Summary: CRISPR-associated protein Cse2 (CRISPR_cse2)
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CRISPR Edit Wikipedia article
|Cascade (CRISPR-associated complex for antiviral defense)|
|Part of a series on|
|Genetically modified organisms|
|History and regulation|
CRISPR (//) (an acronym for clustered regularly interspaced short palindromic repeats) is a family of DNA sequences found in the genomes of prokaryotic organisms such as bacteria and archaea. These sequences are derived from DNA fragments of bacteriophages that had previously infected the prokaryote. They are used to detect and destroy DNA from similar bacteriophages during subsequent infections. Hence these sequences play a key role in the antiviral (i.e. anti-phage) defense system of prokaryotes and provide a form of acquired immunity. CRISPR are found in approximately 50% of sequenced bacterial genomes and nearly 90% of sequenced archaea.
Cas9 (or "CRISPR-associated protein 9") is an enzyme that uses CRISPR sequences as a guide to recognize and cleave specific strands of DNA that are complementary to the CRISPR sequence. Cas9 enzymes together with CRISPR sequences form the basis of a technology known as CRISPR-Cas9 that can be used to edit genes within organisms. This editing process has a wide variety of applications including basic biological research, development of biotechnological products, and treatment of diseases. The development of the CRISPR-Cas9 genome editing technique was recognized by the Nobel Prize in Chemistry in 2020 which was awarded to Emmanuelle Charpentier and Jennifer Doudna.
The discovery of clustered DNA repeats took place independently in three parts of the world. The first description of what would later be called CRISPR is from Osaka University researcher Yoshizumi Ishino and his colleagues in 1987. They accidentally cloned part of a CRISPR sequence together with the "iap" gene (isozyme conversion of alkaline phosphatase) from the genome of Escherichia coli that was their target. The organization of the repeats was unusual. Repeated sequences are typically arranged consecutively, without interspersed different sequences. They did not know the function of the interrupted clustered repeats.
In 1993, researchers of Mycobacterium tuberculosis in the Netherlands published two articles about a cluster of interrupted direct repeats (DR) in that bacterium. They recognized the diversity of the sequences that intervened the direct repeats among different strains of M. tuberculosis and used this property to design a typing method that was named spoligotyping, which is still in use today.
Francisco Mojica at the University of Alicante in Spain studied repeats observed in the archaeal organisms of Haloferax and Haloarcula species, and their function. Mojica's supervisor surmised at the time that the clustered repeats had a role in correctly segregating replicated DNA into daughter cells during cell division because plasmids and chromosomes with identical repeat arrays could not coexist in Haloferax volcanii. Transcription of the interrupted repeats was also noted for the first time; this was the first full characterization of CRISPR. By 2000, Mojica performed a survey of scientific literature and one of his students performed a search in published genomes with a program devised by himself. They identified interrupted repeats in 20 species of microbes as belonging to the same family. Because those sequences were interspaced, Mojica initially called these sequences "short regularly spaced repeats" (SRSR). In 2001, Mojica and Ruud Jansen, who were searching for additional interrupted repeats, proposed the acronym CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) to alleviate the confusion stemming from the numerous acronyms used to describe the sequences in the scientific literature. In 2002, Tang, et al. showed evidence that CRISPR repeat regions from the genome of Archaeoglobus fulgidus were transcribed into long RNA molecules that were subsequently processed into unit-length small RNAs, plus some longer forms of 2, 3, or more spacer-repeat units.
In 2005, yogurt researcher Rodolphe Barrangou, discovered that Streptococcus thermophilus, after iterative phage challenges, develops increased phage resistance, and this enhanced resistance is due to incorporation of additional CRISPR spacer sequences. The Danish food company Danisco, which at that time Barrangou worked for, then developed phage resistant S. thermophilus strains for use in yogurt production. Danisco was later bought out by DuPont, which "owns about 50 percent of the global dairy culture market" and the technology went mainstream.
A major addition to the understanding of CRISPR came with Jansen's observation that the prokaryote repeat cluster was accompanied by a set of homologous genes that make up CRISPR-associated systems or cas genes. Four cas genes (cas 1â€“4) were initially recognized. The Cas proteins showed helicase and nuclease motifs, suggesting a role in the dynamic structure of the CRISPR loci. In this publication the acronym CRISPR was used as the universal name of this pattern. However, the CRISPR function remained enigmatic.
In 2005, three independent research groups showed that some CRISPR spacers are derived from phage DNA and extrachromosomal DNA such as plasmids. In effect, the spacers are fragments of DNA gathered from viruses that previously tried to attack the cell. The source of the spacers was a sign that the CRISPR/cas system could have a role in adaptive immunity in bacteria. All three studies proposing this idea were initially rejected by high-profile journals, but eventually appeared in other journals.
The first publication proposing a role of CRISPR-Cas in microbial immunity, by Mojica and collaborators at the University of Alicante, predicted a role for the RNA transcript of spacers on target recognition in a mechanism that could be analogous to the RNA interference system used by eukaryotic cells. Koonin and colleagues extended this RNA interference hypothesis by proposing mechanisms of action for the different CRISPR-Cas subtypes according to the predicted function of their proteins.
Experimental work by several groups revealed the basic mechanisms of CRISPR-Cas immunity. In 2007, the first experimental evidence that CRISPR was an adaptive immune system was published. A CRISPR region in Streptococcus thermophilus acquired spacers from the DNA of an infecting bacteriophage. The researchers manipulated the resistance of S. thermophilus to different types of phage by adding and deleting spacers whose sequence matched those found in the tested phages. In 2008, Brouns and Van der Oost identified a complex of Cas proteins (called Cascade) that in E. coli cut the CRISPR RNA precursor within the repeats into mature spacer-containing RNA molecules called CRISPR RNA (crRNA), which remained bound to the protein complex. Moreover, it was found that Cascade, crRNA and a helicase/nuclease (Cas3) were required to provide a bacterial host with immunity against infection by a DNA virus. By designing an anti-virus CRISPR, they demonstrated that two orientations of the crRNA (sense/antisense) provided immunity, indicating that the crRNA guides were targeting dsDNA. That year Marraffini and Sontheimer confirmed that a CRISPR sequence of S. epidermidis targeted DNA and not RNA to prevent conjugation. This finding was at odds with the proposed RNA-interference-like mechanism of CRISPR-Cas immunity, although a CRISPR-Cas system that targets foreign RNA was later found in Pyrococcus furiosus. A 2010 study showed that CRISPR-Cas cuts both strands of phage and plasmid DNA in S. thermophilus.
Researchers studied a simpler CRISPR system from Streptococcus pyogenes that relies on the protein Cas9. The Cas9 endonuclease is a four-component system that includes two small molecules: crRNA and trans-activating CRISPR RNA (tracrRNA). Jennifer Doudna and Emmanuelle Charpentier re-engineered the Cas9 endonuclease into a more manageable two-component system by fusing the two RNA molecules into a "single-guide RNA" that, when combined with Cas9, could find and cut the DNA target specified by the guide RNA. This contribution was so significant that it was recognized by the Nobel Prize in Chemistry in 2020. By manipulating the nucleotide sequence of the guide RNA, the artificial Cas9 system could be programmed to target any DNA sequence for cleavage. Another group of collaborators comprising Virginijus Å ikÅ¡nys together with GasiÅ«nas, Barrangou and Horvath showed that Cas9 from the S. thermophilus CRISPR system can also be reprogrammed to target a site of their choosing by changing the sequence of its crRNA. These advances fueled efforts to edit genomes with the modified CRISPR-Cas9 system.
Groups led by Feng Zhang and George Church simultaneously published descriptions of genome editing in human cell cultures using CRISPR-Cas9 for the first time. It has since been used in a wide range of organisms, including baker's yeast (Saccharomyces cerevisiae), the opportunistic pathogen Candida albicans, zebrafish (Danio rerio), fruit flies (Drosophila melanogaster), ants (Harpegnathos saltator and Ooceraea biroi), mosquitoes (Aedes aegypti), nematodes (Caenorhabditis elegans), plants, mice (Mus musculus domesticus), monkeys and human embryos.
The CRISPR-Cas9 system has shown to make effective gene edits in Human tripronuclear zygotes first described in a 2015 paper by Chinese scientists P. Liang and Y. Xu. The system made a successful cleavage of mutant Beta-Hemoglobin (HBB) in 28 out of 54 embryos. 4 out of the 28 embryos were successfully recombined using a donor template given by the scientists. The scientists showed that during DNA recombination of the cleaved strand, the homologous endogenous sequence HBD competes with the exogenous donor template. DNA repair in human embryos is much more complicated and particular than in derived stem cells.
Cas12a (formerly Cpf1)
In 2015, the nuclease Cas12a (formerly known as Cpf1) was characterized in the CRISPR/Cpf1 system of the bacterium Francisella novicida. Its original name, from a TIGRFAMs protein family definition built in 2012, reflects the prevalence of its CRISPR-Cas subtype in the Prevotella and Francisella lineages. Cas12a showed several key differences from Cas9 including: causing a 'staggered' cut in double stranded DNA as opposed to the 'blunt' cut produced by Cas9, relying on a 'T rich' PAM (providing alternative targeting sites to Cas9) and requiring only a CRISPR RNA (crRNA) for successful targeting. By contrast Cas9 requires both crRNA and a transactivating crRNA (tracrRNA).
These differences may give Cas12a some advantages over Cas9. For example, Cas12a's small crRNAs are ideal for multiplexed genome editing, as more of them can be packaged in one vector than can Cas9's sgRNAs. As well, the sticky 5â€² overhangs left by Cas12a can be used for DNA assembly that is much more target-specific than traditional Restriction Enzyme cloning. Finally, Cas12a cleaves DNA 18â€“23 base pairs downstream from the PAM site. This means there is no disruption to the recognition sequence after repair, and so Cas12a enables multiple rounds of DNA cleavage. By contrast, since Cas9 cuts only 3 base pairs upstream of the PAM site, the NHEJ pathway results in indel mutations that destroy the recognition sequence, thereby preventing further rounds of cutting. In theory, repeated rounds of DNA cleavage should cause an increased opportunity for the desired genomic editing to occur. A distinctive feature of Cas12a, as compared to Cas9, is that after cutting its target, Cas12a remains bound to the target and then cleaves other ssDNA molecules non-discriminately. This property is called "collateral cleavage" or "trans-cleavage" activity and has been exploited for the development of various diagnostic technologies.
Cas13 (formerly C2c2)
In 2016, the nuclease Cas13a (formerly known as C2c2) from the bacterium Leptotrichia shahii was characterized. Cas13 is an RNA-guided RNA endonuclease, which means that it does not cleave DNA, but only single-stranded RNA. Cas13 is guided by its crRNA to a ssRNA target and binds and cleaves the target. Similar to Cas12a, the Cas13 remains bound to the target and then cleaves other ssRNA molecules non-discriminately. This collateral cleavage property has been exploited for the development of various diagnostic technologies.
Repeats and spacers
The CRISPR array is made up of an AT-rich leader sequence followed by short repeats that are separated by unique spacers. CRISPR repeats typically range in size from 28 to 37 base pairs (bps), though there can be as few as 23 bp and as many as 55 bp. Some show dyad symmetry, implying the formation of a secondary structure such as a stem-loop ('hairpin') in the RNA, while others are designed to be unstructured. The size of spacers in different CRISPR arrays is typically 32 to 38 bp (range 21 to 72 bp). New spacers can appear rapidly as part of the immune response to phage infection. There are usually fewer than 50 units of the repeat-spacer sequence in a CRISPR array.
CRISPR RNA structures
Cas genes and CRISPR subtypes
Small clusters of cas genes are often located next to CRISPR repeat-spacer arrays. Collectively the 93 cas genes are grouped into 35 families based on sequence similarity of the encoded proteins. 11 of the 35 families form the cas core, which includes the protein families Cas1 through Cas9. A complete CRISPR-Cas locus has at least one gene belonging to the cas core.
CRISPR-Cas systems fall into two classes. Class 1 systems use a complex of multiple Cas proteins to degrade foreign nucleic acids. Class 2 systems use a single large Cas protein for the same purpose. Class 1 is divided into types I, III, and IV; class 2 is divided into types II, V, and VI. The 6 system types are divided into 19 subtypes. Each type and most subtypes are characterized by a "signature gene" found almost exclusively in the category. Classification is also based on the complement of cas genes that are present. Most CRISPR-Cas systems have a Cas1 protein. The phylogeny of Cas1 proteins generally agrees with the classification system, but exceptions exist due to module shuffling. Many organisms contain multiple CRISPR-Cas systems suggesting that they are compatible and may share components. The sporadic distribution of the CRISPR/Cas subtypes suggests that the CRISPR/Cas system is subject to horizontal gene transfer during microbial evolution.
This table is missing information about UniProt and InterPro cross-reference.(October 2020)
|Class||Cas type||Cas subtype||Signature protein||Function||Reference|
|1||I||â€”||Cas3||Single-stranded DNA nuclease (HD domain) and ATP-dependent helicase|||
|I-A||Cas8a, Cas5||Cas8 is a Subunit of the interference module that is important in targeting of invading DNA by recognizing the PAM sequence. Cas5 is required for processing and stability of crRNAs|||
|I-D||Cas10d||contains a domain homologous to the palm domain of nucleic acid polymerases and nucleotide cyclases|||
|I-F||Csy1, Csy2, Csy3||Not determined|||
|III||â€”||Cas10||Homolog of Cas10d and Cse1. Binds CRISPR target RNA and promotes stability of the interference complex|||
|III-C||Cas10 or Csx11|||
|2||II||â€”||Cas9||Nucleases RuvC and HNH together produce DSBs, and separately can produce single-strand breaks. Ensures the acquisition of functional spacers during adaptation.|||
|II-A||Csn2||Ring-shaped DNA-binding protein. Involved in primed adaptation in Type II CRISPR system.|||
|II-B||Cas4||Endonuclease that works with cas1 and cas2 to generate spacer sequences|||
|II-C||Characterized by the absence of either Csn2 or Cas4|||
|V||â€”||Cas12||Nuclease RuvC. Lacks HNH.|||
|V-F||Cas12f (Cas14, C2c10)|||
|V-K[Note 2]||Cas12k (C2c5)|||
|V-U||C2c4, C2c8, C2c9|||
CRISPR-Cas immunity is a natural process of bacteria and archaea. CRISPR-Cas prevents bacteriophage infection, conjugation and natural transformation by degrading foreign nucleic acids that enter the cell.
When a microbe is invaded by a bacteriophage, the first stage of the immune response is to capture phage DNA and insert it into a CRISPR locus in the form of a spacer. Cas1 and Cas2 are found in both types of CRISPR-Cas immune systems, which indicates that they are involved in spacer acquisition. Mutation studies confirmed this hypothesis, showing that removal of cas1 or cas2 stopped spacer acquisition, without affecting CRISPR immune response.
Multiple Cas1 proteins have been characterised and their structures resolved. Cas1 proteins have diverse amino acid sequences. However, their crystal structures are similar and all purified Cas1 proteins are metal-dependent nucleases/integrases that bind to DNA in a sequence-independent manner. Representative Cas2 proteins have been characterised and possess either (single strand) ssRNA- or (double strand) dsDNA- specific endoribonuclease activity.
In the I-E system of E. coli Cas1 and Cas2 form a complex where a Cas2 dimer bridges two Cas1 dimers. In this complex Cas2 performs a non-enzymatic scaffolding role, binding double-stranded fragments of invading DNA, while Cas1 binds the single-stranded flanks of the DNA and catalyses their integration into CRISPR arrays. New spacers are usually added at the beginning of the CRISPR next to the leader sequence creating a chronological record of viral infections. In E. coli a histone like protein called integration host factor (IHF), which binds to the leader sequence, is responsible for the accuracy of this integration. IHF also enhances integration efficiency in the type I-F system of Pectobacterium atrosepticum. but in other systems different host factors may be required
Protospacer adjacent motifs
Bioinformatic analysis of regions of phage genomes that were excised as spacers (termed protospacers) revealed that they were not randomly selected but instead were found adjacent to short (3â€“5 bp) DNA sequences termed protospacer adjacent motifs (PAM). Analysis of CRISPR-Cas systems showed PAMs to be important for type I and type II, but not type III systems during acquisition. 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, thus maintaining the regularity of the spacer size in the CRISPR array. The conservation of the PAM sequence differs between CRISPR-Cas systems and appears to be evolutionarily linked to Cas1 and the leader sequence.
New spacers are added to a CRISPR array in a directional manner, occurring preferentially, but not exclusively, adjacent to the leader sequence. Analysis of the type I-E system from E. coli 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.
The PAM sequence appears to be important during spacer insertion in type I-E systems. That sequence contains a strongly conserved final nucleotide (nt) adjacent to the first nt of the protospacer. This nt becomes the final base in the first direct repeat. 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 in other organisms do not show the same level of conservation in the final position. 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.
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.
Multiple CRISPRs contain many spacers to the same phage. The mechanism that causes this phenomenon was discovered in the type I-E system of E. coli. A significant enhancement in spacer acquisition was detected where spacers already target the phage, even mismatches to the protospacer. This â€˜primingâ€™ requires the Cas proteins involved in both 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 priming spacer. This observation led to the hypothesis that the acquisition machinery slides along the foreign DNA after priming to find a new protospacer.
CRISPR-RNA (crRNA), which later guides the Cas nuclease to the target during the interference step, must be generated from the CRISPR sequence. The crRNA is initially transcribed as part of a single long transcript encompassing much of the CRISPR array. This transcript is then cleaved by Cas proteins to form crRNAs. The mechanism to produce crRNAs differs among CRISPR/Cas systems. In type I-E and type I-F systems, the proteins Cas6e and Cas6f respectively, recognise stem-loops created by the pairing of identical repeats that flank the crRNA. These Cas proteins cleave the longer transcript at the edge of the paired region, leaving a single crRNA along with a small remnant of the paired repeat region.
Type III systems also use Cas6, however their repeats do not produce stem-loops. Cleavage instead occurs by the longer transcript wrapping around the Cas6 to allow cleavage just upstream of the repeat sequence.
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 crRNA (tracrRNA). 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, which is instead truncated at one end.
CrRNAs associate with Cas proteins to form ribonucleotide complexes that recognize foreign nucleic acids. CrRNAs show no preference between the coding and non-coding strands, which is indicative of an RNA-guided DNA-targeting system. The type I-E complex (commonly referred to as Cascade) requires five Cas proteins bound to a single crRNA.
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. 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 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 six or seven Cas proteins binding to crRNAs. The type III systems analysed from S. solfataricus and P. furiosus both target the mRNA of phages rather than phage DNA genome, which may make these systems uniquely capable of targeting RNA-based phage genomes. Type III systems were also found to target DNA in addition to RNA using a different Cas protein in the complex, Cas10. The DNA cleavage was shown to be transcription dependent.
The mechanism for distinguishing self from foreign DNA during interference is built into the crRNAs and is therefore likely common to all three systems. Throughout 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. RNA-guided CRISPR enzymes are classified as type V restriction enzymes.
|CRISPR associated protein Cas2 (adaptation RNase)|
|CRISPR-associated protein CasA/Cse1 (Type I effector DNase)|
|CRISPR associated protein CasC/Cse3/Cas6 (Type I effector RNase)|
The cas genes in the adaptor and effector modules of the CRISPR-Cas system are believed to have evolved from two different ancestral modules. A transposon-like element called casposon encoding the Cas1-like integrase and potentially other components of the adaptation module was inserted next to the ancestral effector module, which likely functioned as an independent innate immune system. The highly conserved cas1 and cas2 genes of the adaptor module evolved from the ancestral module while a variety of class 1 effector cas genes evolved from the ancestral effector module. The evolution of these various class 1 effector module cas genes was guided by various mechanisms, such as duplication events. On the other hand, each type of class 2 effector module arose from subsequent independent insertions of mobile genetic elements. These mobile genetic elements took the place of the multiple gene effector modules to create single gene effector modules that produce large proteins which perform all the necessary tasks of the effector module. The spacer regions of CRISPR-Cas systems are taken directly from foreign mobile genetic elements and thus their long term evolution is hard to trace. The non-random evolution of these spacer regions has been found to be highly dependent on the environment and the particular foreign mobile genetic elements it contains.
CRISPR/Cas can immunize bacteria against certain phages and thus halt transmission. For this reason, Koonin described CRISPR/Cas as a Lamarckian inheritance mechanism. However, this was disputed by a critic who noted, "We should remember [Lamarck] for the good he contributed to science, not for things that resemble his theory only superficially. Indeed, thinking of CRISPR and other phenomena as Lamarckian only obscures the simple and elegant way evolution really works". But as more recent studies have been conducted, it has become apparent that the acquired spacer regions of CRISPR-Cas systems are indeed a form of Lamarckian evolution because they are genetic mutations that are acquired and then passed on. On the other hand, the evolution of the Cas gene machinery that facilitates the system evolves through classic Darwinian evolution.
Analysis of CRISPR sequences revealed coevolution of host and viral genomes. 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 interaction with eukaryotic hosts. For example, 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.
The basic model of CRISPR evolution is newly incorporated spacers driving phages to mutate their genomes to avoid the bacterial immune response, creating diversity in both the phage and host populations. To resist a phage infection, the sequence of the CRISPR spacer must correspond perfectly to the sequence of the target phage gene. Phages can continue to infect their hosts given point mutations in the spacer. Similar stringency is required in PAM or the bacterial strain remains phage sensitive.
A study of 124 S. thermophilus strains showed that 26% of all spacers were unique and that different CRISPR loci showed different rates of spacer acquisition. Some CRISPR loci evolve more rapidly than others, which allowed the strains' phylogenetic relationships to be determined. A comparative genomic analysis showed that E. coli and S. enterica evolve much more slowly than S. thermophilus. The latter's strains that diverged 250 thousand years ago still contained the same spacer complement.
Metagenomic analysis of two acid-mine-drainage biofilms showed that one of the analyzed CRISPRs contained extensive deletions and spacer additions versus the other biofilm, suggesting a higher phage activity/prevalence in one community than the other. In the oral cavity, a temporal study determined that 7â€“22% of spacers were shared over 17 months within an individual while less than 2% were shared across individuals.
From the same environment a single strain was tracked using PCR primers specific to its CRISPR system. Broad-level results of spacer presence/absence showed significant diversity. However, this CRISPR added 3 spacers over 17 months, suggesting that even in an environment with significant CRISPR diversity some loci evolve slowly.
CRISPRs were analysed from the metagenomes produced for the human microbiome project. Although most were body-site specific, some within a body site are widely shared among individuals. One of these loci originated from streptococcal species and contained â‰ˆ15,000 spacers, 50% of which were unique. Similar to the targeted studies of the oral cavity, some showed little evolution over time.
CRISPR evolution was studied in chemostats using S. thermophilus to directly examine spacer acquisition rates. In one week, S. thermophilus strains acquired up to three spacers when challenged with a single phage. During the same interval the phage developed single nucleotide polymorphisms that became fixed in the population, suggesting that targeting had prevented phage replication absent these mutations.
Another S. thermophilus experiment showed that phages can infect and replicate in hosts that have only one targeting spacer. Yet another showed that sensitive hosts can exist in environments with high phage titres. The chemostat and observational studies suggest many nuances to CRISPR and phage (co)evolution.
CRISPRs are widely distributed among bacteria and archaea and show some sequence similarities. 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 repeats decreases the likelihood of a false positive match.
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 many reference genomes are available, polymerase chain reaction (PCR) can be used to amplify CRISPR arrays and analyse spacer content. However, this approach yields information only for specifically targeted CRISPRs and for organisms with sufficient representation in public databases to design reliable polymerase chain reaction (PCR) primers. Degenerate repeat-specific primers can be used to amplify CRISPR spacers directly from environmental samples; amplicons containing two or three spacers can be then computationally assembled to reconstruct long CRISPR arrays.
The alternative is to extract and reconstruct CRISPR arrays from shotgun metagenomic data. This 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 appearing in a single read. CRISPR identification in raw reads has been achieved using purely de novo identification or by using direct repeat sequences in partially assembled CRISPR arrays from contigs (overlapping DNA segments that together represent a consensus region of DNA) and direct repeat sequences from published genomes as a hook for identifying direct repeats in individual reads.
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 a bacterial chromosome upon phage infection and can inhibit phage replication. PICIs are induced, excised, replicated and finally packaged into small capsids by certain staphylococcal temperate phages. PICIs use several mechanisms to block phage reproduction. In first mechanism PICI-encoded Ppi differentially blocks phage maturation by binding or interacting specifically with phage TerS, hence blocks phage TerS/TerL complex formation responsible for phage DNA packaging. In second mechanism PICI CpmAB redirect the phage capsid morphogenetic protein to make 95% of SaPI-sized capsid and phage DNA can package only 1/3rd of their genome in these small capsid and hence become nonviable phage. The third mechanism involves two proteins, PtiA and PtiB, that target the LtrC, which is responsible for the production of virion and lysis proteins. This interference mechanism is modulated by a modulatory protein, PtiM, binds to one of the interference-mediating proteins, PtiA, and hence achieving the required level of interference.
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 I-F system found in Yersinia pestis. Moreover, like the bacterial CRISPR/Cas system, ICP1 CRISPR/Cas can acquire new sequences, which allows phage and host to co-evolve.
Certain archaeal viruses were shown to carry mini-CRISPR arrays containing one or two spacers. It has been shown that spacers within the virus-borne CRISPR arrays target other viruses and plasmids, suggesting that mini-CRISPR arrays represent a mechanism of heterotypic superinfection exclusion and participate in interviral conflicts.
CRISPR gene editing
CRISPR technology has been applied in the food and farming industries to engineer probiotic cultures and to immunize industrial cultures (for yogurt, for instance) against infections. It is also being used in crops to enhance yield, drought tolerance and nutritional value.
By the end of 2014 some 1000 research papers had been published that mentioned CRISPR. The technology had been used to functionally inactivate genes in human cell lines and cells, to study Candida albicans, to modify yeasts used to make biofuels and to genetically modify crop strains. Hsu and his colleagues state that the ability to manipulate the genetic sequences allows for reverse engineering that can positively affect biofuel production  CRISPR can also be used to change mosquitos so they cannot transmit diseases such as malaria. CRISPR-based approaches utilizing Cas12a have recently been utilized in the successful modification of a broad number of plant species.
In February 2020, progress was made on HIV treatments with 60-80% of the integrated viral DNA removed in mice and some being completely free from the virus after edits involving both LASER ART, a new anti-retroviral therapy, and CRISPR.
In the future, CRISPR gene editing could potentially be used to create new species or revive extinct species from closely related ones.
CRISPR-based re-evaluations of claims for gene-disease relationships have led to the discovery of potentially important anomalies.
CRISPR as diagnostic tool
CRISPR associated nucleases have shown to be useful as a tool for molecular testing due to their ability to specifically target nucleic acid sequences in a high background of non-target sequences. In 2016, the Cas9 nuclease was used to deplete unwanted nucleotide sequences in next-generation sequencing libraries while requiring only 250 picograms of initial RNA input. Beginning in 2017, CRISPR associated nucleases were also used for direct diagnostic testing of nucleic acids, down to single molecule sensitivity.
By coupling CRISPR-based diagnostics to additional enzymatic processes, the detection of molecules beyond nucleic acids is possible. One example of a coupled technology is SHERLOCK-based Profiling of IN vitro Transcription (SPRINT). SPRINT can be used to detect a variety of substances, such as metabolites in patient samples or contaminants in environmental samples, with high throughput or with portable point-of-care devices. CRISPR/Cas platforms are also being explored for detection  and inactivation of SARS-CoV-2, the virus that causes COVID-19.
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- Haurwitz RE, Jinek M, Wiedenheft B, Zhou K, Doudna JA (September 2010). "Sequence- and structure-specific RNA processing by a CRISPR endonuclease". Science. 329 (5997): 1355â€“1358. Bibcode:2010Sci...329.1355H. doi:10.1126/science.1192272. PMCÂ 3133607. PMIDÂ 20829488.
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- Wang R, Preamplume G, Terns MP, Terns RM, Li H (February 2011). "Interaction of the Cas6 riboendonuclease with CRISPR RNAs: recognition and cleavage". Structure. 19 (2): 257â€“264. doi:10.1016/j.str.2010.11.014. PMCÂ 3154685. PMIDÂ 21300293.
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- Gudbergsdottir S, Deng L, Chen Z, Jensen JV, Jensen LR, She Q, Garrett RA (January 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): 35â€“49. doi:10.1111/j.1365-2958.2010.07452.x. PMCÂ 3025118. PMIDÂ 21166892.
- Manica A, Zebec Z, Teichmann D, Schleper C (April 2011). "In vivo activity of CRISPR-mediated virus defence in a hyperthermophilic archaeon". Molecular Microbiology. 80 (2): 481â€“491. doi:10.1111/j.1365-2958.2011.07586.x. PMIDÂ 21385233. S2CIDÂ 41442419.
- Jore MM, Lundgren M, van Duijn E, Bultema JB, Westra ER, Waghmare SP, etÂ al. (May 2011). "Structural basis for CRISPR RNA-guided DNA recognition by Cascade" (PDF). Nature Structural & Molecular Biology. 18 (5): 529â€“536. doi:10.1038/nsmb.2019. PMIDÂ 21460843. S2CIDÂ 10987554.
- Wiedenheft B, Lander GC, Zhou K, Jore MM, Brouns SJ, van der Oost J, Doudna JA, Nogales E (September 2011). "Structures of the RNA-guided surveillance complex from a bacterial immune system". Nature. 477 (7365): 486â€“489. Bibcode:2011Natur.477..486W. doi:10.1038/nature10402. PMCÂ 4165517. PMIDÂ 21938068.
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- Hale CR, Zhao P, Olson S, Duff MO, Graveley BR, Wells L, Terns RM, Terns MP (November 2009). "RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex". Cell. 139 (5): 945â€“956. doi:10.1016/j.cell.2009.07.040. PMCÂ 2951265. PMIDÂ 19945378.
- Estrella MA, Kuo FT, Bailey S (2016). "RNA-activated DNA cleavage by the Type III-B CRISPRâ€“Cas effector complex". Genes & Development. 30 (4): 460â€“470. doi:10.1101/gad.273722.115. PMCÂ 4762430. PMIDÂ 26848046.
- Samai P, Pyenson N, Jiang W, Goldberg GW, Hatoum-Aslan A, Marraffini LA (2015). "Co-transcriptional DNA and RNA Cleavage during Type III CRISPR-Cas Immunity". Cell. 161 (5): 1164â€“1174. doi:10.1016/j.cell.2015.04.027. PMCÂ 4594840. PMIDÂ 25959775.
- Marraffini LA, Sontheimer EJ (January 2010). "Self versus non-self discrimination during CRISPR RNA-directed immunity". Nature. 463 (7280): 568â€“571. Bibcode:2010Natur.463..568M. doi:10.1038/nature08703. PMCÂ 2813891. PMIDÂ 20072129.
- Krupovic M, BÃ©guin P, Koonin EV (August 2017). "Casposons: mobile genetic elements that gave rise to the CRISPR-Cas adaptation machinery". Current Opinion in Microbiology. 38: 36â€“43. doi:10.1016/j.mib.2017.04.004. PMCÂ 5665730. PMIDÂ 28472712.
- Koonin EV, Makarova KS (May 2013). "CRISPR-Cas: evolution of an RNA-based adaptive immunity system in prokaryotes". RNA Biology. 10 (5): 679â€“686. doi:10.4161/rna.24022. PMCÂ 3737325. PMIDÂ 23439366.
- Koonin EV, Makarova KS, Zhang F (June 2017). "Diversity, classification and evolution of CRISPR-Cas systems". Current Opinion in Microbiology. 37: 67â€“78. doi:10.1016/j.mib.2017.05.008. PMCÂ 5776717. PMIDÂ 28605718.
- Shmakov S, Smargon A, Scott D, Cox D, Pyzocha N, Yan W, Abudayyeh OO, Gootenberg JS, Makarova KS, Wolf YI, Severinov K, Zhang F, Koonin EV (March 2017). "Diversity and evolution of class 2 CRISPR-Cas systems". Nature Reviews. Microbiology. 15 (3): 169â€“182. doi:10.1038/nrmicro.2016.184. PMCÂ 5851899. PMIDÂ 28111461.
- Kupczok A, Bollback JP (February 2013). "Probabilistic models for CRISPR spacer content evolution". BMC Evolutionary Biology. 13 (1): 54. doi:10.1186/1471-2148-13-54. PMCÂ 3704272. PMIDÂ 23442002.
- Sternberg SH, Richter H, Charpentier E, Qimron U (March 2016). "Adaptation in CRISPR-Cas Systems". Molecular Cell. 61 (6): 797â€“808. doi:10.1016/j.molcel.2016.01.030. hdl:21.11116/0000-0003-E74E-2. PMIDÂ 26949040.
- Koonin EV, Wolf YI (November 2009). "Is evolution Darwinian or/and Lamarckian?". Biology Direct. 4: 42. doi:10.1186/1745-6150-4-42. PMCÂ 2781790. PMIDÂ 19906303.
- Weiss A (October 2015). "Lamarckian Illusions". Trends in Ecology & Evolution. 30 (10): 566â€“568. doi:10.1016/j.tree.2015.08.003. PMIDÂ 26411613.
- Koonin EV, Wolf YI (February 2016). "Just how Lamarckian is CRISPR-Cas immunity: the continuum of evolvability mechanisms". Biology Direct. 11 (1): 9. doi:10.1186/s13062-016-0111-z. PMCÂ 4765028. PMIDÂ 26912144.
- Heidelberg JF, Nelson WC, Schoenfeld T, Bhaya D (2009). Ahmed N (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. Bibcode:2009PLoSO...4.4169H. doi:10.1371/journal.pone.0004169. PMCÂ 2612747. PMIDÂ 19132092.
- Sampson TR, Saroj SD, Llewellyn AC, Tzeng YL, Weiss DS (May 2013). "A CRISPR/Cas system mediates bacterial innate immune evasion and virulence". Nature. 497 (7448): 254â€“257. Bibcode:2013Natur.497..254S. doi:10.1038/nature12048. PMCÂ 3651764. PMIDÂ 23584588.
- Touchon M, Rocha EP (June 2010). Randau L (ed.). "The small, slow and specialized CRISPR and anti-CRISPR of Escherichia and Salmonella". PLOS ONE. 5 (6): e11126. Bibcode:2010PLoSO...511126T. doi:10.1371/journal.pone.0011126. PMCÂ 2886076. PMIDÂ 20559554.
- Rho M, Wu YW, Tang H, Doak TG, Ye Y (2012). "Diverse CRISPRs evolving in human microbiomes". PLOS Genetics. 8 (6): e1002441. doi:10.1371/journal.pgen.1002441. PMCÂ 3374615. PMIDÂ 22719260.
- Sun CL, Barrangou R, Thomas BC, Horvath P, Fremaux C, Banfield JF (February 2013). "Phage mutations in response to CRISPR diversification in a bacterial population". Environmental Microbiology. 15 (2): 463â€“470. doi:10.1111/j.1462-2920.2012.02879.x. PMIDÂ 23057534.
- Kuno S, Sako Y, Yoshida T (May 2014). "Diversification of CRISPR within coexisting genotypes in a natural population of the bloom-forming cyanobacterium Microcystis aeruginosa". Microbiology. 160 (Pt 5): 903â€“916. doi:10.1099/mic.0.073494-0. PMIDÂ 24586036.
- Sorek R, Kunin V, Hugenholtz P (March 2008). "CRISPRâ€”a widespread system that provides acquired resistance against phages in bacteria and archaea". Nature Reviews. Microbiology. 6 (3): 181â€“186. doi:10.1038/nrmicro1793. PMIDÂ 18157154. S2CIDÂ 3538077.
Table 1: Web resources for CRISPR analysis
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- Held NL, Herrera A, Whitaker RJ (November 2013). "Reassortment of CRISPR repeat-spacer loci in Sulfolobus islandicus". Environmental Microbiology. 15 (11): 3065â€“3076. doi:10.1111/1462-2920.12146. PMIDÂ 23701169.
- Held NL, Herrera A, Cadillo-Quiroz H, Whitaker RJ (September 2010). "CRISPR associated diversity within a population of Sulfolobus islandicus". PLOS ONE. 5 (9): e12988. Bibcode:2010PLoSO...512988H. doi:10.1371/journal.pone.0012988. PMCÂ 2946923. PMIDÂ 20927396.
- Medvedeva S, Liu Y, Koonin EV, Severinov K, Prangishvili D, Krupovic M (November 2019). "Virus-borne mini-CRISPR arrays are involved in interviral conflicts". Nature Communications. 10 (1): 5204. Bibcode:2019NatCo..10.5204M. doi:10.1038/s41467-019-13205-2. PMCÂ 6858448. PMIDÂ 31729390.
- Skennerton CT, Imelfort M, Tyson GW (May 2013). "Crass: identification and reconstruction of CRISPR from unassembled metagenomic data". Nucleic Acids Research. 41 (10): e105. doi:10.1093/nar/gkt183. PMCÂ 3664793. PMIDÂ 23511966.
- Stern A, Mick E, Tirosh I, Sagy O, Sorek R (October 2012). "CRISPR targeting reveals a reservoir of common phages associated with the human gut microbiome". Genome Research. 22 (10): 1985â€“1994. doi:10.1101/gr.138297.112. PMCÂ 3460193. PMIDÂ 22732228.
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- Ram G, Chen J, Ross HF, Novick RP (October 2014). "Precisely modulated pathogenicity island interference with late phage gene transcription". Proceedings of the National Academy of Sciences of the United States of America. 111 (40): 14536â€“14541. Bibcode:2014PNAS..11114536R. doi:10.1073/pnas.1406749111. PMCÂ 4209980. PMIDÂ 25246539.
- Seed KD, Lazinski DW, Calderwood SB, Camilli A (February 2013). "A bacteriophage encodes its own CRISPR/Cas adaptive response to evade host innate immunity". Nature. 494 (7438): 489â€“491. Bibcode:2013Natur.494..489S. doi:10.1038/nature11927. PMCÂ 3587790. PMIDÂ 23446421.
- Boyd CM, Angermeyer A, Hays SG, Barth ZK, Patel KM, Seed KD (September 2021). "Bacteriophage ICP1: A Persistent Predator of Vibrio cholerae". Annual Review of Virology. 8 (1): 285â€“304. doi:10.1146/annurev-virology-091919-072020. ISSNÂ 2327-056X. PMIDÂ 34314595.
- "What is CRISPR and How does it work?". Livescience.Tech. 30 April 2018. Retrieved 2019-12-14.
- Verma AK, Mandal S, Tiwari A, Monachesi C, Catassi GN, Srivastava A, Gatti S, Lionetti E, Catassi C (2 October 2021). "Current Status and Perspectives on the Application of CRISPR/Cas9 Gene-Editing System to Develop a Low-Gluten, Non-Transgenic Wheat Variety". Foods. 10 (2351): 2351. doi:10.3390/foods10102351. PMCÂ 8534962. PMIDÂ 34681400.
- Doudna JA, Charpentier E (November 2014). "Genome editing. The new frontier of genome engineering with CRISPR-Cas9". Science. 346 (6213): 1258096. doi:10.1126/science.1258096. PMIDÂ 25430774. S2CIDÂ 6299381.
- Ledford H (June 2015). "CRISPR, the disruptor". Nature. 522 (7554): 20â€“24. Bibcode:2015Natur.522...20L. doi:10.1038/522020a. PMIDÂ 26040877.
- Hsu PD, Lander ES, Zhang F (June 2014). "Development and applications of CRISPR-Cas9 for genome engineering". Cell. 157 (6): 1262â€“1278. doi:10.1016/j.cell.2014.05.010. PMCÂ 4343198. PMIDÂ 24906146.
- Alphey L (February 2016). "Can CRISPR-Cas9 gene drives curb malaria?". Nature Biotechnology. 34 (2): 149â€“150. doi:10.1038/nbt.3473. PMIDÂ 26849518. S2CIDÂ 10014014.
- BernabÃ©-Orts JM, Casas-Rodrigo I, Minguet EG, Landolfi V, Garcia-Carpintero V, Gianoglio S, etÂ al. (October 2019). "Assessment of Cas12a-mediated gene editing efficiency in plants". Plant Biotechnology Journal. 17 (10): 1971â€“1984. doi:10.1111/pbi.13113. PMCÂ 6737022. PMIDÂ 30950179.
- "In A 1st, Doctors In U.S. Use CRISPR Tool To Treat Patient With Genetic Disorder". NPR.org. Retrieved 2019-07-31.
- National Institute on Drug Abuse (2020-02-14). "Antiretroviral Therapy Combined With CRISPR Gene Editing Can Eliminate HIV Infection in Mice". National Institute on Drug Abuse. Retrieved 2020-11-15.
- "In A 1st, Scientists Use Revolutionary Gene-Editing Tool To Edit Inside A Patient". NPR.org.
- The-Crispr (2019-07-15). "Listen Radiolab CRISPR podcast". The Crispr. Retrieved 2019-07-15.
- Ledford H (2017). "CRISPR studies muddy results of older gene research". Nature. doi:10.1038/nature.2017.21763. S2CIDÂ 90757972.
- Gu W, Crawford ED, O'Donovan BD, Wilson MR, Chow ED, Retallack H, DeRisi JL (March 2016). "Depletion of Abundant Sequences by Hybridization (DASH): using Cas9 to remove unwanted high-abundance species in sequencing libraries and molecular counting applications". Genome Biology. 17 (1): 41. doi:10.1186/s13059-016-0904-5. PMCÂ 4778327. PMIDÂ 26944702.
- Chen JS, Ma E, Harrington LB, Da Costa M, Tian X, Palefsky JM, Doudna JA (April 2018). "CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity". Science. 360 (6387): 436â€“439. Bibcode:2018Sci...360..436C. doi:10.1126/science.aar6245. PMCÂ 6628903. PMIDÂ 29449511.
- Iwasaki RS, Batey RT (September 2020). "SPRINT: a Cas13a-based platform for detection of small molecules". Nucleic Acids Research. 48 (17): e101. doi:10.1093/nar/gkaa673. PMCÂ 7515716. PMIDÂ 32797156.
- Joung J, Ladha A, Saito M, Kim NG, Woolley AE, Segel M, etÂ al. (October 2020). "Detection of SARS-CoV-2 with SHERLOCK One-Pot Testing". The New England Journal of Medicine. 383 (15): 1492â€“1494. doi:10.1056/NEJMc2026172. PMCÂ 7510942. PMIDÂ 32937062.
- Dhamad AE, Abdal Rhida MA (2020). "COVID-19: molecular and serological detection methods". PeerJ. 8: e10180. doi:10.7717/peerj.10180. PMCÂ 7547594. PMIDÂ 33083156.
- Patchsung M, Jantarug K, Pattama A, Aphicho K, Suraritdechachai S, Meesawat P, etÂ al. (December 2020). "Clinical validation of a Cas13-based assay for the detection of SARS-CoV-2 RNA". Nature Biomedical Engineering. 4 (12): 1140â€“1149. doi:10.1038/s41551-020-00603-x. PMIDÂ 32848209.
- Konwarh R (September 2020). "Can CRISPR/Cas Technology Be a Felicitous Stratagem Against the COVID-19 Fiasco? Prospects and Hitches". Frontiers in Molecular Biosciences. 7: 557377. doi:10.3389/fmolb.2020.557377. PMCÂ 7511716. PMIDÂ 33134311.
- Doudna J, Mali P (23 March 2016). CRISPR-Cas: A Laboratory Manual. New York: Cold Spring Harbor Laboratory Press. ISBNÂ 978-1-62182-131-1.
- Mohanraju P, Makarova KS, Zetsche B, Zhang F, Koonin EV, van der Oost J (August 2016). "Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems" (PDF). Science. 353 (6299): aad5147. doi:10.1126/science.aad5147. hdl:1721.1/113195. PMIDÂ 27493190. S2CIDÂ 11086282.
- Sander JD, Joung JK (April 2014). "CRISPR-Cas systems for editing, regulating and targeting genomes". Nature Biotechnology. 32 (4): 347â€“355. doi:10.1038/nbt.2842. PMCÂ 4022601. PMIDÂ 24584096.
- Slaymaker IM, Gao L, Zetsche B, Scott DA, Yan WX, Zhang F (January 2016). "Rationally engineered Cas9 nucleases with improved specificity". Science. 351 (6268): 84â€“88. Bibcode:2016Sci...351...84S. doi:10.1126/science.aad5227. PMCÂ 4714946. PMIDÂ 26628643.
- Terns RM, Terns MP (March 2014). "CRISPR-based technologies: prokaryotic defense weapons repurposed". Trends in Genetics. 30 (3): 111â€“118. doi:10.1016/j.tig.2014.01.003. PMCÂ 3981743. PMIDÂ 24555991.
- Westra ER, Buckling A, Fineran PC (May 2014). "CRISPR-Cas systems: beyond adaptive immunity". Nature Reviews Microbiology. 12 (5): 317â€“326. doi:10.1038/nrmicro3241. PMIDÂ 24704746. S2CIDÂ 36575361.
- Andersson AF, Banfield JF (May 2008). "Virus population dynamics and acquired virus resistance in natural microbial communities". Science. 320 (5879): 1047â€“1050. Bibcode:2008Sci...320.1047A. doi:10.1126/science.1157358. PMIDÂ 18497291. S2CIDÂ 26209623.
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- van der Ploeg JR (June 2009). "Analysis of CRISPR in Streptococcus mutans suggests frequent occurrence of acquired immunity against infection by M102-like bacteriophages" (PDF). Microbiology. 155 (Pt 6): 1966â€“1976. doi:10.1099/mic.0.027508-0. PMIDÂ 19383692.
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- Karginov FV, Hannon GJ (January 2010). "The CRISPR system: small RNA-guided defense in bacteria and archaea". Molecular Cell. 37 (1): 7â€“19. doi:10.1016/j.molcel.2009.12.033. PMCÂ 2819186. PMIDÂ 20129051.
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|Wikimedia Commons has media related to CRISPR.|
|Scholia has a topic profile for CRISPR.|
- Advanced Gene Editing: CRISPR-Cas9 Congressional Research Service
- Jennifer Doudna talk: Genome Engineering with CRISPR-Cas9: Birth of a Breakthrough Technology
- Overview of all the structural information available in the PDB for UniProt: Q46901 (CRISPR system Cascade subunit CasA) at the PDBe-KB.
- Overview of all the structural information available in the PDB for UniProt: P76632 (CRISPR system Cascade subunit CasB) at the PDBe-KB.
- Overview of all the structural information available in the PDB for UniProt: Q46899 (CRISPR system Cascade subunit CasC) at the PDBe-KB.
- Overview of all the structural information available in the PDB for UniProt: Q46898 (CRISPR system Cascade subunit CasD) at the PDBe-KB.
- Overview of all the structural information available in the PDB for UniProt: Q46897 (CRISPR system Cascade subunit CasE) at the PDBe-KB.
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 Cse2 (CRISPR_cse2) Provide feedback
Clusters of short DNA repeats with non-homologous spacers, which are found at regular intervals in the genomes of phylogenetically distinct prokaryotic species, comprise a family with recognisable features. This family is known as CRISPR (short for Clustered, Regularly Interspaced Short Palindromic Repeats). A number of protein families appear only in association with these repeats and are designated Cas (CRISPR-Associated) proteins. This family of proteins, represented by CT1973 from Chlorobaculum tepidum, is encoded by genes found in the CRISPR/Cas subtype Ecoli regions of many bacteria (most of which are mesophiles), and not in Archaea. It is designated Cse2.
This tab holds annotation information from the InterPro database.
InterPro entry IPR013382
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 an endoribonuclease 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 Cse2 family of Cas proteins, which includes CT1973 from Chlorobium tepidum. These proteins are found in the CRISPR/Cas subtype Ecoli regions of many bacteria (most of which are mesophiles), and not in Archaea [ PUBMED:16292354 ]. This is also known as CasB or Cse2 Type I-E [ PUBMED:21552286 ].
Below is a listing of the unique domain organisations or architectures in which this domain is found. More...
The graphic that is shown by default represents the longest sequence with a given architecture. Each row contains the following information:
- the number of sequences which exhibit this architecture
a textual description of the architecture, e.g. Gla, EGF x 2, Trypsin.
This example describes an architecture with one
Gladomain, followed by two consecutive
EGFdomains, and finally a single
- a link to the page in the Pfam site showing information about the sequence that the graphic describes
- the UniProt description of the protein sequence
- the number of residues in the sequence
- the Pfam graphic itself.
Note that you can see the family page for a particular domain by clicking on the graphic. You can also choose to see all sequences which have a given architecture by clicking on the Show link in each row.
Finally, because some families can be found in a very large number of architectures, we load only the first fifty architectures by default. If you want to see more architectures, click the button at the bottom of the page to load the next set.
Loading domain graphics...
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 and the UniProtKB sequence database. More...
There are various ways to view or download the sequence alignments that we store. We provide several sequence viewers and a plain-text Stockholm-format file for download.
We make a range of alignments for each Pfam-A family:
- the curated alignment from which the HMM for the family is built
- the alignment generated by searching the sequence database using the HMM
- Representative Proteomes (RPs) at 15%, 35%, 55% and 75% co-membership thresholds
- alignment generated by searching the UniProtKB sequence database using the family HMM
You can see the alignments as HTML or in three different sequence viewers:
- a Java applet developed at the University of Dundee. You will need Java installed before running jalview
- an HTML page showing the whole alignment.Please note: full Pfam alignments can be very large. These HTML views are extremely large and often cause problems for browsers. Please use either jalview or the Pfam viewer if you have trouble viewing the HTML version
- an HTML-based representation of the alignment, coloured according to the posterior-probability (PP) values from the HMM. As for the standard HTML view, heatmap alignments can also be very large and slow to render.
You can download (or view in your browser) a text representation of a Pfam alignment in various formats:
You can also change the order in which sequences are listed in the alignment, change how insertions are represented, alter the characters that are used to represent gaps in sequences and, finally, choose whether to download the alignment or to view it in your browser directly.
You may find that large alignments cause problems for the viewers and the reformatting tool, so we also provide all alignments in Stockholm format. You can download either the plain text alignment, or a gzipped version of it.
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.
1Cannot generate PP/Heatmap alignments for seeds; no PP data available
Key: available, not generated, — not available.
Format an alignment
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.
You can also download a FASTA format file containing the full-length sequences for all sequences in the full alignment.
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...
If you find these logos useful in your own work, please consider citing the following article:
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.
|Author:||TIGRFAMs, Coggill P|
|Number in seed:||74|
|Number in full:||885|
|Average length of the domain:||148.20 aa|
|Average identity of full alignment:||22 %|
|Average coverage of the sequence by the domain:||72.68 %|
|HMM build commands:||
build method: hmmbuild -o /dev/null HMM SEED
search method: hmmsearch -Z 61295632 -E 1000 --cpu 4 HMM pfamseq
|Family (HMM) version:||13|
|Download:||download the raw HMM for this family|
Weight segments by...
Change the size of the sunburst
selected sequences to HMM
a FASTA-format file
- 0 sequences
- 0 species
This visualisation provides a simple graphical representation of the distribution of this family across species. You can find the original interactive tree in the More....
This chart is a modified "sunburst" visualisation of the species tree for this family. It shows each node in the tree as a separate arc, arranged radially with the superkingdoms at the centre and the species arrayed around the outermost ring.
How the sunburst is generated
The tree is built by considering the taxonomic lineage of each sequence that has a match to this family. For each node in the resulting tree, we draw an arc in the sunburst. The radius of the arc, its distance from the root node at the centre of the sunburst, shows the taxonomic level ("superkingdom", "kingdom", etc). The length of the arc represents either the number of sequences represented at a given level, or the number of species that are found beneath the node in the tree. The weighting scheme can be changed using the sunburst controls.
In order to reduce the complexity of the representation, we reduce the number of taxonomic levels that we show. We consider only the following eight major taxonomic levels:
Colouring and labels
Segments of the tree are coloured approximately according to their superkingdom. For example, archeal branches are coloured with shades of orange, eukaryotes in shades of purple, etc. The colour assignments are shown under the sunburst controls. Where space allows, the name of the taxonomic level will be written on the arc itself.
As you move your mouse across the sunburst, the current node will be highlighted. In the top section of the controls panel we show a summary of the lineage of the currently highlighed node. If you pause over an arc, a tooltip will be shown, giving the name of the taxonomic level in the title and a summary of the number of sequences and species below that node in the tree.
Anomalies in the taxonomy tree
There are some situations that the sunburst tree cannot easily handle and for which we have work-arounds in place.
Missing taxonomic levels
Some species in the taxonomic tree may not have one or more of the main eight levels that we display. For example, Bos taurus is not assigned an order in the NCBI taxonomic tree. In such cases we mark the omitted level with, for example, "No order", in both the tooltip and the lineage summary.
Unmapped species names
The tree is built by looking at each sequence in the full alignment for the family. We take the name of the species given by UniProt and try to map that to the full taxonomic tree from NCBI. In some cases, the name chosen by UniProt does not map to any node in the NCBI tree, perhaps because the chosen name is listed as a synonym or a misspelling in the NCBI taxonomy.
So that these nodes are not simply omitted from the sunburst tree, we group them together in a separate branch (or segment of the sunburst tree). Since we cannot determine the lineage for these unmapped species, we show all levels between the superkingdom and the species as "uncategorised".
Since we reduce the species tree to only the eight main taxonomic levels, sequences that are mapped to the sub-species level in the tree would not normally be shown. Rather than leave out these species, we map them instead to their parent species. So, for example, for sequences belonging to one of the Vibrio cholerae sub-species in the NCBI taxonomy, we show them instead as belonging to the species Vibrio cholerae.
Too many species/sequences
For large species trees, you may see blank regions in the outer layers of the sunburst. These occur when there are large numbers of arcs to be drawn in a small space. If an arc is less than approximately one pixel wide, it will not be drawn and the space will be left blank. You may still be able to get some information about the species in that region by moving your mouse across the area, but since each arc will be very small, it will be difficult to accurately locate a particular species.
The tree shows the occurrence of this domain across different species. More...
We show the species tree in one of two ways. For smaller trees we try to show an interactive representation, which allows you to select specific nodes in the tree and view them as an alignment or as a set of Pfam domain graphics.
Unfortunately we have found that there are problems viewing the interactive tree when the it becomes larger than a certain limit. Furthermore, we have found that Internet Explorer can become unresponsive when viewing some trees, regardless of their size. We therefore show a text representation of the species tree when the size is above a certain limit or if you are using Internet Explorer to view the site.
If you are using IE you can still load the interactive tree by clicking the "Generate interactive tree" button, but please be aware of the potential problems that the interactive species tree can cause.
For all of the domain matches in a full alignment, we count the number that are found on all sequences in the alignment. This total is shown in the purple box.
We also count the number of unique sequences on which each domain is found, which is shown in green. Note that a domain may appear multiple times on the same sequence, leading to the difference between these two numbers.
Finally, we group sequences from the same organism according to the NCBI code that is assigned by UniProt, allowing us to count the number of distinct sequences on which the domain is found. This value is shown in the pink boxes.
We use the NCBI species tree to group organisms according to their taxonomy and this forms the structure of the displayed tree. Note that in some cases the trees are too large (have too many nodes) to allow us to build an interactive tree, but in most cases you can still view the tree in a plain text, non-interactive representation. Those species which are represented in the seed alignment for this domain are highlighted.
You can use the tree controls to manipulate how the interactive tree is displayed:
- show/hide the summary boxes
- highlight species that are represented in the seed alignment
- expand/collapse the tree or expand it to a given depth
- select a sub-tree or a set of species within the tree and view them graphically or as an alignment
- save a plain text representation of the tree
Please note: for large trees this can take some time. While the tree is loading, you can safely switch away from this tab but if you browse away from the family page entirely, the tree will not be loaded.
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_Cse2 domain has been found. There are 31 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 sequence.
Loading structure mapping...
AlphaFold Structure Predictions
The list of proteins below match this family and have AlphaFold predicted structures. Click on the protein accession to view the predicted structure.
|Protein||Predicted structure||External Information|
|P76632||View 3D Structure||Click here|
|Q53VY0||View 3D Structure||Click here|