Summary: CRISPR-associated protein Cse1 (CRISPR_cse1)
Pfam includes annotations and additional family information from a range of different sources. These sources can be accessed via the tabs below.
This is the Wikipedia entry entitled "CRISPR". More...
The Wikipedia text that you see displayed here is a download from Wikipedia. This means that the information we display is a copy of the information from the Wikipedia database. The button next to the article title ("Edit Wikipedia article") takes you to the edit page for the article directly within Wikipedia. You should be aware you are not editing our local copy of this information. Any changes that you make to the Wikipedia article will not be displayed here until we next download the article from Wikipedia. We currently download new content on a nightly basis.
Does Pfam agree with the content of the Wikipedia entry ?
Pfam has chosen to link families to Wikipedia articles. In some case we have created or edited these articles but in many other cases we have not made any direct contribution to the content of the article. The Wikipedia community does monitor edits to try to ensure that (a) the quality of article annotation increases, and (b) vandalism is very quickly dealt with. However, we would like to emphasise that Pfam does not curate the Wikipedia entries and we cannot guarantee the accuracy of the information on the Wikipedia page.
Editing Wikipedia articles
Before you edit for the first time
Wikipedia is a free, online encyclopedia. Although anyone can edit or contribute to an article, Wikipedia has some strong editing guidelines and policies, which promote the Wikipedia standard of style and etiquette. Your edits and contributions are more likely to be accepted (and remain) if they are in accordance with this policy.
You should take a few minutes to view the following pages:
How your contribution will be recorded
Anyone can edit a Wikipedia entry. You can do this either as a new user or you can register with Wikipedia and log on. When you click on the "Edit Wikipedia article" button, your browser will direct you to the edit page for this entry in Wikipedia. If you are a registered user and currently logged in, your changes will be recorded under your Wikipedia user name. However, if you are not a registered user or are not logged on, your changes will be logged under your computer's IP address. This has two main implications. Firstly, as a registered Wikipedia user your edits are more likely seen as valuable contribution (although all edits are open to community scrutiny regardless). Secondly, if you edit under an IP address you may be sharing this IP address with other users. If your IP address has previously been blocked (due to being flagged as a source of 'vandalism') your edits will also be blocked. You can find more information on this and creating a user account at Wikipedia.
If you have problems editing a particular page, contact us at email@example.com and we will try to help.
The community annotation is a new facility of the Pfam web site. If you have problems editing or experience problems with these pages please contact us.
CRISPR Edit Wikipedia article
Clustered regularly interspaced short palindromic repeats (CRISPR, //) are segments of prokaryotic DNA containing short, repetitive base sequences. These play a key role in a bacterial defence system, and form the basis of a genome editing technology known as CRISPR/Cas9 that allows permanent modification of genes within organisms. In a palindromic repeat, the sequence of nucleotides is the same in both directions. Each repetition is followed by short segments of spacer DNA from previous exposures to foreign DNA (e.g., a virus or plasmid). Small clusters of cas (CRISPR-associated system) genes are located next to CRISPR sequences.
The CRISPR/Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements such as those present within plasmids and phages that provides a form of acquired immunity. RNA harboring the spacer sequence helps Cas proteins recognize and cut exogenous DNA. Other RNA-guided Cas proteins cut foreign RNA. CRISPRs are found in approximately 40% of sequenced bacterial genomes and 90% of sequenced archaea.[note 1]
A simple version of the CRISPR/Cas system, CRISPR/Cas9, has been modified to edit genomes. By delivering the Cas9 nuclease complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be cut at a desired location, allowing existing genes to be removed and/or new ones added. The Cas9-gRNA complex corresponds with the CAS III crRNA complex in the above diagram.
CRISPR/Cas genome editing techniques have many potential applications, including medicine and crop seed enhancement. The use of CRISPR/Cas9-gRNA complex for genome editing was the AAAS's choice for breakthrough of the year in 2015. Bioethical concerns have been raised about the prospect of using CRISPR for germline editing.
- 1 History
- 2 Locus structure
- 3 Mechanism
- 4 Evolution
- 5 Identification
- 6 Use by phages
- 7 Applications
- 8 Patents and commercialization
- 9 Society and culture
- 10 See also
- 11 Notes
- 12 References
- 13 Further reading
- 14 External links
|Cascade (CRISPR-associated complex for antiviral defense)|
Structure of crRNA-guided E. coli Cascade complex (Cas, blue) bound to single-stranded DNA (orange).
The discovery of clustered DNA repeats began independently in three parts of the world.
The first description of what would later be called CRISPR was from Osaka University researcher Yoshizumi Ishino in 1987, who accidentally cloned part of a CRISPR together with the iap gene, the target of interest. The organization of the repeats was unusual because repeated sequences are typically arranged consecutively along DNA. The function of the interrupted clustered repeats was not known at the time.
In 1993 researchers of Mycobacterium tuberculosis in the Netherlands published two articles about a cluster of interrupted direct repeats (DR) in this bacterium. These researchers recognized the diversity of the DR-intervening sequences 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.
At the same time, repeats were observed in the archaeal organisms Haloferax and Haloarcula species, and their function was studied by Francisco Mojica at the University of Alicante in Spain. Although his hypothesis turned out to be wrong, Mojica 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. By 2000, Mojica's group had identified interrupted repeats in 20 species of microbes. In 2001, Mojica and Ruud Jansen, who was 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.
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 1 to 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 coined 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's group, 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 that hypothesis by proposing mechanisms of action for the different CRISPR-Cas subtypes according to the predicted function of their proteins. Others hypothesized that CRISPR sequences directed Cas enzymes to degrade viral DNA.
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 phage by adding and deleting spacers whose sequence matched those found in the tested phages. In 2008, Brouns and colleagues identified a complex of Cas protein that in E. coli cut the CRISPR RNA within the repeats into spacer-containing RNA molecules, which remained bound to the protein complex. That year Marraffini and Sontheimer showed 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 RNA molecules named CRISPR RNA (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 mixed with Cas9, could find and cut the DNA target specified by the guide RNA. 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 Šikšnys together with Gašiūnas, 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.
Feng Zhang's and George Church's groups simultaneously described 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), zebrafish (D. rerio), fruit flies (Drosophila melanogaster), nematodes (C. elegans), plants, mice, monkeys and human embryos.
In 2015, the nuclease Cpf1 was discovered in the CRISPR/Cpf1 system of the bacterium Francisella novicida. Cpf1 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 alternate 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)).
In the early 2000s, researchers developed zinc finger nucleases, synthetic proteins whose DNA-binding domains enable them to create double-stranded breaks in DNA at specific points. In 2010, synthetic nucleases called transcription activator-like effector nucleases (TALENs) provided an easier way to target a double-stranded break to a specific location on the DNA strand. Both zinc finger nucleases and TALENs require the creation of a custom protein for each targeted DNA sequence, which is a more difficult and time-consuming process than that for guide RNAs. CRISPRs are much easier to design because the process requires making only a short RNA sequence.
Repeats and spacers
The CRISPR array comprises 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 hairpin in the RNA, while others are predicted 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.
Cas genes and CRISPR subtypes
Small clusters of cas genes are often located next to CRISPR repeat-spacer arrays. Collectively there are 93 cas genes that 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. 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.
|Class||Cas type||Signature protein||Function||Reference|
|1||I||Cas3||Single-stranded DNA nuclease (HD domain) and ATP-dependent helicase|||
|IA||Cas8a, Cas5||Subunit of the interference module. Important in targeting of invading DNA by recognizing the PAM sequence|||
|ID||Cas10d||contains a domain homologous to the palm domain of nucleic acid polymerases and nucleotide cyclases|||
|IF||Csy1, Csy2, Csy3||Not determined|||
|III||Cas10||Homolog of Cas10d and Cse1|||
|IIIC||Cas10 or Csx11|||
|2||II||Cas9||Nucleases RuvC and HNH together produce DSBs, and separately can produce single-strand breaks. Ensures the acquisition of functional spacers during adaptation.|||
|IIA||Csn2||Ring-shaped DNA-binding protein. Involved in primed adaptation in Type II CRISPR system.|||
|IIC||Characterized by the absence of either Csn2 or Cas4|||
|V||Cpf1, C2c1, C2c3||Nuclease RuvC. Lacks HNH.|||
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 virus, the first stage of the immune response is to capture viral DNA and insert it into a CRISPR locus in the form of a spacer. Cas1 and Cas2 are found in all three 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.
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 (3 – 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 ‘priming’ 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.
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|
crystal structure of a crispr-associated protein from Thermus thermophilus
|CRISPR associated protein Cas2|
crystal structure of a hypothetical protein tt1823 from Thermus thermophilus
|CRISPR-associated protein Cse1|
|CRISPR-associated protein Cse2|
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".
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 fight off 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 7-22% 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 ~15,000 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. Three programs used for CRISPR repeat identification search for regularly interspaced repeats in long sequences: CRT, PILER-CR and CRISPRfinder.
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.
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. 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 phage and host to co-evolve.
By the end of 2014 some 600 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. CRISPR can also be used to change mosquitos so they cannot transmit diseases such as malaria.
CRISPR-based re-evaluations of claims for gene-disease relationships have led to the discovery of potentially important anomalies
CRISPR/Cas9 genome editing is carried out with a Type II CRISPR system. When utilized for genome editing, this system includes Cas9, crRNA, tracrRNA along with an optional section of DNA repair template that is utilized in either Non-Homologous End Joining (NHEJ) or Homology Directed Repair (HDR).
|crRNA||Contains the guide RNA that locates the correct section of host DNA along with a region that binds to tracrRNA (generally in a hairpin loop form) forming an active complex.|
|tracrRNA||Binds to crRNA and forms an active complex.|
|sgRNA||Single guide RNAs are a combined RNA consisting of a tracrRNA and at least one crRNA|
|Cas9||Protein whose active form is able to modify DNA. Many variants exist with differing functions (i.e. single strand nicking, double strand break, DNA binding) due to Cas9's DNA site recognition function.|
|Repair template||DNA that guides the cellular repair process allowing insertion of a specific DNA sequence|
CRISPR/Cas9 often employs a plasmid to transfect the target cells. The main components of this plasmid are displayed in the image and listed in the table. The crRNA needs to be designed for each application as this is the sequence that Cas9 uses to identify and directly bind to the cell's DNA. The crRNA must bind only where editing is desired. The repair template is designed for each application, as it must overlap with the sequences on either side of the cut and code for the insertion sequence.
Multiple crRNAs and the tracrRNA can be packaged together to form a single-guide RNA (sgRNA). This sgRNA can be joined together with the Cas9 gene and made into a plasmid in order to be transfected into cells.
CRISPR/Cas9 offers a high degree of fidelity and relatively simple construction. It depends on two factors for its specificity: the target sequence and the PAM. The target sequence is 20 bases long as part of each CRISPR locus in the crRNA array. A typical crRNA array has multiple unique target sequences. Cas9 proteins select the correct location on the host's genome by utilizing the sequence to bond with base pairs on the host DNA. The sequence is not part of the Cas9 protein and as a result is customizable and can be independently synthesized.
The PAM sequence on the host genome is recognized by Cas9. Cas9 cannot be easily modified to recognize a different PAM sequence. However this is not too limiting as it is a short sequence and nonspecific (e.g. the SpCas9 PAM sequence is 5'-NGG-3' and in the human genome occurs roughly every 8 to 12 base pairs).
Once these have been assembled into a plasmid and transfected into cells the Cas9 protein with the help of the crRNA finds the correct sequence in the host cell's DNA and – depending on the Cas9 variant – creates a single or double strand break in the DNA.
Properly spaced single strand breaks in the host DNA can trigger homology directed repair, which is less error prone than the non-homologous end joining that typically follows a double strand break. Providing a DNA repair template allows for the insertion of a specific DNA sequence at an exact location within the genome. The repair template should extend 40 to 90 base pairs beyond the Cas9 induced DNA break. The goal is for the cell's HDR process to utilize the provided repair template and thereby incorporate the new sequence into the genome. Once incorporated, this new sequence is now part of the cell's genetic material and passes into its daughter cells.
Many online tools are available to aid in designing effective sgRNA sequences.
Scientists can use viral or non-viral systems for delivery of the Cas9 and sgRNA into target cells. Electroporation of DNA, RNA or ribonucleocomplexes is the most common and cheapest system. This technique was used to edit CXCR4 and PD-1, knocking in new sequences to replace specific genetic "letters" in these proteins. The group was then able to sort the cells, using cell surface markers, to help identify successfully edited cells. Deep sequencing of a target site confirmed that knock-in genome modifications had occurred with up to ∼20% efficiency, which accounted for up to approximately one-third of total editing events. However, hard-to-transfect cells (stem cells, neurons, hematopoietic cells, etc.) require more efficient delivery systems such as those based on lentivirus (LVs), adenovirus (AdV) and adeno-associated virus (AAV).
CRISPRs have been used to cut five to 62 genes at once: pig cells have been engineered to inactivate all 62 Porcine Endogenous Retroviruses in the pig genome, which eliminated transinfection from the pig to human cells in culture. CRISPR's low cost compared to alternatives is widely seen as revolutionary.
- Immunization of industrially important bacteria, including some used in food production and large-scale fermentation
- Cellular or organism RNA-guided genome engineering. Proof of concept studies demonstrated examples both in vitro and in vivo
- Bacterial strain discrimination by comparison of spacer sequences
Controlled genome editing
Several variants of CRISPR/Cas9 allow gene activation or genome editing with an external trigger such as light or small molecules. These include photoactivatable CRISPR systems developed by fusing light-responsive protein partners with an activator domain and a dCas9 for gene activation, or fusing similar light responsive domains with two constructs of split-Cas9, or by incorporating caged unnatural amino acids into Cas9, or by modifying the guide RNAs with photocleavable complements for genome editing.
Methods to control genome editing with small molecules include an allosteric Cas9, with no detectable background editing, that will activate binding and cleavage upon the addition of 4-hydroxytamoxifen (4-HT), 4-HT responsive intein-linked Cas9s or a Cas9 that is 4-HT responsive when fused to four ERT2 domains. Intein-inducible split-Cas9 allows dimerization of Cas9 fragments and Rapamycin-inducible split-Cas9 system developed by fusing two constructs of split Cas9 with FRB and FKBP fragments. Furthermore, other studies have shown to induce transcription of Cas9 with a small molecule, doxycyline. Small molecules can also be used to improve Homology Directed Repair (HDR), often by inhibiting the Non-Homologous End Joining (NHEJ) pathway. These systems allow conditional control of CRISPR activity for improved precision, efficiency and spatiotemporal control.
Using "dead" versions of Cas9 (dCas9) eliminates CRISPR's DNA-cutting ability, while preserving its ability to target desirable sequences. Multiple groups added various regulatory factors to dCas9s, enabling them to turn almost any gene on or off or adjust its level of activity. Like RNAi, CRISPR interference (CRISPRi) turns off genes in a reversible fashion by targeting, but not cutting a site. The targeted site is methylated, epigenetically modifying the gene. This modification inhibits transcription. Cas9 is an effective way of targeting and silencing specific genes at the DNA level. In bacteria, the presence of Cas9 alone is enough to block transcription. For mammalian applications, a section of protein is added. Its guide RNA targets regulatory DNA sequences called promoters that immediately precede the target gene.
Cas9 was used to carry synthetic transcription factors that activated specific human genes. The technique achieved a strong effect by targeting multiple CRISPR constructs to slightly different locations on the gene's promoter.
In 2016 researchers demonstrated that CRISPR from an ordinary mouth bacterium could be used to edit RNA. The researchers searched databases containing hundreds of millions of genetic sequences for those that resembled Crispr genes. They considered the fusobacteria Leptotrichia shahii. It had a group of genes that resembled CRISPR genes, but with important differences. When the researchers equipped other bacteria with these genes, which they called C2c2, they found that the organisms gained a novel defense.
Many viruses encode their genetic information in RNA rather than DNA that they repurpose to make new viruses. HIV and poliovirus are such viruses. Bacteria with C2c2 make molecules that can dismember RNA, destroying the virus. Tailoring these genes opened any RNA molecule to editing.
CRISPR simplifies creation of animals for research that mimic disease or show what happens when a gene is knocked down or mutated. CRISPR may be used at the germline level to create animals where the gene is changed everywhere, or it may be targeted at non-germline cells.
CRISPR can be utilized to create human cellular models of disease. For instance, applied to human pluripotent stem cells CRISPR introduced targeted mutations in genes relevant to polycystic kidney disease (PKD) and focal segmental glomerulosclerosis (FSG). These CRISPR-modified pluripotent stem cells were subsequently grown into human kidney organoids that exhibited disease-specific phenotypes. Kidney organoids from stem cells with PKD populations formed large, translucent cyst structures from kidney tubules. Kidney organoids with mutations in a gene linked to FSG developed junctional defects between podocytes, the filtering cells affected in that disease. Importantly, these disease phenotypes were absent in control organoids of identical genetic background, but lacking the CRISPR modifications.
A similar approach was taken to model long QT syndrome in cardiomyocytes derived from pluripotent stem cells. These CRISPR-generated cellular models, with isogenic controls, provide a new way to study human disease and test drugs.
In 2003 evolutionary biologist Austin Burt envisioned attaching a gene that coded for a desired trait to "selfish" DNA elements that could copy themselves from one chromosome position to another. That would bias daughter cells to inherit it, quickly spreading it throughout a population. In 2015 a U.S. team used CRISPR to create a "mutagenic chain reaction" that drove a pigmentation trait in lab-grown Drosophila to the next generation with 97% efficiency. With another research group they created a gene drive in mosquitoes that spread genes that prevented the insects from harboring malaria parasites. Weeks later, the team reported a second drive with genes that rendered female mosquitoes infertile and could quickly wipe out a population.
CRISPR/Cas-based "RNA-guided nucleases" can be used to target virulence factors, genes encoding antibiotic resistance and other medically relevant sequences of interest. This technology thus represents a novel form of antimicrobial therapy and a strategy by which to manipulate bacterial populations. Some of the affected genes are tied to human diseases, including those involved in muscle differentiation, cancer, inflammation and fetal hemoglobin.
Research suggests that CRISPR is an effective way to limit replication of multiple herpesviruses. It was able to eradicate viral DNA in the case of Epstein-Barr virus (EBV). Anti-herpesvirus CRISPRs have promising applications such as removing cancer-causing EBV from tumor cells, helping rid donated organs for immunocompromised patients of viral invaders, or preventing cold sore outbreaks and recurrent eye infections by blocking HSV-1 reactivation. As of August 2016, these were awaiting testing. CRISPR is being appied to develop tissue-based treatments for cancer and other diseases.
CRISPR may revive the concept of transplanting animal organs into people. Retroviruses present in animal genomes could harm transplant recipients. In 2015 a team eliminated 62 copies of a retrovirus's DNA from the pig genome.
CRISPR may have applications in tissue engineering and regenerative medicine, such as by creating human blood vessels that lack expression of MHC class II proteins, which often cause transplant rejection.
In 2015, multiple studies attempted to systematically disable each individual human gene, in an attempt to identify which genes were essential to human biology. Between 1,600 and 1,800 genes passed this test—of the 20,000 or so known human genes. Such genes are more strongly activated, and unlikely to carry disabling mutations. They are more likely to have indispensable counterparts in other species. They build proteins that unite to form larger collaborative complexes. The studies also catalogued the essential genes in four cancer-cell lines and identified genes that are expendable in healthy cells, but crucial in specific tumor types and drugs that could target these rogue genes.
The specific functions of some 18 percent of the essential genes are unidentified. In one 2015 targeting experiment, disabling individual genes in groups of cells attempted to identify those involved in resistance to a melanoma drug. Each such gene manipulation is itself a separate "drug", potentially opening the entire genome to CRISPR-based regulation.
In vitro genetic depletion
Unenriched sequencing libraries often have abundant undesired sequences. Cas9 can specifically deplete the undesired sequences with double strand breakage with up to 99% efficiency and without significant off-target effects as seen with restriction enzymes. Treatment with Cas9 can deplete abundant rRNA while increasing pathogen sensitivity in RNA-seq libraries.
Patents and commercialization
As of December 2014, patent rights to CRISPR were contested. Several companies formed to develop related drugs and research tools. As companies ramp up financing, doubts as to whether CRISPR can be quickly monetized were raised. In February 2017 the US patent office ruled on a patent interference case brought by University of California with respect to patents issued to the Broad Institute, and found that the Broad patents, with claims covering the application of CRISPR/cas9 in eukaryotic cells, were distinct from the inventions claimed by University of California.
As of November 2013, SAGE Labs (now part of Horizon Discovery group) had exclusive rights from one of those companies to produce and sell genetically engineered rats and non-exclusive rights for mouse and rabbit models. By 2015[update], Thermo Fisher Scientific had licensed intellectual property from ToolGen to develop CRISPR reagent kits.
Society and culture
Human germline modification
At least four labs in the US, labs in China and the UK, and a US biotechnology company called Ovascience announced plans or ongoing research to apply CRISPR to human embryos. Scientists, including a CRISPR co-inventor, urged a worldwide moratorium on applying CRISPR to the human germline, especially for clinical use. They said "scientists should avoid even attempting, in lax jurisdictions, germline genome modification for clinical application in humans" until the full implications "are discussed among scientific and governmental organizations". These scientists support basic research on CRISPR and do not see CRISPR as developed enough for any clinical use in making heritable changes to humans.
In April 2015, Chinese scientists reported results of an attempt to alter the DNA of non-viable human embryos using CRISPR to correct a mutation that causes beta thalassemia, a lethal heritable disorder. The study had previously been rejected by both Nature and Science in part because of ethical concerns. The experiments resulted in changing only some genes, and had off-target effects on other genes. The researchers stated that CRISPR is not ready for clinical application in reproductive medicine. In April 2016 Chinese scientists were reported to have made a second unsuccessful attempt to alter the DNA of non-viable human embryos using CRISPR - this time to alter the CCR5 gene to make the embryo HIV resistant.
In December 2015, an International Summit on Human Gene Editing took place in Washington under the guidance of David Baltimore. Members of national scientific academies of America, Britain and China discussed the ethics of germline modification. They agreed to support basic and clinical research under appropriate legal and ethical guidelines. A specific distinction was made between somatic cells, where the effects of edits are limited to a single individual, versus germline cells, where genome changes could be inherited by future generations. Heritable modifications could have unintended and far-reaching consequences for human evolution, genetically (e.g. gene/environment interactions) and culturally (e.g. Social Darwinism). Altering of gametocytes and embryos to generate inheritable changes in humans was defined to be irresponsible. The group agreed to initiate an international forum to address such concerns and harmonize regulations across countries.
Policy barriers to genetic engineering
Policy regulations for the CRISPR/cas9 system vary around the globe. In February 2016, British scientists were given permission by regulators to genetically modify human embryos by using CRISPR-Cas9 and related techniques. However, researchers were forbidden from implanting the embryos and the embryos were to be destroyed after seven days.
The US has an elaborate, interdepartmental regulatory system to evaluate new genetically modified foods and crops. For example, the Agriculture Risk Protection Act of 2000 gives the USDA the authority to oversee the detection, control, eradication, suppression, prevention, or retardation of the spread of plant pests or noxious weeds to protect the agriculture, environment and economy of the US. The act regulates any genetically modified organism that utilizes the genome of a predefined 'plant pest' or any plant not previously categorized. In 2015, Yang successfully deactivated 16 specific genes in the white button mushroom. Since he had not added any foreign DNA to his organism, the mushroom could not be regulated under by the USDA under Section 340.2. Yang's white button mushroom was the first organism genetically modified with the Crispr/cas9 protein system to pass US regulation. In 2016, the USDA sponsored a committee to consider future regulatory policy for upcoming genetic modification techniques. With the help of the US National Academies of Sciences, Engineering and Medicine, special interests groups met on April 15 to contemplate the possible advancements in genetic engineering within the next 5 years and potential policy regulations that would need to come into play. With the emergence of rogue genetic engineers employing the technology, the FDA has begun issuing new regulations.
In 2012 and 2013, CRISPR was a runner-up in Science Magazine's Breakthrough of the Year award. In 2015, it was the winner of that award. CRISPR was named as one of MIT Technology Review's 10 breakthroughs technologies in 2014 and 2016.
- Horvath P, Barrangou R (January 2010). "CRISPR/Cas, the immune system of bacteria and archaea". Science. 327 (5962): 167–70. Bibcode:2010Sci...327..167H. PMID 20056882. doi:10.1126/Science.1179555.
- Sawyer E (9 February 2013). "Editing Genomes with the Bacterial Immune System". Scitable. Nature Publishing Group. Retrieved 6 April 2015.
- Barrangou R (2015). "The roles of CRISPR-Cas systems in adaptive immunity and beyond". Current Opinion in Immunology. 32: 36–41. PMID 25574773. doi:10.1016/j.coi.2014.12.008.
- Zhang F, Wen Y, Guo X (2014). "CRISPR/Cas9 for genome editing: progress, implications and challenges". Human Molecular Genetics. 23 (R1): R40–6. PMID 24651067. doi:10.1093/hmg/ddu125.
- Marraffini LA, Sontheimer EJ (March 2010). "CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea". Nature Reviews Genetics. 11 (3): 181–90. PMC . PMID 20125085. doi:10.1038/nrg2749.
- Redman M, King A, Watson C, King D (August 2016). "What is CRISPR/Cas9?". Archives of Disease in Childhood. Education and Practice Edition. 101 (4): 213–5. PMID 27059283. doi:10.1136/archdischild-2016-310459.
- Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, Romero DA, Horvath P (March 2007). "CRISPR provides acquired resistance against viruses in prokaryotes". Science. 315 (5819): 1709–12. Bibcode:2007Sci...315.1709B. PMID 17379808. doi:10.1126/science.1138140. (registration required)
- Marraffini LA, Sontheimer EJ (December 2008). "CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA". Science. 322 (5909): 1843–5. Bibcode:2008Sci...322.1843M. PMC . PMID 19095942. doi:10.1126/science.1165771.
- Mohanraju P, Makarova KS, Zetsche B, Zhang F, Koonin EV, van der Oost J (2016). "Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems". Science. 353 (6299): aad5147. PMID 27493190. doi:10.1126/science.aad5147.
- Grissa I, Vergnaud G, Pourcel C (May 2007). "The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats". BMC Bioinformatics. 8: 172. PMC . PMID 17521438. doi:10.1186/1471-2105-8-172.
- Ledford, Heidi (3 June 2015). "CRISPR, the disruptor". News Feature. Nature. 522 (7554).
- Snyder B (21 August 2014). "New technique accelerates genome editing process". research news @ Vanderbilt. Nashville, Tennessee: Vanderbilt University.
- Hendel A, Bak RO, Clark JT, Kennedy AB, Ryan DE, Roy S, Steinfeld I, Lunstad BD, Kaiser RJ, Wilkens AB, Bacchetta R, Tsalenko A, Dellinger D, Bruhn L, Porteus MH (September 2015). "Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells". Nature Biotechnology. 33 (9): 985–9. PMC . PMID 26121415. doi:10.1038/nbt.3290.
- Ledford H (March 2016). "CRISPR: gene editing is just the beginning". Nature. 531 (7593): 156–9. PMID 26961639. doi:10.1038/531156a.
- Maxmen A (August 2015). "The Genesis Engine". WIRED. Retrieved 2016-06-05.
- Travis J (17 December 2015). "Breakthrough of the Year: CRISPR makes the cut". Science Magazine. American Association for the Advancement of Science.
- Ledford H (June 2015). "CRISPR, the disruptor". Nature. 522 (7554): 20–4. PMID 26040877. doi:10.1038/522020a.
- Ishino Y, Shinagawa H, Makino K, Amemura M, Nakata A (December 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): 5429–33. PMC . PMID 3316184.
- Hsu PD, Lander ES, Zhang F (June 2014). "Development and applications of CRISPR-Cas9 for genome engineering". Cell. 157 (6): 1262–78. PMC . PMID 24906146. doi:10.1016/j.cell.2014.05.010.
- van Soolingen D, de Haas PE, Hermans PW, Groenen PM, van Embden JD (August 1993). "Comparison of various repetitive DNA elements as genetic markers for strain differentiation and epidemiology of Mycobacterium tuberculosis". Journal of Clinical Microbiology. 31 (8): 1987–95. PMC . PMID 7690367.
- Groenen PM, Bunschoten AE, van Soolingen D, van Embden JD (December 1993). "Nature of DNA polymorphism in the direct repeat cluster of Mycobacterium tuberculosis; application for strain differentiation by a novel typing method". Molecular Microbiology. 10 (5): 1057–65. PMID 7934856. doi:10.1111/j.1365-2958.1993.tb00976.x.
- Mojica FJ, Montoliu L (2016). "On the Origin of CRISPR-Cas Technology: From Prokaryotes to Mammals". Trends in Microbiology. 24 (10): 811–20. PMID 27401123. doi:10.1016/j.tim.2016.06.005.
- Mojica FJ, Rodriguez-Valera F (2016). "The discovery of CRISPR in archaea and bacteria". The FEBS Journal. 283 (17): 3162–9. PMID 27234458. doi:10.1111/febs.13766.
- Mojica FJ, Díez-Villaseñor C, Soria E, Juez G (April 2000). "Biological significance of a family of regularly spaced repeats in the genomes of Archaea, Bacteria and mitochondria". Molecular Microbiology. 36 (1): 244–6. PMID 10760181. doi: .
- Barrangou R, van der Oost J (2013). CRISPR-Cas Systems : RNA-mediated Adaptive Immunity in Bacteria and Archaea. Heidelberg: Springer. p. 6. ISBN 978-3-642-34656-9.
- Jansen R, Embden JD, Gaastra W, Schouls LM (March 2002). "Identification of genes that are associated with DNA repeats in prokaryotes". Molecular Microbiology. 43 (6): 1565–75. PMID 11952905. doi:10.1046/j.1365-2958.2002.02839.x.
- Pourcel C, Salvignol G, Vergnaud G (March 2005). "CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies". Microbiology. 151 (Pt 3): 653–63. PMID 15758212. doi:10.1099/mic.0.27437-0.
- Mojica FJ, Díez-Villaseñor C, García-Martínez J, Soria E (February 2005). "Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements". Journal of Molecular Evolution. 60 (2): 174–82. PMID 15791728. doi:10.1007/s00239-004-0046-3.
- Bolotin A, Quinquis B, Sorokin A, Ehrlich SD (August 2005). "Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin". Microbiology. 151 (Pt 8): 2551–61. PMID 16079334. doi: .
- Morange M (June 2015). "What history tells us XXXVII. CRISPR-Cas: The discovery of an immune system in prokaryotes". Journal of Biosciences. 40 (2): 221–3. PMID 25963251. doi:10.1007/s12038-015-9532-6.
- Lander ES (January 2016). "The Heroes of CRISPR". Cell. 164 (1–2): 18–28. PMID 26771483. doi:10.1016/j.cell.2015.12.041.
- Makarova KS, Grishin NV, Shabalina SA, Wolf YI, Koonin EV (March 2006). "A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action". Biology Direct. 1: 7. PMC . PMID 16545108. doi:10.1186/1745-6150-1-7.
- Marraffini LA (October 2015). "CRISPR-Cas immunity in prokaryotes". Nature. 526 (7571): 55–61. PMID 26432244. doi:10.1038/nature15386.
- Pennisi E (August 2013). "The CRISPR craze". News Focus. Science. 341 (6148): 833–6. PMID 23970676. doi:10.1126/science.341.6148.833.
- Garneau JE, Dupuis MÈ, Villion M, Romero DA, Barrangou R, Boyaval P, Fremaux C, Horvath P, Magadán AH, Moineau S (November 2010). "The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA". Nature. 468 (7320): 67–71. PMID 21048762. doi:10.1038/nature09523.
- Barrangou R (November 2015). "Diversity of CRISPR-Cas immune systems and molecular machines". Genome Biology. 16: 247. PMC . PMID 26549499. doi:10.1186/s13059-015-0816-9.
- Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (August 2012). "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity". Science. 337 (6096): 816–21. Bibcode:2012Sci...337..816J. PMID 22745249. doi:10.1126/science.1225829.
- Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F (February 2013). "Multiplex genome engineering using CRISPR/Cas systems". Science. 339 (6121): 819–23. PMC . PMID 23287718. doi:10.1126/science.1231143.
- Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM (February 2013). "RNA-guided human genome engineering via Cas9". Science. 339 (6121): 823–6. PMC . PMID 23287722. doi:10.1126/science.1232033.
- DiCarlo JE, Norville JE, Mali P, Rios X, Aach J, Church GM (April 2013). "Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems". Nucleic Acids Research. 41 (7): 4336–43. PMC . PMID 23460208. doi:10.1093/nar/gkt135.
- Zhang GC, Kong II, Kim H, Liu JJ, Cate JH, Jin YS (December 2014). "Construction of a quadruple auxotrophic mutant of an industrial polyploid saccharomyces cerevisiae strain by using RNA-guided Cas9 nuclease". Applied and Environmental Microbiology. 80 (24): 7694–701. PMC . PMID 25281382. doi:10.1128/AEM.02310-14.
- Liu JJ, Kong II, Zhang GC, Jayakody LN, Kim H, Xia PF, Kwak S, Sung BH, Sohn JH, Walukiewicz HE, Rao CV, Jin YS (April 2016). "Metabolic Engineering of Probiotic Saccharomyces boulardii". Applied and Environmental Microbiology. 82 (8): 2280–7. PMC . PMID 26850302. doi:10.1128/AEM.00057-16.
- Hwang WY, Fu Y, Reyon D, Maeder ML, Tsai SQ, Sander JD, Peterson RT, Yeh JR, Joung JK (March 2013). "Efficient genome editing in zebrafish using a CRISPR-Cas system". Nature Biotechnology. 31 (3): 227–9. PMC . PMID 23360964. doi:10.1038/nbt.2501.
- Gratz SJ, Cummings AM, Nguyen JN, Hamm DC, Donohue LK, Harrison MM, Wildonger J, O'Connor-Giles KM (August 2013). "Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease". Genetics. 194 (4): 1029–35. PMC . PMID 23709638. doi:10.1534/genetics.113.152710.
- Friedland AE, Tzur YB, Esvelt KM, Colaiácovo MP, Church GM, Calarco JA (August 2013). "Heritable genome editing in C. elegans via a CRISPR-Cas9 system". Nature Methods. 10 (8): 741–3. PMC . PMID 23817069. doi:10.1038/nmeth.2532.
- Jiang W, Zhou H, Bi H, Fromm M, Yang B, Weeks DP (November 2013). "Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice". Nucleic Acids Research. 41 (20): e188. PMC . PMID 23999092. doi:10.1093/nar/gkt780.
- Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch R (May 2013). "One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering". Cell. 153 (4): 910–8. PMC . PMID 23643243. doi:10.1016/j.cell.2013.04.025.
- Guo X, Li XJ (July 2015). "Targeted genome editing in primate embryos". Cell Research. 25 (7): 767–8. PMC . PMID 26032266. doi:10.1038/cr.2015.64.
- Baltimore D, Berg P, Botchan M, Carroll D, Charo RA, Church G, Corn JE, Daley GQ, Doudna JA, Fenner M, Greely HT, Jinek M, Martin GS, Penhoet E, Puck J, Sternberg SH, Weissman JS, Yamamoto KR (April 2015). "Biotechnology. A prudent path forward for genomic engineering and germline gene modification". Science. 348 (6230): 36–8. Bibcode:2015Sci...348...36B. PMC . PMID 25791083. doi:10.1126/science.aab1028.
- Larson MH, Gilbert LA, Wang X, Lim WA, Weissman JS, Qi LS (November 2013). "CRISPR interference (CRISPRi) for sequence-specific control of gene expression". Nature Protocols. 8 (11): 2180–96. PMC . PMID 24136345. doi:10.1038/nprot.2013.132.
- Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS, Essletzbichler P, Volz SE, Joung J, van der Oost J, Regev A, Koonin EV, Zhang F (October 2015). "Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system". Cell. 163 (3): 759–71. PMID 26422227. doi:10.1016/j.cell.2015.09.038.
- Fonfara I, Richter H, Bratovič M, Le Rhun A, Charpentier E (April 2016). "The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA". Nature. 532 (7600): 517–21. PMID 27096362. doi:10.1038/nature17945.
- Young S (11 February 2014). "CRISPR and Other Genome Editing Tools Boost Medical Research and Gene Therapy's Reach". MIT Technology Review. Cambridge, Massachusetts: Massachusetts Institute of Technology. Retrieved 2014-04-13.
- Hille F, Charpentier E (November 2016). "CRISPR-Cas: biology, mechanisms and relevance". Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences. 371 (1707): 20150496. PMC . PMID 27672148. doi:10.1098/rstb.2015.0496.
- Barrangou R, Marraffini LA (April 2014). "CRISPR-Cas systems: Prokaryotes upgrade to adaptive immunity". Molecular Cell. 54 (2): 234–44. PMC . PMID 24766887. doi:10.1016/j.molcel.2014.03.011.
- Tyson GW, Banfield JF (January 2008). "Rapidly evolving CRISPRs implicated in acquired resistance of microorganisms to viruses". Environmental Microbiology. 10 (1): 200–7. PMID 17894817. doi:10.1111/j.1462-2920.2007.01444.x.
- Makarova KS, Wolf YI, Alkhnbashi OS, Costa F, Shah SA, Saunders SJ, Barrangou R, Brouns SJ, Charpentier E, Haft DH, Horvath P, Moineau S, Mojica FJ, Terns RM, Terns MP, White MF, Yakunin AF, Garrett RA, van der Oost J, Backofen R, Koonin EV (November 2015). "An updated evolutionary classification of CRISPR-Cas systems". Nature Reviews. Microbiology. 13 (11): 722–36. PMID 26411297. doi:10.1038/nrmicro3569.
- Wright AV, Nuñez JK, Doudna JA (January 2016). "Biology and Applications of CRISPR Systems: Harnessing Nature's Toolbox for Genome Engineering". Cell. 164 (1–2): 29–44. PMID 26771484. doi:10.1016/j.cell.2015.12.035.
- Westra ER, Dowling AJ, Broniewski JM, van Houte S (November 2016). "Evolution and Ecology of CRISPR". Annual Review of Ecology, Evolution, and Systematics. 47 (1): 307–331. doi:10.1146/annurev-ecolsys-121415-032428.
- Wiedenheft B, Sternberg SH, Doudna JA (February 2012). "RNA-guided genetic silencing systems in bacteria and archaea". Nature. 482 (7385): 331–8. Bibcode:2012Natur.482..331W. PMID 22337052. doi:10.1038/nature10886.
- Deng L, Garrett RA, Shah SA, Peng X, She Q (March 2013). "A novel interference mechanism by a type IIIB CRISPR-Cmr module in Sulfolobus". Molecular Microbiology. 87 (5): 1088–99. PMID 23320564. doi: .
- Sinkunas T, Gasiunas G, Fremaux C, Barrangou R, Horvath P, Siksnys V (April 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. PMC . PMID 21343909. doi:10.1038/emboj.2011.41.
- Huo Y, Nam KH, Ding F, Lee H, Wu L, Xiao Y, Farchione MD, Zhou S, Rajashankar K, Kurinov I, Zhang R, Ke A (September 2014). "Structures of CRISPR Cas3 offer mechanistic insights into Cascade-activated DNA unwinding and degradation". Nature Structural & Molecular Biology. 21 (9): 771–7. PMC . PMID 25132177. doi:10.1038/nsmb.2875.
- Chylinski K, Makarova KS, Charpentier E, Koonin EV (June 2014). "Classification and evolution of type II CRISPR-Cas systems". Nucleic Acids Research. 42 (10): 6091–105. PMC . PMID 24728998. doi:10.1093/nar/gku241.
- Makarova KS, Aravind L, Wolf YI, Koonin EV (July 2011). "Unification of Cas protein families and a simple scenario for the origin and evolution of CRISPR-Cas systems". Biology Direct. 6: 38. PMC . PMID 21756346. doi:10.1186/1745-6150-6-38.
- Gasiunas G, Barrangou R, Horvath P, Siksnys V (September 2012). "Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria". Proceedings of the National Academy of Sciences of the United States of America. 109 (39): E2579–86. Bibcode:2012PNAS..109E2579G. PMC . PMID 22949671. doi:10.1073/pnas.1208507109.
- Heler R, Samai P, Modell JW, Weiner C, Goldberg GW, Bikard D, Marraffini LA (March 2015). "Cas9 specifies functional viral targets during CRISPR-Cas adaptation". Nature. 519 (7542): 199–202. Bibcode:2015Natur.519..199H. PMC . PMID 25707807. doi:10.1038/nature14245.
- Nam KH, Kurinov I, Ke A (September 2011). "Crystal structure of clustered regularly interspaced short palindromic repeats (CRISPR)-associated Csn2 protein revealed Ca2+-dependent double-stranded DNA binding activity". The Journal of Biological Chemistry. 286 (35): 30759–68. PMC . PMID 21697083. doi:10.1074/jbc.M111.256263.
- Chylinski K, Le Rhun A, Charpentier E (May 2013). "The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems". RNA Biology. 10 (5): 726–37. PMC . PMID 23563642. doi:10.4161/rna.24321.
- Aliyari R, Ding SW (January 2009). "RNA-based viral immunity initiated by the Dicer family of host immune receptors". Immunological Reviews. 227 (1): 176–88. PMC . PMID 19120484. doi:10.1111/j.1600-065X.2008.00722.x.
- Dugar G, Herbig A, Förstner KU, Heidrich N, Reinhardt R, Nieselt K, Sharma CM (May 2013). "High-resolution transcriptome maps reveal strain-specific regulatory features of multiple Campylobacter jejuni isolates". PLoS Genetics. 9 (5): e1003495. PMC . PMID 23696746. doi:10.1371/journal.pgen.1003495.
- Hatoum-Aslan A, Maniv I, Marraffini LA (December 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. Bibcode:2011PNAS..10821218H. PMC . PMID 22160698. doi:10.1073/pnas.1112832108.
- Yosef I, Goren MG, Qimron U (July 2012). "Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli". Nucleic Acids Research. 40 (12): 5569–76. PMC . PMID 22402487. doi:10.1093/nar/gks216.
- Swarts DC, Mosterd C, van Passel MW, Brouns SJ (2012). "CRISPR interference directs strand specific spacer acquisition". PloS One. 7 (4): e35888. Bibcode:2012PLoSO...735888S. PMC . PMID 22558257. doi:10.1371/journal.pone.0035888.
- 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 EV, Savchenko A, Emili A, Greenblatt J, Edwards AM, Yakunin AF (January 2011). "A dual function of the CRISPR-Cas system in bacterial antivirus immunity and DNA repair". Molecular Microbiology. 79 (2): 484–502. PMC . PMID 21219465. doi:10.1111/j.1365-2958.2010.07465.x.
- Han D, Lehmann K, Krauss G (June 2009). "SSO1450--a CAS1 protein from Sulfolobus solfataricus P2 with high affinity for RNA and DNA". FEBS Letters. 583 (12): 1928–32. PMID 19427858. doi:10.1016/j.febslet.2009.04.047.
- Wiedenheft B, Zhou K, Jinek M, Coyle SM, Ma W, Doudna JA (June 2009). "Structural basis for DNase activity of a conserved protein implicated in CRISPR-mediated genome defense". Structure. 17 (6): 904–12. PMID 19523907. doi:10.1016/j.str.2009.03.019.
- Beloglazova N, Brown G, Zimmerman MD, Proudfoot M, Makarova KS, Kudritska M, Kochinyan S, Wang S, Chruszcz M, Minor W, Koonin EV, Edwards AM, Savchenko A, Yakunin AF (July 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. PMC . PMID 18482976. doi:10.1074/jbc.M803225200.
- Samai P, Smith P, Shuman S (December 2010). "Structure of a CRISPR-associated protein Cas2 from Desulfovibrio vulgaris". Acta Crystallographica Section F. 66 (Pt 12): 1552–6. PMC . PMID 21139194. doi:10.1107/S1744309110039801.
- Nam KH, Ding F, Haitjema C, Huang Q, DeLisa MP, Ke A (October 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. PMC . PMID 22942283. doi:10.1074/jbc.M112.382598.
- Nuñez JK, Kranzusch PJ, Noeske J, Wright AV, Davies CW, Doudna JA (June 2014). "Cas1-Cas2 complex formation mediates spacer acquisition during CRISPR-Cas adaptive immunity". Nature Structural & Molecular Biology. 21 (6): 528–34. PMC . PMID 24793649. doi:10.1038/nsmb.2820.
- Nuñez JK, Lee AS, Engelman A, Doudna JA (March 2015). "Integrase-mediated spacer acquisition during CRISPR-Cas adaptive immunity". Nature. 519 (7542): 193–8. PMC . PMID 25707795. doi:10.1038/nature14237.
- Wang J, Li J, Zhao H, Sheng G, Wang M, Yin M, Wang Y (November 2015). "Structural and Mechanistic Basis of PAM-Dependent Spacer Acquisition in CRISPR-Cas Systems". Cell. 163 (4): 840–53. PMID 26478180. doi:10.1016/j.cell.2015.10.008.
- Nuñez JK, Harrington LB, Kranzusch PJ, Engelman AN, Doudna JA (November 2015). "Foreign DNA capture during CRISPR-Cas adaptive immunity". Nature. 527 (7579): 535–8. PMID 26503043. doi:10.1038/nature15760.
- Horvath P, Romero DA, Coûté-Monvoisin AC, Richards M, Deveau H, Moineau S, Boyaval P, Fremaux C, Barrangou R (February 2008). "Diversity, activity, and evolution of CRISPR loci in Streptococcus thermophilus". Journal of Bacteriology. 190 (4): 1401–12. PMC . PMID 18065539. doi:10.1128/JB.01415-07.
- Deveau H, Barrangou R, Garneau JE, Labonté J, Fremaux C, Boyaval P, Romero DA, Horvath P, Moineau S (February 2008). "Phage response to CRISPR-encoded resistance in Streptococcus thermophilus". Journal of Bacteriology. 190 (4): 1390–400. PMC . PMID 18065545. doi:10.1128/JB.01412-07.
- Mojica FJ, Díez-Villaseñor C, García-Martínez J, Almendros C (March 2009). "Short motif sequences determine the targets of the prokaryotic CRISPR defence system". Microbiology. 155 (Pt 3): 733–40. PMID 19246744. doi:10.1099/mic.0.023960-0.
- Lillestøl RK, Shah SA, Brügger K, Redder P, Phan H, Christiansen J, Garrett RA (April 2009). "CRISPR families of the crenarchaeal genus Sulfolobus: bidirectional transcription and dynamic properties". Molecular Microbiology. 72 (1): 259–72. PMID 19239620. doi:10.1111/j.1365-2958.2009.06641.x.
- Shah SA, Hansen NR, Garrett RA (February 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): 23–8. PMID 19143596. doi:10.1042/BST0370023.
- Díez-Villaseñor C, Guzmán NM, Almendros C, García-Martínez J, Mojica FJ (May 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. PMC . PMID 23445770. doi:10.4161/rna.24023.
- Erdmann S, Garrett RA (September 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. PMC . PMID 22834906. doi:10.1111/j.1365-2958.2012.08171.x.
- Shah SA, Erdmann S, Mojica FJ, Garrett RA (May 2013). "Protospacer recognition motifs: mixed identities and functional diversity". RNA Biology. 10 (5): 891–9. PMC . PMID 23403393. doi:10.4161/rna.23764.
- Andersson AF, Banfield JF (May 2008). "Virus population dynamics and acquired virus resistance in natural microbial communities". Science. 320 (5879): 1047–50. Bibcode:2008Sci...320.1047A. PMID 18497291. doi:10.1126/science.1157358.
- Pride DT, Sun CL, Salzman J, Rao N, Loomer P, Armitage GC, Banfield JF, Relman DA (January 2011). "Analysis of streptococcal CRISPRs from human saliva reveals substantial sequence diversity within and between subjects over time". Genome Research. 21 (1): 126–36. PMC . PMID 21149389. doi:10.1101/gr.111732.110.
- Goren MG, Yosef I, Auster O, Qimron U (October 2012). "Experimental definition of a clustered regularly interspaced short palindromic duplicon in Escherichia coli". Journal of Molecular Biology. 423 (1): 14–6. PMID 22771574. doi:10.1016/j.jmb.2012.06.037.
- Datsenko KA, Pougach K, Tikhonov A, Wanner BL, Severinov K, Semenova E (July 2012). "Molecular memory of prior infections activates the CRISPR/Cas adaptive bacterial immunity system". Nature Communications. 3: 945. Bibcode:2012NatCo...3E.945D. PMID 22781758. doi:10.1038/ncomms1937.
- Gesner EM, Schellenberg MJ, Garside EL, George MM, Macmillan AM (June 2011). "Recognition and maturation of effector RNAs in a CRISPR interference pathway". Nature Structural & Molecular Biology. 18 (6): 688–92. PMID 21572444. doi:10.1038/nsmb.2042.
- Sashital DG, Jinek M, Doudna JA (June 2011). "An RNA-induced conformational change required for CRISPR RNA cleavage by the endoribonuclease Cse3". Nature Structural & Molecular Biology. 18 (6): 680–7. PMID 21572442. doi:10.1038/nsmb.2043.
- 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): 1355–8. Bibcode:2010Sci...329.1355H. PMC . PMID 20829488. doi:10.1126/science.1192272.
- Kunin V, Sorek R, Hugenholtz P (2007). "Evolutionary conservation of sequence and secondary structures in CRISPR repeats". Genome Biology. 8 (4): R61. PMC . PMID 17442114. doi:10.1186/gb-2007-8-4-r61.
- Carte J, Wang R, Li H, Terns RM, Terns MP (December 2008). "Cas6 is an endoribonuclease that generates guide RNAs for invader defense in prokaryotes". Genes & Development. 22 (24): 3489–96. PMC . PMID 19141480. doi:10.1101/gad.1742908.
- 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): 257–64. PMC . PMID 21300293. doi:10.1016/j.str.2010.11.014.
- Niewoehner O, Jinek M, Doudna JA (January 2014). "Evolution of CRISPR RNA recognition and processing by Cas6 endonucleases". Nucleic Acids Research. 42 (2): 1341–53. PMC . PMID 24150936. doi:10.1093/nar/gkt922.
- Semenova E, Jore MM, Datsenko KA, Semenova A, Westra ER, Wanner B, van der Oost J, Brouns SJ, Severinov K (June 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. Bibcode:2011PNAS..10810098S. PMC . PMID 21646539. doi:10.1073/pnas.1104144108.
- 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): 35–49. PMC . PMID 21166892. doi:10.1111/j.1365-2958.2010.07452.x.
- 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): 481–91. PMID 21385233. doi:10.1111/j.1365-2958.2011.07586.x.
- Jore MM, Lundgren M, van Duijn E, Bultema JB, Westra ER, Waghmare SP, Wiedenheft B, Pul U, Wurm R, Wagner R, Beijer MR, Barendregt A, Zhou K, Snijders AP, Dickman MJ, Doudna JA, Boekema EJ, Heck AJ, van der Oost J, Brouns SJ (May 2011). "Structural basis for CRISPR RNA-guided DNA recognition by Cascade". Nature Structural & Molecular Biology. 18 (5): 529–36. PMID 21460843. doi:10.1038/nsmb.2019.
- 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): 486–9. Bibcode:2011Natur.477..486W. PMC . PMID 21938068. doi:10.1038/nature10402.
- Zhang J, Rouillon C, Kerou M, Reeks J, Brugger K, Graham S, Reimann J, Cannone G, Liu H, Albers SV, Naismith JH, Spagnolo L, White MF (February 2012). "Structure and mechanism of the CMR complex for CRISPR-mediated antiviral immunity". Molecular Cell. 45 (3): 303–13. PMC . PMID 22227115. doi:10.1016/j.molcel.2011.12.013.
- 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): 945–56. PMC . PMID 19945378. doi:10.1016/j.cell.2009.07.040.
- Marraffini LA, Sontheimer EJ (January 2010). "Self versus non-self discrimination during CRISPR RNA-directed immunity". Nature. 463 (7280): 568–71. Bibcode:2010Natur.463..568M. PMC . PMID 20072129. doi:10.1038/nature08703.
- Koonin EV, Wolf YI (November 2009). "Is evolution Darwinian or/and Lamarckian?". Biology Direct. 4: 42. PMC . PMID 19906303. doi:10.1186/1745-6150-4-42.
- Weiss A (October 2015). "Lamarckian Illusions". Trends in Ecology & Evolution. 30 (10): 566–8. PMID 26411613. doi:10.1016/j.tree.2015.08.003.
- 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. PMC . PMID 19132092. doi:10.1371/journal.pone.0004169.
- 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): 254–7. Bibcode:2013Natur.497..254S. PMC . PMID 23584588. doi:10.1038/nature12048.
- 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. PMC . PMID 20559554. doi:10.1371/journal.pone.0011126.
- Rho M, Wu YW, Tang H, Doak TG, Ye Y (2012). "Diverse CRISPRs evolving in human microbiomes". PLoS Genetics. 8 (6): e1002441. PMC . PMID 22719260. doi:10.1371/journal.pgen.1002441.
- 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): 463–70. PMID 23057534. doi:10.1111/j.1462-2920.2012.02879.x.
- 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): 903–16. PMID 24586036. doi:10.1099/mic.0.073494-0.
- Bland C, Ramsey TL, Sabree F, Lowe M, Brown K, Kyrpides NC, Hugenholtz P (June 2007). "CRISPR recognition tool (CRT): a tool for automatic detection of clustered regularly interspaced palindromic repeats". BMC Bioinformatics. 8: 209. PMC . PMID 17577412. doi:10.1186/1471-2105-8-209.
- Edgar RC (January 2007). "PILER-CR: fast and accurate identification of CRISPR repeats". BMC Bioinformatics. 8: 18. PMC . PMID 17239253. doi:10.1186/1471-2105-8-18.
- Grissa I, Vergnaud G, Pourcel C (July 2007). "CRISPRFinder: a web tool to identify clustered regularly interspaced short palindromic repeats". Nucleic Acids Research. 35 (Web Server issue): W52–7. PMC . PMID 17537822. doi:10.1093/nar/gkm360.
- Pride DT, Salzman J, Relman DA (September 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. PMC . PMID 22583485. doi:10.1111/j.1462-2920.2012.02775.x.
- Held NL, Herrera A, Whitaker RJ (November 2013). "Reassortment of CRISPR repeat-spacer loci in Sulfolobus islandicus". Environmental Microbiology. 15 (11): 3065–76. PMID 23701169. doi:10.1111/1462-2920.12146.
- 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. PMC . PMID 20927396. doi:10.1371/journal.pone.0012988.
- Skennerton CT, Imelfort M, Tyson GW (May 2013). "Crass: identification and reconstruction of CRISPR from unassembled metagenomic data". Nucleic Acids Research. 41 (10): e105. PMC . PMID 23511966. doi:10.1093/nar/gkt183.
- 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): 1985–94. PMC . PMID 22732228. doi:10.1101/gr.138297.112.
- Novick RP, Christie GE, Penadés JR (August 2010). "The phage-related chromosomal islands of Gram-positive bacteria". Nature Reviews. Microbiology. 8 (8): 541–51. PMC . PMID 20634809. doi:10.1038/nrmicro2393.
- 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): 489–91. Bibcode:2013Natur.494..489S. PMC . PMID 23446421. doi:10.1038/nature11927.
- Ledford H (June 2015). "CRISPR, the disruptor". Nature. 522 (7554): 20–4. Bibcode:2015Natur.522...20L. PMID 26040877. doi:10.1038/522020a.
- Alphey L (2016). "Can CRISPR-Cas9 gene drives curb malaria?". Nature Biotechnology. 34 (2): 149–50. PMID 26849518. doi:10.1038/nbt.3473.
- Ledford H (5 April 2017). "CRISPR studies muddy results of older gene research". Nature'.
- "CRISPR/Cas9 Plasmids". www.systembio.com. Retrieved 2015-12-17.
- "CRISPR Cas9 Genome Editing". www.origene.com. OriGene. Retrieved 2015-12-17.
- Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F (November 2013). "Genome engineering using the CRISPR-Cas9 system". Nature Protocols. 8 (11): 2281–308. PMC . PMID 24157548. doi:10.1038/nprot.2013.143.
- Ly, Joseph (2013). Discovering Genes Responsible for Kidney Diseases (Ph.D.). University of Toronto. Retrieved 26 December 2016.
- Horvath P, Barrangou R (January 2010). "CRISPR/Cas, the immune system of bacteria and archaea". Science. 327 (5962): 167–70. PMID 20056882. doi:10.1126/science.1179555.
- Bialk P, Rivera-Torres N, Strouse B, Kmiec EB (2015-06-08). "Regulation of Gene Editing Activity Directed by Single-Stranded Oligonucleotides and CRISPR/Cas9 Systems". PloS One. 10 (6): e0129308. PMC . PMID 26053390. doi:10.1371/journal.pone.0129308.
- Robert Sanders (12 November 2015). "CRISPR-Cas9 gene editing: check three times, cut once". University of California, Berkeley. Archived from the original on 26 December 2016. Retrieved 26 December 2016.
- "Optimized CRISPR Design". crispr.mit.edu. Retrieved 2015-12-20.
- "Scientists successfully edit human immune-system T cells | KurzweilAI". www.kurzweilai.net. Retrieved 2016-01-01.
- Schumann K, Lin S, Boyer E, Simeonov DR, Subramaniam M, Gate RE, Haliburton GE, Ye CJ, Bluestone JA, Doudna JA, Marson A (August 2015). "Generation of knock-in primary human T cells using Cas9 ribonucleoproteins". Proceedings of the National Academy of Sciences of the United States of America. 112 (33): 10437–42. PMC . PMID 26216948. doi:10.1073/pnas.1512503112.
- Zimmerman C (Oct 15, 2015). "Editing of Pig DNA May Lead to More Organs for People". NY Times.
- Hale CR, Majumdar S, Elmore J, Pfister N, Compton M, Olson S, Resch AM, Glover CV, Graveley BR, Terns RM, Terns MP (February 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. PMC . PMID 22227116. doi:10.1016/j.molcel.2011.10.023.
- Sorek R, Kunin V, Hugenholtz P (March 2008). "CRISPR--a widespread system that provides acquired resistance against phages in bacteria and archaea". Nature Reviews. Microbiology. 6 (3): 181–6. PMID 18157154. doi:10.1038/nrmicro1793.
- Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F (February 2013). "Multiplex genome engineering using CRISPR/Cas systems". Science. 339 (6121): 819–23. Bibcode:2013Sci...339..819C. PMC . PMID 23287718. doi:10.1126/science.1231143.
- Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM (February 2013). "RNA-guided human genome engineering via Cas9". Science. 339 (6121): 823–6. Bibcode:2013Sci...339..823M. PMC . PMID 23287722. doi:10.1126/science.1232033.
Hou Z, Zhang Y, Propson NE, Howden SE, Chu LF, Sontheimer EJ, Thomson JA (September 2013). "Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis". Proceedings of the National Academy of Sciences of the United States of America. 110 (39): 15644–9. Bibcode:2013PNAS..11015644H. PMC . PMID 23940360. doi:10.1073/pnas.1313587110.
- Oakes BL, Nadler DC, Flamholz A, Fellmann C, Staahl BT, Doudna JA, Savage DF (June 2016). "Profiling of engineering hotspots identifies an allosteric CRISPR-Cas9 switch". Nature Biotechnology. 34 (6): 646–51. PMC . PMID 27136077. doi:10.1038/nbt.3528.
- Nuñez JK, Harrington LB, Doudna JA (March 2016). "Chemical and Biophysical Modulation of Cas9 for Tunable Genome Engineering". ACS Chemical Biology. 11 (3): 681–8. PMID 26857072. doi:10.1021/acschembio.5b01019.
- Zhou W, Deiters A (April 2016). "Conditional Control of CRISPR/Cas9 Function". Angewandte Chemie. 55 (18): 5394–9. PMID 26996256. doi:10.1002/anie.201511441.
- Polstein LR, Gersbach CA (March 2015). "A light-inducible CRISPR-Cas9 system for control of endogenous gene activation". Nature Chemical Biology. 11 (3): 198–200. PMC . PMID 25664691. doi:10.1038/nchembio.1753.
- Nihongaki Y, Yamamoto S, Kawano F, Suzuki H, Sato M (February 2015). "CRISPR-Cas9-based photoactivatable transcription system". Chemistry & Biology. 22 (2): 169–74. PMID 25619936. doi:10.1016/j.chembiol.2014.12.011.
- Wright AV, Sternberg SH, Taylor DW, Staahl BT, Bardales JA, Kornfeld JE, Doudna JA (March 2015). "Rational design of a split-Cas9 enzyme complex". Proceedings of the National Academy of Sciences of the United States of America. 112 (10): 2984–9. PMC . PMID 25713377. doi:10.1073/pnas.1501698112.
- Nihongaki Y, Kawano F, Nakajima T, Sato M (July 2015). "Photoactivatable CRISPR-Cas9 for optogenetic genome editing". Nature Biotechnology. 33 (7): 755–60. PMID 26076431. doi:10.1038/nbt.3245.
- Hemphill J, Borchardt EK, Brown K, Asokan A, Deiters A (May 2015). "Optical Control of CRISPR/Cas9 Gene Editing". Journal of the American Chemical Society. 137 (17): 5642–5. PMC . PMID 25905628. doi:10.1021/ja512664v.
- Jain PK, Ramanan V, Schepers AG, Dalvie NS, Panda A, Fleming HE, Bhatia SN (September 2016). "Development of Light-Activated CRISPR Using Guide RNAs with Photocleavable Protectors". Angewandte Chemie. 55 (40): 12440–4. PMID 27554600. doi:10.1002/anie.201606123.
- Davis KM, Pattanayak V, Thompson DB, Zuris JA, Liu DR (May 2015). "Small molecule-triggered Cas9 protein with improved genome-editing specificity". Nature Chemical Biology. 11 (5): 316–8. PMC . PMID 25848930. doi:10.1038/nchembio.1793.
- Liu KI, Ramli MN, Woo CW, Wang Y, Zhao T, Zhang X, Yim GR, Chong BY, Gowher A, Chua MZ, Jung J, Lee JH, Tan MH (November 2016). "A chemical-inducible CRISPR-Cas9 system for rapid control of genome editing". Nature Chemical Biology. 12 (11): 980–987. PMID 27618190. doi:10.1038/nchembio.2179.
- Truong DJ, Kühner K, Kühn R, Werfel S, Engelhardt S, Wurst W, Ortiz O (July 2015). "Development of an intein-mediated split-Cas9 system for gene therapy". Nucleic Acids Research. 43 (13): 6450–8. PMC . PMID 26082496. doi:10.1093/nar/gkv601.
- Zetsche B, Volz SE, Zhang F (February 2015). "A split-Cas9 architecture for inducible genome editing and transcription modulation". Nature Biotechnology. 33 (2): 139–42. PMC . PMID 25643054. doi:10.1038/nbt.3149.
- González F, Zhu Z, Shi ZD, Lelli K, Verma N, Li QV, Huangfu D (August 2014). "An iCRISPR platform for rapid, multiplexable, and inducible genome editing in human pluripotent stem cells". Cell Stem Cell. 15 (2): 215–26. PMC . PMID 24931489. doi:10.1016/j.stem.2014.05.018.
- Dow LE, Fisher J, O'Rourke KP, Muley A, Kastenhuber ER, Livshits G, Tschaharganeh DF, Socci ND, Lowe SW (April 2015). "Inducible in vivo genome editing with CRISPR-Cas9". Nature Biotechnology. 33 (4): 390–4. PMC . PMID 25690852. doi:10.1038/nbt.3155.
- Yu C, Liu Y, Ma T, Liu K, Xu S, Zhang Y, Liu H, La Russa M, Xie M, Ding S, Qi LS (February 2015). "Small molecules enhance CRISPR genome editing in pluripotent stem cells". Cell Stem Cell. 16 (2): 142–7. PMC . PMID 25658371. doi:10.1016/j.stem.2015.01.003.
- Maruyama T, Dougan SK, Truttmann MC, Bilate AM, Ingram JR, Ploegh HL (May 2015). "Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining". Nature Biotechnology. 33 (5): 538–42. PMC . PMID 25798939. doi:10.1038/nbt.3190.
- Science News Staff (December 17, 2015). "And Science's Breakthrough of the Year is ...". news.sciencemag.org. Retrieved 2015-12-21.
- Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelsen TS, Heckl D, Ebert BL, Root DE, Doench JG, Zhang F (January 2014). "Genome-scale CRISPR-Cas9 knockout screening in human cells". Science. 343 (6166): 84–7. PMC . PMID 24336571. doi:10.1126/science.1247005.
- Zimmer C (2016-06-03). "Scientists Find Form of Crispr Gene Editing With New Capabilities". The New York Times. ISSN 0362-4331. Retrieved 2016-06-10.
- van Erp PB, Bloomer G, Wilkinson R, Wiedenheft B (June 2015). "The history and market impact of CRISPR RNA-guided nucleases". Current Opinion in Virology. 12: 85–90. PMC . PMID 25914022. doi:10.1016/j.coviro.2015.03.011.
- Maggio I, Gonçalves MA (May 2015). "Genome editing at the crossroads of delivery, specificity, and fidelity". Trends in Biotechnology. 33 (5): 280–91. PMID 25819765. doi:10.1016/j.tibtech.2015.02.011.
- Rath D, Amlinger L, Rath A, Lundgren M (October 2015). "The CRISPR-Cas immune system: biology, mechanisms and applications". Biochimie. 117: 119–28. PMID 25868999. doi:10.1016/j.biochi.2015.03.025.
- Freedman BS, Brooks CR, Lam AQ, Fu H, Morizane R, Agrawal V, Saad AF, Li MK, Hughes MR, Werff RV, Peters DT, Lu J, Baccei A, Siedlecki AM, Valerius MT, Musunuru K, McNagny KM, Steinman TI, Zhou J, Lerou PH, Bonventre JV (October 2015). "Modelling kidney disease with CRISPR-mutant kidney organoids derived from human pluripotent epiblast spheroids". Nature Communications. 6: 8715. PMC . PMID 26493500. doi:10.1038/ncomms9715.
- Bellin M, Casini S, Davis RP, D'Aniello C, Haas J, Ward-van Oostwaard D, Tertoolen LG, Jung CB, Elliott DA, Welling A, Laugwitz KL, Moretti A, Mummery CL (December 2013). "Isogenic human pluripotent stem cell pairs reveal the role of a KCNH2 mutation in long-QT syndrome". The EMBO Journal. 32 (24): 3161–75. PMC . PMID 24213244. doi:10.1038/emboj.2013.240.
- Gomaa AA, Klumpe HE, Luo ML, Selle K, Barrangou R, Beisel CL (January 2014). "Programmable removal of bacterial strains by use of genome-targeting CRISPR-Cas systems". mBio. 5 (1): e00928–13. PMC . PMID 24473129. doi:10.1128/mBio.00928-13.
- Citorik RJ, Mimee M, Lu TK (November 2014). "Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases". Nature Biotechnology. 32 (11): 1141–5. PMC . PMID 25240928. doi:10.1038/nbt.3011.
- van Diemen FR, Kruse EM, Hooykaas MJ, Bruggeling CE, Schürch AC, van Ham PM, Imhof SM, Nijhuis M, Wiertz EJ, Lebbink RJ (2016). "CRISPR/Cas9-Mediated Genome Editing of Herpesviruses Limits Productive and Latent Infections". PLoS Pathogens. 12 (6): e1005701. PMC . PMID 27362483. doi:10.1371/journal.ppat.1005701. Lay summary – PLOS Media YouTube Channel.
- Liu Y, Zhan Y, Chen Z, He A, Li J, Wu H, Liu L, Zhuang C, Lin J, Guo X, Zhang Q, Huang W, Cai Z (September 2016). "Directing cellular information flow via CRISPR signal conductors". Nature Methods. 13: 938–944. PMID 27595406. doi:10.1038/nmeth.3994.
- Abrahimi P, Chang WG, Kluger MS, Qyang Y, Tellides G, Saltzman WM, Pober JS (July 2015). "Efficient gene disruption in cultured primary human endothelial cells by CRISPR/Cas9". Circulation Research. 117 (2): 121–8. PMID 25940550. doi:10.1161/CIRCRESAHA.117.306290.
- Yong E (2015-11-25). "The Revolutionary Gene-Editing Technique That Reveals Cancer's Weaknesses". The Atlantic. Retrieved 2016-02-21.
- 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: 41. PMC . PMID 26944702. doi:10.1186/s13059-016-0904-5.
- "Who Owns the Biggest Biotech Discovery of the Century? There's a bitter fight over the patents for CRISPR, a breakthrough new form of DNA editing.". MIT Technology Review. Retrieved 25 February 2015.
CRISPR Patents Spark Fight to Control Genome Editing
- Fye S. "Genetic Rough Draft: Editas and CRISPR". The Atlas Business Journal. Retrieved 19 January 2016.
- Pollack, Andrew (15 February 2017). "Harvard and M.I.T. Scientists Win Gene-Editing Patent Fight". The New York Times.
- Akst, Jef (February 15, 2017). "Broad Wins CRISPR Patent Interference Case". The Scientist Magazine.
- Noonan, Kevin E. (February 16, 2017). "PTAB Decides CRISPR Interference in Favor of Broad Institute -- Their Reasoning". Patent Docs.
- "CRISPR Madness". GEN.
- Staff (1 April 2015). "News: Products & Services". Genetic Engineering & Biotechnology News (Paper). 35 (7): 8.
- Antonio Regalado for MIT Technology Review, March 5, 2015 Engineering the Perfect Baby
- Lanphier E, Urnov F, Haecker SE, Werner M, Smolenski J (March 2015). "Don't edit the human germ line". Nature. 519 (7544): 410–1. Bibcode:2015Natur.519..410L. PMID 25810189. doi:10.1038/519410a.
- Wade N (19 March 2015). "Scientists Seek Ban on Method of Editing the Human Genome". The New York Times. Retrieved 20 March 2015.
The biologists writing in Science support continuing laboratory research with the technique, and few if any scientists believe it is ready for clinical use.
- Liang P, Xu Y, Zhang X, Ding C, Huang R, Zhang Z, Lv J, Xie X, Chen Y, Li Y, Sun Y, Bai Y, Songyang Z, Ma W, Zhou C, Huang J (May 2015). "CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes". Protein & Cell. 6 (5): 363–72. PMC . PMID 25894090. doi:10.1007/s13238-015-0153-5.
- Kolata G (23 April 2015). "Chinese Scientists Edit Genes of Human Embryos, Raising Concerns". The New York Times. Retrieved 24 April 2015.
- "Chinese scientists genetically modify human embryos". Nature. 22 April 2015.
- Regalado, Antonio (2016-05-08). "Chinese Researchers Experiment with Making HIV-Proof Embryos". MIT Technology Review. Retrieved 2016-06-10.
- "International Summit on Gene Editing". National Academies of Sciences, Engineering, and Medicine. 3 December 2015. Retrieved 3 December 2015.
- Ewen Callaway (1 February 2016). "UK scientists gain licence to edit genes in human embryos". Nature. Archived from the original on 26 December 2016. Retrieved 26 December 2016.
- McHughen A, Smyth S (January 2006). "US regulatory system for genetically modified [genetically modified organism (GMO), rDNA or transgenic] crop cultivars". Plant Biotechnol J. 6 (1): 2–12. PMID 17956539. doi:10.1111/j.1467-7652.2007.00300.x.
- USDA. "Re: Request to confirm" (PDF).
- "Gene-edited CRISPR mushroom escapes US regulation".
- "Gene-editing surges as US rethinks regulations".
- "The FDA Is Cracking Down On Rogue Genetic Engineers", Kristen V. Brown. Gizmodo. February 1, 2017. Retrieved 5 feb 2017
- Talbot D (2016). "Precise Gene Editing in Plants/ 10 Breakthrough Technologies 2016". MIT Technology review. Massachusetts Institute of Technology. Retrieved 18 March 2016.
- Larson C, Schaffer A (2014). "Genome Editing/ 10 Breakthrough Technologies 2014". Massachusetts Institute of Technology. Retrieved 18 March 2016.
- CRISPR-Cas: A Laboratory Manual Edited by Jennifer Doudna, University of California, Berkeley; Prashant Mali, University of California, San Diego
- 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". Science. 353 (6299): aad5147. PMID 27493190. doi:10.1126/science.aad5147.
- Sander JD, Joung JK (April 2014). "CRISPR-Cas systems for editing, regulating and targeting genomes". Nature Biotechnology. 32 (4): 347–55. PMID 24584096. doi:10.1038/nbt.2842.
- Slaymaker IM, Gao L, Zetsche B, Scott DA, Yan WX, Zhang F (January 2016). "Rationally engineered Cas9 nucleases with improved specificity". Science. 351 (6268): 84–8. PMC . PMID 26628643. doi:10.1126/science.aad5227.
- Terns RM, Terns MP (March 2014). "CRISPR-based technologies: prokaryotic defense weapons repurposed". Trends in Genetics. 30 (3): 111–8. PMID 24555991. doi:10.1016/j.tig.2014.01.003.
- Westra ER, Buckling A, Fineran PC (May 2014). "CRISPR-Cas systems: beyond adaptive immunity". Nature Reviews. Microbiology. 12 (5): 317–26. PMID 24704746. doi:10.1038/nrmicro3241.
- Andersson AF, Banfield JF (May 2008). "Virus population dynamics and acquired virus resistance in natural microbial communities". Science. 320 (5879): 1047–50. Bibcode:2008Sci...320.1047A. PMID 18497291. doi:10.1126/science.1157358.
- Hale C, Kleppe K, Terns RM, Terns MP (December 2008). "Prokaryotic silencing (psi)RNAs in Pyrococcus furiosus". RNA. 14 (12): 2572–9. PMC . PMID 18971321. doi:10.1261/rna.1246808.
- van der Ploeg JR (June 2009). "Analysis of CRISPR in Streptococcus mutans suggests frequent occurrence of acquired immunity against infection by M102-like bacteriophages". Microbiology. 155 (Pt 6): 1966–76. PMID 19383692. doi:10.1099/mic.0.027508-0.
- van der Oost J, Brouns SJ (November 2009). "RNAi: prokaryotes get in on the act". Cell. 139 (5): 863–5. PMID 19945373. doi:10.1016/j.cell.2009.11.018.
- Karginov FV, Hannon GJ (January 2010). "The CRISPR system: small RNA-guided defense in bacteria and archaea". Molecular Cell. 37 (1): 7–19. PMC . PMID 20129051. doi:10.1016/j.molcel.2009.12.033.
- Pul U, Wurm R, Arslan Z, Geissen R, Hofmann N, Wagner R (March 2010). "Identification and characterization of E. coli CRISPR-cas promoters and their silencing by H-NS". Molecular Microbiology. 75 (6): 1495–512. PMID 20132443. doi:10.1111/j.1365-2958.2010.07073.x.
- Díez-Villaseñor C, Almendros C, García-Martínez J, Mojica FJ (May 2010). "Diversity of CRISPR loci in Escherichia coli". Microbiology. 156 (Pt 5): 1351–61. PMID 20133361. doi:10.1099/mic.0.036046-0.
- Deveau H, Garneau JE, Moineau S (2010). "CRISPR/Cas system and its role in phage-bacteria interactions". Annual Review of Microbiology. 64: 475–93. PMID 20528693. doi:10.1146/annurev.micro.112408.134123.
- Koonin EV, Makarova KS (December 2009). "CRISPR-Cas: an adaptive immunity system in prokaryotes". F1000 Biology Reports. 1: 95. PMC . PMID 20556198. doi:10.3410/B1-95.
- "The age of the red pen". The Economist. August 22, 2015. ISSN 0013-0613. Retrieved 2015-08-25.
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 Cse1 (CRISPR_cse1) 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 entry, represented by CT1972 from Chlorobaculum tepidum, is found in the CRISPR/Cas subtype Ecoli regions of many bacteria (most of which are mesophiles), and not in Archaea. It is designated Cse1.
This tab holds annotation information from the InterPro database.
InterPro entry IPR013381
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 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 Cse1 family of Cas proteins, which includes CT1972 from Chlorobium tepidum [PUBMED:16292354]. These proteins are found in the CRISPR/Cas subtype Escherichia coli regions of many bacteria (most of which are mesophiles), and not in Archaea. This is also known as Cse1 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, the UniProtKB sequence database, the NCBI sequence database, and our metagenomics 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
- alignment generated by searching the NCBI sequence database using the family HMM
- alignment generated by searching the metagenomics 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:||107|
|Number in full:||409|
|Average length of the domain:||462.70 aa|
|Average identity of full alignment:||19 %|
|Average coverage of the sequence by the domain:||86.02 %|
|HMM build commands:||
build method: hmmbuild -o /dev/null HMM SEED
search method: hmmsearch -Z 26740544 -E 1000 --cpu 4 HMM pfamseq
|Family (HMM) version:||9|
|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.
There is 1 interaction for this family. More...
We determine these interactions using iPfam, which considers the interactions between residues in three-dimensional protein structures and maps those interactions back to Pfam families. You can find more information about the iPfam algorithm in the journal article that accompanies the website.
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_Cse1 domain has been found. There are 19 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.
Loading structure mapping...