.jpg
crystal structure of a crispr-associated protein from thermus thermophilus
Symbol CRISPR_assoc
Pfam PF08798
Pfam clan CL0362
InterPro IPR010179
CDD cd09727
CRISPR associated protein Cas2
PDB 1zpw EBI.jpg
crystal structure of a hypothetical protein tt1823 from thermus thermophilus
Symbol CRISPR_Cas2
Pfam PF09827
InterPro IPR019199
CDD cd09638
CRISPR-associated protein Cse1
Symbol CRISPR_Cse1
Pfam PF09481
InterPro IPR013381
CDD cd09729
CRISPR-associated protein Cse2
Symbol CRISPR_Cse2
Pfam PF09485
InterPro IPR013382
CDD cd09670


Acquisition of Spacers into CRISPR loci

Capturing invading DNA into a CRISPR locus in the form of a spacer is the first stage in the immune response. The prevalence of cas1 and cas2 was the first clue that they were involved in spacer acquisition as all CRISPRs shared the regular repeating structure. Mutation studies confirmed this hypothesis as removal of cas1 or cas2 abrogated spacer acquisition, without affecting CRISPR immune response.[36][40][41][42][43] The exact function of Cas1 and Cas2 is unknown, however a number of Cas1 proteins have been biochemically characterised and their structures resolved.[44][45][46] Cas1 proteins have very diverse amino acid sequences, however their crystal structures are strikingly similar and all purified Cas1 proteins are metal-dependent nucleases that bind to DNA in a sequence-independent manner.[33] Representative Cas2 proteins have also been characterised and possess either ssRNA [47] or dsDNA [48][49] specific endoribonuclease activity. The functional data and genetic mutation studies suggests that Cas1 and Cas2 excise fragments of invading DNA and insert them into CRISPR arrays.

Bioinformatic analysis of regions of phage genomes that were excised as spacers (termed protospacers) revealed that they were not randomly distributed in but instead were found adjacent to short (3 – 5 bp) DNA sequences termed PAMs (protospacer adjacent motifs). Analysis of CRISPR-Cas systems from the three major divisions have shown PAMs to be important for type I, type II but not type III systems during the spacer acquisition process.[28][50][51][52][53][54] In type I and type II systems, protospacers are excised at positions adjacent to a PAM sequence, with the other end of the spacer cut using a ruler mechanism inherent to the Cas1 protein, thus maintaining the regularity of the spacer size in the CRISPR array.[55][56] The conservation of the PAM sequence differs between CRISPR-Cas systems and appears to be evolutionarily linked to cas1 and the leader sequence.[54][57]

New spacers are added to a CRISPR array in a directional manner, occurring preferentially [50][51][58][59][60] but not exclusively adjacent [53][56] to the leader sequence. Analysis of the type I-E system from E. coli have demonstrated that the first direct repeat, adjacent to the leader sequence is copied, with the newly acquired spacer inserted between the first and second direct repeats.[42][55] The PAM sequence also appears to be important during spacer insertion in type I-E systems. The PAM sequence of the I-E system contains a strongly conserved final nucleotide (adjacent to the first nucleotide of the protospacer) and it has been shown that this nucleotide becomes the final base in the first direct repeat.[43][61][62] This suggests that the spacer acquisition machinery generates single stranded overhangs in the second-to-last position of the direct repeat and in the PAM during spacer insertion. However, not all CRISPR-Cas systems appear to share this mechanism as PAMs characterised in other organisms do not show the same level of conservation in the final position.[57] It is likely that in those systems, a blunt end is generated at the very end of the direct repeat and the protospacer during acquisition. Recent analysis of Sulfolobus solfataricus CRISPRs revealed further complexities to the canonical model of spacer insertion as one of its six CRISPR loci inserted new spacers randomly throughout its CRISPR array, as opposed to inserting closest to the leader sequence.[56]

It has been noted in a number of CRISPRs that they contain many spacers to the same phage. The mechanism that causes this phenomenon has recently been elucidated in the type I-E system of E. coli. A significant enhancement in spacer acquisition has been detected where there are already spacers targeting the phage, even mismatches to the protospacer. This ‘priming’ requires both the Cas proteins involved in acquisition and interference to interact with each other. Newly acquired spacers that result from the priming mechanism are always found on the same strand as the original spacer that caused the priming.[43][61][62] This observation has led to the hypothesis that the acquisition machinery slides along the foreign DNA after priming to find a new protospacer.[62]

Interference stage

The CRISPR immune response occurs through two steps: CRISPR-RNA (crRNA) biogenesis and crRNA-guided interference. A CRISPR array is transcribed from a promoter in the leader into a single long transcript.[36][63][64] This transcript is processed by cleavage inside the repeat sequence to form crRNAs. The mechanisms to produce mature crRNAs differ greatly between the three main CRISPR-Cas systems. In both type I-E and type I-F systems, the proteins Cas6e and Cas6f respectively, recognise stem-loops [65][66][67] created by the palindromic nature of the direct repeats.[26] These proteins cleave the primary transcript at the junction between double-stranded and single-stranded RNA, leaving an 8 nt 5ʹ-handle originating from the repeat on mature crRNAs along with a single spacer sequence. Type III systems also use Cas6, however the repeats found in type III systems do not produce stem-loops, instead cleavage occurs by the primary transcript wrapping around the Cas6 to allow cleavage 8 nt upstream of the repeat spacer junction.[68][69][70] Type II systems lack the Cas6 gene and instead utilize RNaseIII for cleavage. Functional type II systems encode an extra small RNA that is complementary to the repeat sequence, known as a trans-activating RNA (tracrRNA).[40] Transcription of the tracrRNA and the primary CRISPR transcript results in base pairing and the formation of dsRNA at the repeat sequence, which is subsequently targeted by RNaseIII to produce crRNAs. Unlike the other two systems the crRNA does not contain the full spacer but instead is truncated at one end by 10 nt.[38]

crRNAs associate with Cas proteins to form ribonucleotide complexes that recognize foreign nucleic acids. A number of phage and plasmid challenge experiments have shown that crRNAs show no preference between coding and non-coding strand, which is indicative of an RNA-guided DNA-targeting system.[6][36][43][71][72][73][74] The type I-E complex (commonly referred to as Cascade) requires five Cas proteins arranged in a ‘seahorse’ conformation, bound to a single crRNA that runs down the spine.[75][76] During the interference stage in type I systems the PAM sequence is recognized on the crRNA-complementary strand and is required along with crRNA annealing. In type I systems correct base pairing between the crRNA and the protospacer signals a conformational change in Cascade that recruits Cas3 for DNA degradation.

Type II systems rely on a single multifunctional protein, Cas9, for the interference step.[38] Cas9 requires both the crRNA and the tracrRNA to function and cleaves DNA using its dual HNH and RuvC/RNaseH-like endonuclease domains. Basepairing between the PAM and the phage genome is also required in type II systems, however the PAM is recognized on the same strand as the crRNA (the opposite strand to type I systems).

Type III systems, like type I require a multi-protein complex to associate with the crRNA. Biochemical and structural analyses of complexes from S. solfataricus and Pyrococcus furiosus have elucidated that six or seven cas proteins bind to crRNAs, respectively.[77][78] Surprisingly, the type III systems analysed from S. solfataricus and P. furiosus have both target the mRNA of phage/plasmids,[34][78] which may make these systems uniquely capable of targeting RNA based phage genomes.[33]

The mechanism for distinguishing self from foreign DNA during interference is built into the crRNAs and is therefore inferred to be common to all three systems. Even through the distinctive maturation process of each major type, all crRNAs contain a spacer sequence and some portion of the repeat at one or both ends. It is the partial repeat sequence that prevents the CRISPR-Cas system from targeting the chromosome as base pairing beyond the spacer sequence signals self and prevents DNA cleavage of the chromosome.[79] RNA-guided CRISPR enzymes are classified as type V restriction enzymes.

Evolution and diversity

Studies of Streptococcus thermophilus first indicated how CRISPRs drive phage and bacterial evolution. A CRISPR spacer must correspond perfectly to the sequence of the target phage gene. Phages can continue to infect their hosts where there are point mutations in the spacer.[79] Similar stringency is required in PAM or the strain will remain phage sensitive.[51][79] The basic model of CRISPR evolution is one where newly incorporated spacers drive phages to mutate their genomes creating diversity in both the phage and host populations.

CRISPR evolution has been studied using comparative genomics of many strains of S. thermophilus, Escherichia coli and Salmonella enterica. A study of 124 strains of S. thermophilus showed that 26% of all spacers were unique and that different CRISPR loci showed different rates of new spacer acquisition.[50] The results showed that particular CRISPR loci evolve more rapidly than others, which allowed the strains' phylogenetic relationships to be determined. A similar analysis of E. coli and S. enterica strains revealed that they evolved much slower than S. thermophilus. The latter's strains that had diverged 250 thousand years ago still contained the same spacer complement.[80]

CRISPR diversity was studied in multiple environmental communities using metagenomics. Analysis of two acid mine drainage biofilms showed that one of the analyzed CRISPRs contained extensive deletions and spacer additions in comparison to the other biofilm, suggesting a higher phage activity/prevalence in one community compared to the other.[59] In the oral cavity, a temporal study determined that 7-22% of spacers were shared between timepoints over 17 months within an individual and less than 2% of spacers were shared between different individuals at any single timepoint.[60] From the same environment a single strain was tracked using PCR primers specific to its CRISPR. Unlike the broad-level results of spacer presence/absence, which showed significant diversity, this CRISPR added 3 spacers over 17 months,[60] suggesting that even in an environment with significant CRISPR diversity some loci evolve slowly. CRISPRs have also been analysed from the metagenomes produced for the human microbiome project.[81] Although most CRISPRs were body-site specific, some CRISPRs within a body site are widely shared among individuals. One of these CRISPR loci originated from streptococcal species and contained ~15,000 spacers, 50% of which were unique. Similar to the targeted studies of the oral cavity, some of the CRISPRs showed little evolution between timepoints.[81]

CRISPR evolution has been studied in chemostats using S. thermophilus to explicitly examine the rate of spacer acquisition. Over a period of one week, strains of S. thermophilus acquired up to three spacers when challenged with a single phage.[82] During the same time period the phage developed a number of single nucleotide polymorphisms that became fixed in the population, suggesting that CRISPR targeting had prevented all other phage types from replicating if they did not contain these mutations.[82] Other experiments, also with S. thermophilus, showed that phages can still infect and replicate in hosts that have only one targeting spacer and that sensitive hosts can exist in environments with high phage titres.[83] The chemostat results combined with the observational studies of CRISPRs suggest many nuances to the outcome of CRISPR and phage evolution.

Bioinformatic identification of CRISPRs in genomes and metagenomes

CRISPRs are widely distributed amongst the bacteria and archaea [32] and show some sequence similarities,[26] however their most notable characteristic is their repeating spacers and direct repeats. This characteristic makes CRISPRs easily identifiable in long sequences of DNA, since the number of repeat copies decreases the likelihood of a false positive match. There are currently three programs used for CRISPR repeat identification that search for regularly interspaced repeats in long sequences: CRT,[84] PILER-CR [85] and CRISPRfinder.[86]

Analysis of CRISPRs in metagenomic data is more challenging, as CRISPR loci do not typically assemble due to their repetitive nature or through strain variation, which confuses assembly algorithms. Where there are many reference genomes available, PCR can be used to amplify CRISPR arrays and analyse spacer content.[50][60][87][88][89] However, this approach will only yield information for CRISPRs specifically targeted and for organisms with sufficient representation in public databases to design reliable PCR primers.

The alternative approach is to extract and reconstruct CRISPR arrays from shotgun metagenomic data. Identification of CRISPR arrays from metagenomic reads is computationally more difficult, particularly with second generation sequencing technologies (e.g. 454, Illumina), as the short read lengths prevent more than two or three repeat units being present in a single read. CRISPR identification in raw reads has been achieved using purely denovo identification [90] or by using direct repeat sequences in partially assembled CRISPR arrays from contigs [81] and direct repeat sequences from published genomes [91] as a hook for identifying direct repeats in individual reads.

Evolutionary significance

A bioinformatic study showed that CRISPRs are evolutionarily conserved and cluster into related types. Many show signs of a conserved secondary structure.[26]

Through the CRISPR/Cas mechanism, bacteria can acquire immunity to certain phages and thus halt further transmission of targeted phages. For this reason, CRISPR/Cas can be described as a Lamarckian inheritance mechanism.[92] Others investigated the coevolution of host and viral genomes by analysis of CRISPR sequences.[93]

Cas9 proteins are highly enriched in pathogenic and commensal bacteria. CRISPR/Cas-mediated gene regulation may contribute to the regulation of endogenous bacterial genes, particularly during bacterial interaction with eukaryotic hosts. For example, Cas protein Cas9 of Francisella novicida uses a unique, small, CRISPR/Cas-associated RNA (scaRNA) to repress an endogenous transcript encoding a bacterial lipoprotein that is critical for F. novicida to dampen host response and promote virulence.[94]


The proof-of-principle demonstration of selective engineered redirection of the CRISPR/Cas system in 2012[95] provided a first step toward realization of proposals for CRISPR-derived biotechnology:[96]

  • Artificial immunization against phage by introduction of engineered CRISPR loci in industrially important bacteria, including those used in food production and large-scale fermentation
  • Genome engineering at cellular or organismic level by reprogramming a CRISPR/Cas system to achieve RNA-guided genome engineering. Proof of concept studies demonstrated examples both in vitro[14][97] and in vivo[21][98][99]
  • Discrimination of bacterial strains by comparison of spacer sequences


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 and sickle-cell anemia 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 activates and/or inhibits its expression.[100]

After harvesting blood cell precursors called hematopoietic stem cells from a patient's bone marrow, CRISPR gene surgery would correct the defective gene. Then the gene-­corrected stem cells would be returned to the patient's marrow, which would then produce healthy red blood cells. Replacing 70% of the sickle cells would produce a cure.[24]

Before it can be used clinically, the company must be able to guarantee that only the targeted region will be affected and determine how to deliver the therapy to a patient’s cells.[100]

Other pathologies potentially treatable by CRISPR include Huntington’s disease, aging, schizophrenia and autism, not to mention modifying DNA in living embryos.[24]

Improved targeting is required before CRISPR can be used in medical applications. Current guide RNAs may target sequences that differ by multiple base pairs from the intended sequence.[13]

In 2014, UCSF researchers used CRISPR to create disease-free versions of induced pluripotent stem cells of beta thalassemia patients.[101]

Mouse models

CRISPR simplifies creation of mouse models and reduces the time required to a matter of weeks from months or longer. Knockdown of endogenous genes has been achieved by transfection with a plasmid that contains a CRISPR area with a spacer, which inhibits a target gene. Injecting mouse zygotes with Cas9 and two guide RNAs was able to disable two genes with 80% efficiency. So-called homology-directed repair involves using Cas9 to "nick" DNA, to introduce new gene parts to the zygote.[citation needed]


In 2014, Chinese researcher Gao Caixia filed patents on the creation of a strain of wheat that is resistant to powdery mildew. The strain lacks genes that encode proteins that repress defenses against the mildew. The researchers deleted all three copies of the genes from wheat's hexaploid genome. The strain promises to reduce or eliminate the heavy use of fungicides to control the disease. Gao used the TALENs and CRISPR gene editing tools without adding or changing any other genes. No field trials are yet planned.[102][103]



CRISPRs can add and delete base pairs at specifically targeted DNA loci. CRISPRs have been used to cut as many as five genes at once.[13]

Reversible knockdown

Main article: CRISPR interference

"CRISPRi" like RNAi, turns off genes in a reversible fashion by targeting but not cutting a site. In bacteria, the presence of Cas9 alone is enough to block transcription, but for mammalian applications, a section of protein is added. Its guide RNA targets regulatory DNA, called promoters that immediately precede the gene target.[13]


Main article: CRISPR interference

Cas9 was used to carry synthetic transcription factors (protein fragments that turn on genes) that activated specific human genes. The technique achieved a strong effect by targeting multiple CRISPR constructs to slightly different spots on the gene's promoter.[13]

The genes included some tied to human diseases, including those involved in muscle differentiation, cancer, inflammation and producing fetal hemoglobin.[13]

Use by phages

Another way for bacteria to defend against phage infection is by having chromosomal islands. A subtype of chromosomal islands called phage-inducible chromosomal island (PICI) is excised from bacterial chromosome upon phage infection and can inhibit phage replication.[104] The mechanisms that induce PICI excision and how PICI inhibits phage replication are not well understood. One study showed that lytic ICP1 phage, which specifically targets Vibrio cholerae serogroup O1, has acquired a CRISPR/Cas system that targets a V. cholera PICI-like element. The system has 2 CRISPR loci and 9 Cas genes. It seems to be homologous to the 1-F system found in Yersinia pestis. Moreover, like the bacterial CRISPR/Cas system, ICP1 CRISPR/Cas can acquire new sequences, which allows the phage to co-evolve with its host.[105]

Automation and library support

Free software is available to design RNA to target any desired gene. The Addgene repository offers academics the DNA to make their own CRISPR system for $65. In 2013 Addgene distributed more than 10,000 CRISPR constructs. The facility has received CRISPR-enabling DNA sequences from 11 independent research teams.[13]


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.[106] The patent rights have been assigned by the inventors to the Regents of the University of California and to the University of Vienna.

See also


  1. ^ a b c Horvath, P.; Barrangou, R. (2010). "CRISPR/Cas, the Immune System of Bacteria and Archaea". Science 327 (5962): 167–170. doi:10.1126/science.1179555. PMID 20056882.  edit
  2. ^ a b c Marraffini, L. A.; Sontheimer, E. J. (2010). "CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea". Nature Reviews Genetics 11 (3): 181–190. doi:10.1038/nrg2749. PMC 2928866. PMID 20125085.  edit
  3. ^ 71/79 Archaea, 463/1008 Bacteria CRISPRdb, Date: 19.6.2010
  4. ^ 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.  edit
  5. ^ Barrangou, R.; Fremaux, C.; Deveau, H.; Richards, M.; Boyaval, P.; Moineau, S.; Romero, D. A.; Horvath, P. (2007). "CRISPR Provides Acquired Resistance Against Viruses in Prokaryotes". Science 315 (5819): 1709–1712. doi:10.1126/science.1138140. PMID 17379808.  edit
  6. ^ a b Marraffini, L. A.; Sontheimer, E. J. (2008). "CRISPR Interference Limits Horizontal Gene Transfer in Staphylococci by Targeting DNA". Science 322 (5909): 1843–1845. doi:10.1126/science.1165771. PMC 2695655. PMID 19095942.  edit Cite error: Invalid <ref> tag; name "pmid19095942" defined multiple times with different content (see the help page).
  7. ^ Mali, P; Esvelt, K. M.; Church, G. M. (2013). "Cas9 as a versatile tool for engineering biology". Nature Methods 10 (10): 957–63. doi:10.1038/nmeth.2649. PMC 4051438. PMID 24076990.  edit
  8. ^ Esvelt, Kevin M; Smidler, Andrea L; Catteruccia, Flaminia; Church, George M (July 2014). "Concerning RNA-guided gene drives for the alteration of wild populations". eLife: e03401. doi:10.7554/eLife.03401. PMID 25035423.  edit
  9. ^ Overballe-Petersen, S.; Harms, K.; Orlando, L. A. A.; Mayar, J. V. M.; Rasmussen, S.; Dahl, T. W.; Rosing, M. T.; Poole, A. M.; Sicheritz-Ponten, T.; Brunak, S.; Inselmann, S.; De Vries, J.; Wackernagel, W.; Pybus, O. G.; Nielsen, R.; Johnsen, P. J.; Nielsen, K. M.; Willerslev, E. (2013). "Bacterial natural transformation by highly fragmented and damaged DNA". Proceedings of the National Academy of Sciences 110 (49): 19860–5. doi:10.1073/pnas.1315278110. PMID 24248361.  edit
    "Bacteria incorporate pieces of old DNA in their own genome, scientists discover". KurzweilAI. doi:10.1073/pnas.1315278110. Retrieved 2013-11-26. 
  10. ^ 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". Journal of bacteriology 169 (12): 5429–5433. PMC 213968. PMID 3316184.  edit
  11. ^ Mojica, F. J.; 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". Molecular microbiology 36 (1): 244–246. doi:10.1046/j.1365-2958.2000.01838.x. PMID 10760181.  edit
  12. ^ a b Jansen, R.; Embden, J. D.; Gaastra, W.; Schouls, L. M. (2002). "Identification of genes that are associated with DNA repeats in prokaryotes". Molecular microbiology 43 (6): 1565–1575. doi:10.1046/j.1365-2958.2002.02839.x. PMID 11952905.  edit
  13. ^ a b c d e f g h i j k Pennisi, E. (2013). "The CRISPR Craze". Science 341 (6148): 833–836. doi:10.1126/science.341.6148.833. PMID 23970676.  edit
  14. ^ a b Jinek, M.; Chylinski, K.; Fonfara, I.; Hauer, M.; Doudna, J. A.; Charpentier, E. (2012). "A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity". Science 337 (6096): 816–821. doi:10.1126/science.1225829. PMID 22745249.  edit
  15. ^ "CRISPR gene therapy: Scientists call for more public debate around breakthrough technique - Science - News". The Independent. 2013-11-07. Retrieved 2013-11-25. 
  16. ^ Dicarlo, J. E.; Norville, J. E.; Mali, P; Rios, X; Aach, J; Church, G. M. (2013). "Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems". Nucleic Acids Research 41 (7): 4336–43. doi:10.1093/nar/gkt135. PMC 3627607. PMID 23460208.  edit
  17. ^ Hwang, W. Y.; Fu, Y; Reyon, D; Maeder, M. L.; Tsai, S. Q.; Sander, J. D.; Peterson, R. T.; Yeh, J. R.; Joung, J. K. (2013). "Efficient genome editing in zebrafish using a CRISPR-Cas system". Nature Biotechnology 31 (3): 227–9. doi:10.1038/nbt.2501. PMC 3686313. PMID 23360964.  edit
  18. ^ Gratz, S. J.; Cummings, A. M.; Nguyen, J. N.; Hamm, D. C.; Donohue, L. K.; Harrison, M. M.; Wildonger, J; O'Connor-Giles, K. M. (2013). "Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease". Genetics 194 (4): 1029–35. doi:10.1534/genetics.113.152710. PMC 3730909. PMID 23709638.  edit
  19. ^ Friedland, A. E.; Tzur, Y. B.; Esvelt, K. M.; Colaiácovo, M. P.; Church, G. M.; Calarco, J. A. (2013). "Heritable genome editing in C. Elegans via a CRISPR-Cas9 system". Nature Methods 10 (8): 741–3. doi:10.1038/nmeth.2532. PMC 3822328. PMID 23817069.  edit
  20. ^ Jiang, W; Zhou, H; Bi, H; Fromm, M; Yang, B; Weeks, D. P. (2013). "Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice". Nucleic Acids Research 41 (20): e188. doi:10.1093/nar/gkt780. PMC 3814374. PMID 23999092.  edit
  21. ^ a b Wang, H.; Yang, H.; Shivalila, C. S.; Dawlaty, M. M.; Cheng, A. W.; Zhang, F.; Jaenisch, R. (2013). "One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering". Cell 153 (4): 910–918. doi:10.1016/j.cell.2013.04.025. PMID 23643243.  edit
  22. ^ Larson, M. H.; Gilbert, L. A.; Wang, X; Lim, W. A.; Weissman, J. S.; Qi, L. S. (2013). "CRISPR interference (CRISPRi) for sequence-specific control of gene expression". Nature Protocols 8 (11): 2180–96. doi:10.1038/nprot.2013.132. PMID 24136345.  edit
  23. ^ "Researchers reverse a liver disorder in mice by correcting a mutated gene". PhysOrg. 30 March 2014. Retrieved 31 March 2014. 
  24. ^ a b c Young, Susan (2014-02-11). "CRISPR and Other Genome Editing Tools Boost Medical Research and Gene Therapy’s Reach | MIT Technology Review". Retrieved 2014-04-13. 
  25. ^ a b c Haft, D. H.; Selengut, J.; Mongodin, E. F.; Nelson, K. E. (2005). "A Guild of 45 CRISPR-Associated (Cas) Protein Families and Multiple CRISPR/Cas Subtypes Exist in Prokaryotic Genomes". PLoS Computational Biology 1 (6): e60. doi:10.1371/journal.pcbi.0010060. PMC 1282333. PMID 16292354.  edit Cite error: Invalid <ref> tag; name "pmid16292354" defined multiple times with different content (see the help page).
  26. ^ a b c d Kunin, V.; Sorek, R.; Hugenholtz, P. (2007). "Evolutionary conservation of sequence and secondary structures in CRISPR repeats". Genome Biology 8 (4): R61. doi:10.1186/gb-2007-8-4-r61. PMC 1896005. PMID 17442114.  edit Cite error: Invalid <ref> tag; name "pmid17442114" defined multiple times with different content (see the help page). Cite error: Invalid <ref> tag; name "pmid17442114" defined multiple times with different content (see the help page).
  27. ^ Mojica, F. J. M.; Díez-Villaseñor, C. S.; García-Martínez, J. S.; Soria, E. (2005). "Intervening Sequences of Regularly Spaced Prokaryotic Repeats Derive from Foreign Genetic Elements". Journal of Molecular Evolution 60 (2): 174–182. doi:10.1007/s00239-004-0046-3. PMID 15791728.  edit
  28. ^ a b Bolotin, A.; Quinquis, B.; Sorokin, A.; Ehrlich, S. D. (2005). "Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin". Microbiology 151 (8): 2551–2561. doi:10.1099/mic.0.28048-0. PMID 16079334.  edit Cite error: Invalid <ref> tag; name "pmid16079334" defined multiple times with different content (see the help page).
  29. ^ 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 (3): 653–663. doi:10.1099/mic.0.27437-0. PMID 15758212.  edit
  30. ^ Stern, A.; Keren, L.; Wurtzel, O.; Amitai, G.; Sorek, R. (2010). "Self-targeting by CRISPR: Gene regulation or autoimmunity?". Trends in Genetics 26 (8): 335–340. doi:10.1016/j.tig.2010.05.008. PMC 2910793. PMID 20598393.  edit
  31. ^ Tyson, G. W.; Banfield, J. F. (2007). "Rapidly evolving CRISPRs implicated in acquired resistance of microorganisms to viruses". Environmental Microbiology 10 (1): 200–207. doi:10.1111/j.1462-2920.2007.01444.x. PMID 17894817.  edit
  32. ^ a b c Chylinski, K; Makarova, K. S.; Charpentier, E; Koonin, E. V. (2014). "Classification and evolution of type II CRISPR-Cas systems". Nucleic acids research 42 (10): 6091–105. doi:10.1093/nar/gku241. PMC 4041416. PMID 24728998.  edit
  33. ^ a b c Wiedenheft, B; Sternberg, S. H.; Doudna, J. A. (2012). "RNA-guided genetic silencing systems in bacteria and archaea". Nature 482 (7385): 331–8. doi:10.1038/nature10886. PMID 22337052.  edit
  34. ^ a b Deng, L; Garrett, R. A.; Shah, S. A.; Peng, X; She, Q (2013). "A novel interference mechanism by a type IIIB CRISPR-Cmr module in Sulfolobus". Molecular microbiology 87 (5): 1088–99. doi:10.1111/mmi.12152. PMID 23320564.  edit
  35. ^ Sinkunas, T; Gasiunas, G; Fremaux, C; Barrangou, R; Horvath, P; Siksnys, V (2011). "Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system". The EMBO journal 30 (7): 1335–42. doi:10.1038/emboj.2011.41. PMC 3094125. PMID 21343909.  edit
  36. ^ a b c d Aliyari, R; Ding, S. W. (2009). "RNA-based viral immunity initiated by the Dicer family of host immune receptors". Immunological reviews 227 (1): 176–88. doi:10.1111/j.1600-065X.2008.00722.x. PMC 2676720. PMID 19120484.  edit
  37. ^ a b Makarova, K. S.; Aravind, L; Wolf, Y. I.; Koonin, E. V. (2011). "Unification of Cas protein families and a simple scenario for the origin and evolution of CRISPR-Cas systems". Biology direct 6: 38. doi:10.1186/1745-6150-6-38. PMC 3150331. PMID 21756346.  edit
  38. ^ a b c Gasiunas, G.; Barrangou, R.; Horvath, P.; Siksnys, V. (2012). "Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria". Proceedings of the National Academy of Sciences 109 (39): E2579–E2586. doi:10.1073/pnas.1208507109. PMC 3465414. PMID 22949671.  edit
  39. ^ Chylinski, K; Le Rhun, A; Charpentier, E (2013). "The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems". RNA biology 10 (5): 726–37. doi:10.4161/rna.24321. PMC 3737331. PMID 23563642.  edit
  40. ^ a b Dugar, G; Herbig, A; Förstner, K. U.; Heidrich, N; Reinhardt, R; Nieselt, K; Sharma, C. M. (2013). "High-resolution transcriptome maps reveal strain-specific regulatory features of multiple Campylobacter jejuni isolates". PLoS genetics 9 (5): e1003495. doi:10.1371/journal.pgen.1003495. PMC 3656092. PMID 23696746.  edit
  41. ^ Hatoum-Aslan, A; Maniv, I; Marraffini, L. A. (2011). "Mature clustered, regularly interspaced, short palindromic repeats RNA (crRNA) length is measured by a ruler mechanism anchored at the precursor processing site". Proceedings of the National Academy of Sciences of the United States of America 108 (52): 21218–22. doi:10.1073/pnas.1112832108. PMC 3248500. PMID 22160698.  edit
  42. ^ a b Yosef, I; Goren, M. G.; Qimron, U (2012). "Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli". Nucleic acids research 40 (12): 5569–76. doi:10.1093/nar/gks216. PMC 3384332. PMID 22402487.  edit
  43. ^ a b c d Swarts, D. C.; Mosterd, C; Van Passel, M. W.; Brouns, S. J. (2012). "CRISPR interference directs strand specific spacer acquisition". PloS one 7 (4): e35888. doi:10.1371/journal.pone.0035888. PMC 3338789. PMID 22558257.  edit
  44. ^ Babu, M; Beloglazova, N; Flick, R; Graham, C; Skarina, T; Nocek, B; Gagarinova, A; Pogoutse, O; Brown, G; Binkowski, A; Phanse, S; Joachimiak, A; Koonin, E. V.; Savchenko, A; Emili, A; Greenblatt, J; Edwards, A. M.; Yakunin, A. F. (2011). "A dual function of the CRISPR-Cas system in bacterial antivirus immunity and DNA repair". Molecular microbiology 79 (2): 484–502. doi:10.1111/j.1365-2958.2010.07465.x. PMC 3071548. PMID 21219465.  edit
  45. ^ Han, D; Lehmann, K; Krauss, G (2009). "SSO1450--a CAS1 protein from Sulfolobus solfataricus P2 with high affinity for RNA and DNA". FEBS letters 583 (12): 1928–32. doi:10.1016/j.febslet.2009.04.047. PMID 19427858.  edit
  46. ^ Wiedenheft, B; Zhou, K; Jinek, M; Coyle, S. M.; Ma, W; Doudna, J. A. (2009). "Structural basis for DNase activity of a conserved protein implicated in CRISPR-mediated genome defense". Structure (London, England : 1993) 17 (6): 904–12. doi:10.1016/j.str.2009.03.019. PMID 19523907.  edit
  47. ^ Beloglazova, N; Brown, G; Zimmerman, M. D.; Proudfoot, M; Makarova, K. S.; Kudritska, M; Kochinyan, S; Wang, S; Chruszcz, M; Minor, W; Koonin, E. V.; Edwards, A. M.; Savchenko, A; Yakunin, A. F. (2008). "A novel family of sequence-specific endoribonucleases associated with the clustered regularly interspaced short palindromic repeats". The Journal of biological chemistry 283 (29): 20361–71. doi:10.1074/jbc.M803225200. PMC 2459268. PMID 18482976.  edit
  48. ^ Samai, P; Smith, P; Shuman, S (2010). "Structure of a CRISPR-associated protein Cas2 from Desulfovibrio vulgaris". Acta crystallographica. Section F, Structural biology and crystallization communications 66 (Pt 12): 1552–6. doi:10.1107/S1744309110039801. PMC 2998353. PMID 21139194.  edit
  49. ^ Nam, K. H.; Ding, F; Haitjema, C; Huang, Q; Delisa, M. P.; Ke, A (2012). "Double-stranded endonuclease activity in Bacillus halodurans clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas2 protein". The Journal of biological chemistry 287 (43): 35943–52. doi:10.1074/jbc.M112.382598. PMC 3476262. PMID 22942283.  edit
  50. ^ a b c d Horvath, P.; Romero, D. A.; Coûté-Monvoisin, A. -C.; Richards, M.; Deveau, H.; Moineau, S.; Boyaval, P.; Fremaux, C.; Barrangou, R. (2007). "Diversity, Activity, and Evolution of CRISPR Loci in Streptococcus thermophilus". Journal of Bacteriology 190 (4): 1401–1412. doi:10.1128/JB.01415-07. PMC 2238196. PMID 18065539.  edit
  51. ^ a b c Deveau, H.; Barrangou, R.; Garneau, J. E.; Labonté, J.; Fremaux, C.; Boyaval, P.; Romero, D. A.; Horvath, P.; Moineau, S. (2007). "Phage Response to CRISPR-Encoded Resistance in Streptococcus thermophilus". Journal of Bacteriology 190 (4): 1390–1400. doi:10.1128/JB.01412-07. PMC 2238228. PMID 18065545.  edit
  52. ^ Mojica, F. J. M.; Diez-Villasenor, C.; Garcia-Martinez, J.; Almendros, C. (2009). "Short motif sequences determine the targets of the prokaryotic CRISPR defence system". Microbiology 155 (3): 733–740. doi:10.1099/mic.0.023960-0. PMID 19246744.  edit
  53. ^ a b Lillestøl, R. K.; Shah, S. A.; Brügger, K.; Redder, P.; Phan, H.; Christiansen, J.; Garrett, R. A. (2009). "CRISPR families of the crenarchaeal genus Sulfolobus: Bidirectional transcription and dynamic properties". Molecular Microbiology 72 (1): 259–272. doi:10.1111/j.1365-2958.2009.06641.x. PMID 19239620.  edit
  54. ^ a b Shah, S. A.; Hansen, N. R.; Garrett, R. A. (2009). "Distribution of CRISPR spacer matches in viruses and plasmids of crenarchaeal acidothermophiles and implications for their inhibitory mechanism". Biochemical Society Transactions 37 (Pt 1): 23–28. doi:10.1042/BST0370023. PMID 19143596.  edit
  55. ^ a b Díez-Villaseñor, C; Guzmán, N. M.; Almendros, C; García-Martínez, J; Mojica, F. J. (2013). "CRISPR-spacer integration reporter plasmids reveal distinct genuine acquisition specificities among CRISPR-Cas I-E variants of Escherichia coli". RNA biology 10 (5): 792–802. doi:10.4161/rna.24023. PMC 3737337. PMID 23445770.  edit
  56. ^ a b c Erdmann, S; Garrett, R. A. (2012). "Selective and hyperactive uptake of foreign DNA by adaptive immune systems of an archaeon via two distinct mechanisms". Molecular microbiology 85 (6): 1044–56. doi:10.1111/j.1365-2958.2012.08171.x. PMC 3468723. PMID 22834906.  edit
  57. ^ a b Shah, S. A.; Erdmann, S; Mojica, F. J.; Garrett, R. A. (2013). "Protospacer recognition motifs: Mixed identities and functional diversity". RNA biology 10 (5): 891–9. doi:10.4161/rna.23764. PMC 3737346. PMID 23403393.  edit
  58. ^ Andersson, A. F.; Banfield, J. F. (2008). "Virus Population Dynamics and Acquired Virus Resistance in Natural Microbial Communities". Science 320 (5879): 1047–1050. doi:10.1126/science.1157358. PMID 18497291.  edit
  59. ^ a b Tyson, G. W.; Banfield, J. F. (2007). "Rapidly evolving CRISPRs implicated in acquired resistance of microorganisms to viruses". Environmental Microbiology 10 (1): 200–207. doi:10.1111/j.1462-2920.2007.01444.x. PMID 17894817.  edit
  60. ^ a b c d Pride, D. T.; Sun, C. L.; Salzman, J; Rao, N; Loomer, P; Armitage, G. C.; Banfield, J. F.; Relman, D. A. (2011). "Analysis of streptococcal CRISPRs from human saliva reveals substantial sequence diversity within and between subjects over time". Genome research 21 (1): 126–36. doi:10.1101/gr.111732.110. PMC 3012920. PMID 21149389.  edit
  61. ^ a b Goren, M. G.; Yosef, I; Auster, O; Qimron, U (2012). "Experimental definition of a clustered regularly interspaced short palindromic duplicon in Escherichia coli". Journal of molecular biology 423 (1): 14–6. doi:10.1016/j.jmb.2012.06.037. PMID 22771574.  edit
  62. ^ a b c Datsenko, K. A.; Pougach, K; Tikhonov, A; Wanner, B. L.; Severinov, K; Semenova, E (2012). "Molecular memory of prior infections activates the CRISPR/Cas adaptive bacterial immunity system". Nature communications 3: 945. doi:10.1038/ncomms1937. PMID 22781758.  edit
  63. ^ Tang, T. H.; Bachellerie, J. P.; Rozhdestvensky, T; Bortolin, M. L.; Huber, H; Drungowski, M; Elge, T; Brosius, J; Hüttenhofer, A (2002). "Identification of 86 candidates for small non-messenger RNAs from the archaeon Archaeoglobus fulgidus". Proceedings of the National Academy of Sciences of the United States of America 99 (11): 7536–41. doi:10.1073/pnas.112047299. PMC 124276. PMID 12032318.  edit
  64. ^ Tang, T. H.; Polacek, N; Zywicki, M; Huber, H; Brugger, K; Garrett, R; Bachellerie, J. P.; Hüttenhofer, A (2005). "Identification of novel non-coding RNAs as potential antisense regulators in the archaeon Sulfolobus solfataricus". Molecular microbiology 55 (2): 469–81. doi:10.1111/j.1365-2958.2004.04428.x. PMID 15659164.  edit
  65. ^ Gesner, E. M.; Schellenberg, M. J.; Garside, E. L.; George, M. M.; MacMillan, A. M. (2011). "Recognition and maturation of effector RNAs in a CRISPR interference pathway". Nature structural & molecular biology 18 (6): 688–92. doi:10.1038/nsmb.2042. PMID 21572444.  edit
  66. ^ Sashital, D. G.; Jinek, M; Doudna, J. A. (2011). "An RNA-induced conformational change required for CRISPR RNA cleavage by the endoribonuclease Cse3". Nature structural & molecular biology 18 (6): 680–7. doi:10.1038/nsmb.2043. PMID 21572442.  edit
  67. ^ Haurwitz, R. E.; Jinek, M; Wiedenheft, B; Zhou, K; Doudna, J. A. (2010). "Sequence- and structure-specific RNA processing by a CRISPR endonuclease". Science 329 (5997): 1355–8. doi:10.1126/science.1192272. PMC 3133607. PMID 20829488.  edit
  68. ^ Carte, J.; Wang, R.; Li, H.; Terns, R. M.; Terns, M. P. (2008). "Cas6 is an endoribonuclease that generates guide RNAs for invader defense in prokaryotes". Genes & Development 22 (24): 3489–3496. doi:10.1101/gad.1742908. PMC 2607076. PMID 19141480.  edit
  69. ^ Wang, R; Preamplume, G; Terns, M. P.; Terns, R. M.; Li, H (2011). "Interaction of the Cas6 riboendonuclease with CRISPR RNAs: Recognition and cleavage". Structure (London, England : 1993) 19 (2): 257–64. doi:10.1016/j.str.2010.11.014. PMC 3154685. PMID 21300293.  edit
  70. ^ Niewoehner, O; Jinek, M; Doudna, J. A. (2014). "Evolution of CRISPR RNA recognition and processing by Cas6 endonucleases". Nucleic acids research 42 (2): 1341–53. doi:10.1093/nar/gkt922. PMC 3902920. PMID 24150936.  edit
  71. ^ Garneau, J. E.; Dupuis, Marie-Ève; Villion, M; Romero, D. A.; Barrangou, R; Boyaval, P; Fremaux, C; Horvath, P; Magadán, A. H.; Moineau, S (2010). "The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA". Nature 468 (7320): 67–71. Bibcode:2010Natur.468...67G. doi:10.1038/nature09523. PMID 21048762.  edit
  72. ^ Semenova, E; Jore, M. M.; Datsenko, K. A.; Semenova, A; Westra, E. R.; Wanner, B; Van Der Oost, J; Brouns, S. J.; Severinov, K (2011). "Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence". Proceedings of the National Academy of Sciences of the United States of America 108 (25): 10098–103. doi:10.1073/pnas.1104144108. PMC 3121866. PMID 21646539.  edit
  73. ^ Gudbergsdottir, S; Deng, L; Chen, Z; Jensen, J. V.; Jensen, L. R.; She, Q; Garrett, R. A. (2011). "Dynamic properties of the Sulfolobus CRISPR/Cas and CRISPR/Cmr systems when challenged with vector-borne viral and plasmid genes and protospacers". Molecular microbiology 79 (1): 35–49. doi:10.1111/j.1365-2958.2010.07452.x. PMC 3025118. PMID 21166892.  edit
  74. ^ Manica, A; Zebec, Z; Teichmann, D; Schleper, C (2011). "In vivo activity of CRISPR-mediated virus defence in a hyperthermophilic archaeon". Molecular microbiology 80 (2): 481–91. doi:10.1111/j.1365-2958.2011.07586.x. PMID 21385233.  edit
  75. ^ Jore, M. M.; Lundgren, M; Van Duijn, E; Bultema, J. B.; Westra, E. R.; Waghmare, S. P.; Wiedenheft, B; Pul, U; Wurm, R; Wagner, R; Beijer, M. R.; Barendregt, A; Zhou, K; Snijders, A. P.; Dickman, M. J.; Doudna, J. A.; Boekema, E. J.; Heck, A. J.; Van Der Oost, J; Brouns, S. J. (2011). "Structural basis for CRISPR RNA-guided DNA recognition by Cascade". Nature structural & molecular biology 18 (5): 529–36. doi:10.1038/nsmb.2019. PMID 21460843.  edit
  76. ^ Wiedenheft, B; Lander, G. C.; Zhou, K; Jore, M. M.; Brouns, S. J.; Van Der Oost, J; Doudna, J. A.; Nogales, E (2011). "Structures of the RNA-guided surveillance complex from a bacterial immune system". Nature 477 (7365): 486–9. doi:10.1038/nature10402. PMID 21938068.  edit
  77. ^ Zhang, J; Rouillon, C; Kerou, M; Reeks, J; Brugger, K; Graham, S; Reimann, J; Cannone, G; Liu, H; Albers, S. V.; Naismith, J. H.; Spagnolo, L; White, M. F. (2012). "Structure and mechanism of the CMR complex for CRISPR-mediated antiviral immunity". Molecular cell 45 (3): 303–13. doi:10.1016/j.molcel.2011.12.013. PMC 3381847. PMID 22227115.  edit
  78. ^ a b Hale, C. R.; Zhao, P.; Olson, S.; Duff, M. O.; Graveley, B. R.; Wells, L.; Terns, R. M.; Terns, M. P. (2009). "RNA-Guided RNA Cleavage by a CRISPR RNA-Cas Protein Complex". Cell 139 (5): 945–956. doi:10.1016/j.cell.2009.07.040. PMC 2951265. PMID 19945378.  edit
  79. ^ a b c Marraffini, L. A.; Sontheimer, E. J. (2010). "Self versus non-self discrimination during CRISPR RNA-directed immunity". Nature 463 (7280): 568–571. doi:10.1038/nature08703. PMC 2813891. PMID 20072129.  edit
  80. ^ Touchon, M.; Rocha, E. P. C. (2010). Randau, Lennart, ed. "The Small, Slow and Specialized CRISPR and Anti-CRISPR of Escherichia and Salmonella". PLoS ONE 5 (6): e11126. doi:10.1371/journal.pone.0011126. PMC 2886076. PMID 20559554.  edit
  81. ^ a b c Rho, M; Wu, Y. W.; Tang, H; Doak, T. G.; Ye, Y (2012). "Diverse CRISPRs evolving in human microbiomes". PLoS genetics 8 (6): e1002441. doi:10.1371/journal.pgen.1002441. PMC 3374615. PMID 22719260.  edit
  82. ^ a b Sun, C. L.; Barrangou, R; Thomas, B. C.; Horvath, P; Fremaux, C; Banfield, J. F. (2013). "Phage mutations in response to CRISPR diversification in a bacterial population". Environmental microbiology 15 (2): 463–70. doi:10.1111/j.1462-2920.2012.02879.x. PMID 23057534.  edit
  83. ^ Kuno, S; Sako, Y; Yoshida, T (2014). "Diversification of CRISPR within coexisting genotypes in a natural population of the bloom-forming cyanobacterium Microcystis aeruginosa". Microbiology (Reading, England) 160 (Pt 5): 903–16. doi:10.1099/mic.0.073494-0. PMID 24586036.  edit
  84. ^ Bland, C; Ramsey, T. L.; Sabree, F; Lowe, M; Brown, K; Kyrpides, N. C.; Hugenholtz, P (2007). "CRISPR recognition tool (CRT): A tool for automatic detection of clustered regularly interspaced palindromic repeats". BMC bioinformatics 8: 209. doi:10.1186/1471-2105-8-209. PMC 1924867. PMID 17577412.  edit
  85. ^ Edgar, R. C. (2007). "PILER-CR: Fast and accurate identification of CRISPR repeats". BMC bioinformatics 8: 18. doi:10.1186/1471-2105-8-18. PMC 1790904. PMID 17239253.  edit
  86. ^ Grissa, I; Vergnaud, G; Pourcel, C (2007). "CRISPRFinder: A web tool to identify clustered regularly interspaced short palindromic repeats". Nucleic acids research 35 (Web Server issue): W52–7. doi:10.1093/nar/gkm360. PMC 1933234. PMID 17537822.  edit
  87. ^ Pride, D. T.; Salzman, J; Relman, D. A. (2012). "Comparisons of clustered regularly interspaced short palindromic repeats and viromes in human saliva reveal bacterial adaptations to salivary viruses". Environmental microbiology 14 (9): 2564–76. doi:10.1111/j.1462-2920.2012.02775.x. PMC 3424356. PMID 22583485.  edit
  88. ^ Held, N. L.; Herrera, A; Whitaker, R. J. (2013). "Reassortment of CRISPR repeat-spacer loci in Sulfolobus islandicus". Environmental microbiology. doi:10.1111/1462-2920.12146. PMID 23701169.  edit
  89. ^ Held, N. L.; Herrera, A; Cadillo-Quiroz, H; Whitaker, R. J. (2010). "CRISPR associated diversity within a population of Sulfolobus islandicus". PloS one 5 (9). doi:10.1371/journal.pone.0012988. PMC 2946923. PMID 20927396.  edit
  90. ^ Skennerton, C. T.; Imelfort, M; Tyson, G. W. (2013). "Crass: Identification and reconstruction of CRISPR from unassembled metagenomic data". Nucleic acids research 41 (10): e105. doi:10.1093/nar/gkt183. PMC 3664793. PMID 23511966.  edit
  91. ^ Stern, A; Mick, E; Tirosh, I; Sagy, O; Sorek, R (2012). "CRISPR targeting reveals a reservoir of common phages associated with the human gut microbiome". Genome research 22 (10): 1985–94. doi:10.1101/gr.138297.112. PMC 3460193. PMID 22732228.  edit
  92. ^ Koonin, E. V.; Wolf, Y. I. (2009). "Is evolution Darwinian or/and Lamarckian?". Biology Direct 4: 42. doi:10.1186/1745-6150-4-42. PMC 2781790. PMID 19906303.  edit
  93. ^ Heidelberg, J. F.; Nelson, W. C.; 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.  edit
  94. ^ Sampson, T. R.; Saroj, S. D.; Llewellyn, A. C.; Tzeng, Y. L.; Weiss, D. S. (2013). "A CRISPR/Cas system mediates bacterial innate immune evasion and virulence". Nature 497 (7448): 254–7. doi:10.1038/nature12048. PMC 3651764. PMID 23584588.  edit
  95. ^ Hale, C. R.; Majumdar, S.; Elmore, J.; Pfister, N.; Compton, M.; Olson, S.; Resch, A. M.; Glover Cv, C. V. C.; Graveley, B. R.; Terns, R. M.; Terns, M. P. (2012). "Essential Features and Rational Design of CRISPR RNAs that Function with the Cas RAMP Module Complex to Cleave RNAs". Molecular Cell 45 (3): 292–302. doi:10.1016/j.molcel.2011.10.023. PMC 3278580. PMID 22227116.  edit
  96. ^ Sorek, R.; Kunin, V.; Hugenholtz, P. (2008). "CRISPR — a widespread system that provides acquired resistance against phages in bacteria and archaea". Nature Reviews Microbiology 6 (3): 181–186. doi:10.1038/nrmicro1793. PMID 18157154.  edit
  97. ^ Gasiunas, G.; Barrangou, R.; Horvath, P.; Siksnys, V. (2012). "Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria". Proceedings of the National Academy of Sciences 109 (39): E2579–E2586. doi:10.1073/pnas.1208507109. PMC 3465414. PMID 22949671.  edit
  98. ^ Cong, L.; Ran, F. A.; Cox, D.; Lin, S.; Barretto, R.; Habib, N.; Hsu, P. D.; Wu, X.; Jiang, W.; Marraffini, L. A.; Zhang, F. (2013). "Multiplex Genome Engineering Using CRISPR/Cas Systems". Science 339 (6121): 819–823. doi:10.1126/science.1231143. PMC 3795411. PMID 23287718.  edit
  99. ^ Mali, P.; Yang, L.; Esvelt, K. M.; Aach, J.; Guell, M.; Dicarlo, J. E.; Norville, J. E.; Church, G. M. (2013). "RNA-Guided Human Genome Engineering via Cas9". Science 339 (6121): 823–826. doi:10.1126/science.1232033. PMC 3712628. PMID 23287722.  edit
    Hou, Z.; Zhang, Y.; Propson, N. E.; Howden, S. E.; Chu, L. -F.; Sontheimer, E. J.; Thomson, J. A. (2013). "Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis". Proceedings of the National Academy of Sciences 110 (39): 15644. doi:10.1073/pnas.1313587110.  edit
  100. ^ a b Young, Susan. "Biotech Startup Editas Medicine Wants to Cure Grievous Genetic Diseases with New Genome Editing Technology | MIT Technology Review". Retrieved 2013-11-30. 
  101. ^ "Seamless gene correction of β-thalassemia mutations in patient-specific iPSCs using CRISPR/Cas9 and piggyBac". 
  102. ^ Talbot, David (2014-07-19). "Beijing Researchers Use Gene Editing to Create Disease-Resistant Wheat | MIT Technology Review". Retrieved 2014-07-23. 
  103. ^ Wang, Yanpeng (2014). "Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew". Nature Biotechnology. doi:10.1038/nbt.2969.  edit
  104. ^ Novick, R. P.; Christie, G. E.; Penadés, J. R. (2010). "The phage-related chromosomal islands of Gram-positive bacteria". Nature Reviews Microbiology 8 (8): 541–551. doi:10.1038/nrmicro2393. PMC 3522866. PMID 20634809.  edit
  105. ^ Seed, K. D.; Lazinski, D. W.; Calderwood, S. B.; Camilli, A. (2013). "A bacteriophage encodes its own CRISPR/Cas adaptive response to evade host innate immunity". Nature 494 (7438): 489–491. doi:10.1038/nature11927. PMC 3587790. PMID 23446421.  edit
  106. ^ "Methods And Compositions For Rna-directed Target Dna Modification And For Rna-directed Modulation Of Transcription". Retrieved 2014-04-13. 

Further reading

External links

This page is based on a Wikipedia article. The text is available under the Creative Commons Attribution/Share-Alike License.

This tab holds the annotation information that is stored in the Pfam database. As we move to using Wikipedia as our main source of annotation, the contents of this tab will be gradually replaced by the Wikipedia tab.

CRISPR associated protein Provide feedback

This domain forms an anti-parallel beta strand structure with flanking alpha helical regions.

This tab holds annotation information from the InterPro database.

InterPro entry IPR010179

The CRISPR-Cas system is a prokaryotic defense mechanism against foreign genetic elements. The key elements of this defense system are the Cas proteins and the CRISPR RNA.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) are a family of DNA direct repeats separated by regularly sized non-repetitive spacer sequences that are found in most bacterial and archaeal genomes [PUBMED:17442114]. CRISPRs appear to provide acquired resistance against mobile genetic elements (viruses, transposable elements and conjugative plasmids). CRISPR clusters contain sequences complementary to antecedent mobile elements and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA).

The defense reaction is divided into three stages. In the adaptation stage, the invader DNA is cleaved, and a piece of it is selected to be integrated as a new spacer into the CRISPR locus, where it is stored as an identity tag for future attacks by this invader. During the second stage (the expression stage), the CRISPR RNA (pre-crRNA) is transcribed and subsequently processed into the mature crRNAs. In the third stage (the interference stage), Cas proteins, together with crRNAs, identify and degrade the invader [PUBMED:17379808, PUBMED:16545108, PUBMED:21699496].

The CRISPR-Cas systems have been sorted into three major classes. In CRISPR-Cas types I and III, the mature crRNA is generally generated by a member of the Cas6 protein family. Whereas in system III the Cas6 protein acts alone, in some class I systems it is part of a complex of Cas proteins known as Cascade (CRISPR-associated complex for antiviral defense). The Cas6 protein is necessary for crRNA production whereas the additional Cas proteins that form the Cascade complex are needed for crRNA stability [PUBMED:24459147].

This entry represents the Cse3 (CRISPR/Cas Subtype Ecoli protein 3) family of Cas proteins. The Thermus thermophilus HB8 family member has been crystallised and found to have a structure consisting of two domains with opposing parallel beta-sheets, known as a beta-sheet platform [PUBMED:16672237]. This structure is similar to those found in the sex-lethal protein and poly(A)-binding protein and is consistent with an RNA-binding function.

Domain organisation

Below is a listing of the unique domain organisations or architectures in which this domain is found. More...

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Pfam Clan

This family is a member of clan RAMPS-Cas5-like (CL0362), which has the following description:

This group of families is one of several protein families that are always found associated with prokaryotic CRISPRs, themselves a family of clustered regularly interspaced short palindromic repeats, DNA repeats found in nearly half of all bacterial and archaeal genomes. These DNA repeat regions have a remarkably regular structure: unique sequences of constant size, called spacers, sit between each pair of repeats [1]. It has been shown that the CRISPRs are virus-derived sequences acquired by the host to enable them to resist viral infection. The Cas proteins from the host use the CRISPRs to mediate an antiviral response. After transcription of the CRISPR, a complex of Cas proteins termed Cascade cleaves a CRISPR RNA precursor in each repeat and retains the cleavage products containing the virus-derived sequence. Assisted by the helicase Cas3, these mature CRISPR RNAs then serve as small guide RNAs that enable Cascade to interfere with virus proliferation [2]. Cas5 contains an endonuclease motif, whose inactivation leads to loss of resistance, even in the presence of phage-derived spacers [3].

The clan contains the following 7 members:

Cas6 Cas_Cas5d Cas_Cas6 Cas_Cmr3 CRISPR_assoc DUF2276 RAMPs


We store a range of different sequence alignments for families. As well as the seed alignment from which the family is built, we provide the full alignment, generated by searching the sequence database (reference proteomes) using the family HMM. We also generate alignments using four representative proteomes (RP) sets, the UniProtKB sequence database, the NCBI sequence database, and our metagenomics sequence database. More...

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We make a range of alignments for each Pfam-A family. You can see a description of each above. You can view these alignments in various ways but please note that some types of alignment are never generated while others may not be available for all families, most commonly because the alignments are too large to handle.

Representative proteomes UniProt
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Representative proteomes UniProt

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We make all of our alignments available in Stockholm format. You can download them here as raw, plain text files or as gzip-compressed files.

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You can also download a FASTA format file containing the full-length sequences for all sequences in the full alignment.

HMM logo

HMM logos is one way of visualising profile HMMs. Logos provide a quick overview of the properties of an HMM in a graphical form. You can see a more detailed description of HMM logos and find out how you can interpret them here. More...


This page displays the phylogenetic tree for this family's seed alignment. We use FastTree to calculate neighbour join trees with a local bootstrap based on 100 resamples (shown next to the tree nodes). FastTree calculates approximately-maximum-likelihood phylogenetic trees from our seed alignment.

Note: You can also download the data file for the tree.

Curation and family details

This section shows the detailed information about the Pfam family. You can see the definitions of many of the terms in this section in the glossary and a fuller explanation of the scoring system that we use in the scores section of the help pages.

Curation View help on the curation process

Seed source: pdb_1wj9
Previous IDs: none
Type: Domain
Author: Mistry J
Number in seed: 114
Number in full: 180
Average length of the domain: 218.60 aa
Average identity of full alignment: 24 %
Average coverage of the sequence by the domain: 97.26 %

HMM information View help on HMM parameters

HMM build commands:
build method: hmmbuild -o /dev/null HMM SEED
search method: hmmsearch -Z 11927849 -E 1000 --cpu 4 HMM pfamseq
Model details:
Parameter Sequence Domain
Gathering cut-off 25.0 25.0
Trusted cut-off 25.1 29.9
Noise cut-off 24.3 19.9
Model length: 213
Family (HMM) version: 8
Download: download the raw HMM for this family

Species distribution

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Colour assignments

Archea Archea Eukaryota Eukaryota
Bacteria Bacteria Other sequences Other sequences
Viruses Viruses Unclassified Unclassified
Viroids Viroids Unclassified sequence Unclassified sequence


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This visualisation provides a simple graphical representation of the distribution of this family across species. You can find the original interactive tree in the adjacent tab. More...

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The tree shows the occurrence of this domain across different species. More...


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There is 1 interaction for this family. More...



For those sequences which have a structure in the Protein DataBank, we use the mapping between UniProt, PDB and Pfam coordinate systems from the PDBe group, to allow us to map Pfam domains onto UniProt sequences and three-dimensional protein structures. The table below shows the structures on which the CRISPR_assoc domain has been found. There are 20 instances of this domain found in the PDB. Note that there may be multiple copies of the domain in a single PDB structure, since many structures contain multiple copies of the same protein seqence.

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