Summary: CRISPR associated protein
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CRISPR Edit Wikipedia article
Clustered regularly-interspaced short palindromic repeats (abbreviated as CRISPR, pronounced crisper) are segments of prokaryotic DNA containing short repetitions of base sequences. Each repetition is followed by short segments of "spacer DNA" from previous exposures to a bacterial virus or plasmid.
The CRISPR/Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and phages, and provides a form of acquired immunity. CRISPR spacers recognize and cut these exogenous genetic elements in a manner analogous to RNA interference in eukaryotic organisms. CRISPRs are found in approximately 40% of sequenced bacteria genomes and 90% of sequenced archaea.[note 1]
The CRISPR interference technique has enormous potential application, including altering the germline of humans, animals and other organisms, and modifying the genes of food crops. By delivering the Cas9 protein and appropriate guide RNAs into a cell, the organism's genome can be cut at any desired location. CRISPRs have been used in concert with specific endonuclease enzymes for genome editing and gene regulation in species throughout the tree of life. Ethical concerns have been expressed about the prospect of using this nascent biotechnology for editing the human germline.
- 1 History
- 2 Locus structure
- 3 Mechanism
- 4 Evolution and diversity
- 5 Identification
- 6 Evolutionary significance
- 7 Use by phages
- 8 Applications
- 9 Patents and commercialization
- 10 Society and culture
- 11 See also
- 12 Notes
- 13 References
- 14 Further reading
CRISPR is part of a normally occurring bacterial process. Bacteria may incorporate foreign DNA and even scavenge damaged DNA from their environment.
Clustered repeats were first described in 1987 for the bacterium Escherichia coli by Osaka University researcher Yoshizumi Ishino, but at that time their function was not known. The repeats were observed independently by Spanish researcher Francisco Mojica in 1993. He would later name them short regularly spaced repeats (SRSR) after he found them in many other microbes. In 2000, similar repeats were identified in other bacteria and archaea. SRSR was renamed CRISPR in 2002 after a suggestion from Mojica. CRISPR was proposed to be responsible for the generation of adaptive immunity in microbes. The paper describing this idea was rejected by a number of high profile journals. Papers proposing similar hypotheses by Gilles Vergnaud and, independently, Alexander Bolotin suffered similar fates before being published.  
A set of genes was found to be associated with CRISPR repeats, and was named the cas, or CRISPR-associated, genes. The cas genes encode putative nuclease or helicase proteins, which are enzymes that can cut or unwind DNA, respectively.
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 have 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. Koonin and colleagues proposed that spacers serve as a template for RNA molecules, analogous to a system called RNA interference used by eukaryotic cells.
In 2007, Barrangou, Horvath (food industry scientists at Danisco) and Moineau's group at Université Laval (Canada) showed that they could use spacer DNA to alter the resistance of Streptococcus thermophilus to phage attack.
CRISPR was first shown to work as a genome engineering/editing tool in human cell culture in 2012. It has since been used in a wide range of organisms including baker's yeast (S. cerevisiae), zebrafish (D. rerio), fruit flies (D. melanogaster), axolotl (A. mexicanum), nematodes (C. elegans), plants, mice, monkeys and human embryos.
Jennifer Doudna and Emmanuelle Charpentier had independently been exploring CRISPR-associated proteins to learn how bacteria use spacers in their immune defenses. They jointly studied a simpler CRISPR system that relies on a protein called Cas9. They found that bacteria respond to an invading phage by transcribing spacers and palindromic DNA into a long RNA molecule. The cell then uses tracrRNA and Cas9 to cut this long RNA molecule into pieces called crRNAs.
Cas9 is a nuclease, an enzyme specialized for cutting DNA. It has two active cutting sites (HNH and RuvC), one for each strand of the DNA's double helix. The team demonstrated that they could disable one or both sites while preserving Cas9's ability to home in on its target DNA. In 2012, a group including both Doudna and Charpentier combined tracrRNA and spacer RNA into a "single-guide RNA" molecule that, mixed with Cas9, could find and cut the correct DNA targets. Their study proposed that such synthetic guide RNAs could be used for gene editing.
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 spots. In 2010, synthetic nucleases called TALENs provided an easier way to target a double-strand break to a specific location on the DNA strand. Both zinc-finger nucleases and TALENs require researchers to make 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 it requires only making a short RNA sequence.
Repeats and spacers
CRISPR repeats range in size from 24 to 48 base pairs. They usually show some dyad symmetry, implying the formation of a secondary structure such as a hairpin, but are not truly palindromic. Repeats are separated by spacers of similar length. Some CRISPR spacer sequences exactly match sequences from plasmids and phages,   although some spacers match the prokaryote's genome (self-targeting spacers).  New spacers can be added rapidly as part of the immune response to phage infection.
Cas genes and CRISPR subtypes
Cas genes are often associated with CRISPR repeat-spacer arrays. Comparative genomics identified multiple cas genes; an initial analysis of 200 bacterial and archaeal genomes suggested as many as 45 cas gene families. Only cas1 and cas2 genes are present in all 45 families.
The current CRISPR classification groups cas operons into three major divisions: (I, II, III), each with multiple subdivisions based on cas1 phylogeny and cas operon gene complement. Aside from cas1 and cas2, each major division's operons have a common set of constituent genes. Each subdivision is characterised by a ‘signature gene’ found exclusively in that subdivision. 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.
|Cas type||Signature gene||Function||Reference|
|I||Cas3||Single-stranded DNA nuclease (HD domain) and ATP-dependent helicase|||
|IA||Cas8a||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|||
|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|||
|III||Cas10||Homolog of Cas10d and Cse1|||
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 ssRNA- or 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 from the three major divisions 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 nucleotide of the protospacer. This nucleotide 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 characterised 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.
Recent analysis of Sulfolobus solfataricus CRISPRs revealed further complexities to the canonical model of spacer insertion as one of its six CRISPR loci inserted new spacers randomly throughout its CRISPR array, as opposed to inserting closest to the leader sequence.
Multiple CRISPRs contain many spacers to the same phage. The mechanism that causes this phenomenon was elucidated 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 spacer that caused the priming. This observation led to the hypothesis that the acquisition machinery slides along the foreign DNA after priming to find a new protospacer.
The CRISPR immune response occurs through two steps: CRISPR-RNA (crRNA) biogenesis and crRNA-guided interference.
A CRISPR array is transcribed from a promoter in the leader into a single long transcript. This transcript is processed by cleavage inside the repeat sequence to form crRNAs. The mechanisms to produce mature crRNAs differ greatly between the three main CRISPR-Cas systems. In both type I-E and type I-F systems, the proteins Cas6e and Cas6f respectively, recognise stem-loops created by the palindromic nature of the direct repeats. These proteins cleave the primary transcript at the junction between double-stranded and single-stranded RNA, leaving a "handle" at the 5'-end of the molecule, 8-nucleotides in length[clarification needed] originating from the repeat on mature crRNAs along with a single spacer sequence.
Type III systems also use Cas6, however their repeats do not produce stem-loops. Cleavage instead occurs by the primary transcript wrapping around the Cas6 to allow cleavage 8 nt upstream of the repeat spacer junction.
Type II systems lack the Cas6 gene and instead utilize RNaseIII for cleavage. Functional type II systems encode an extra small RNA that is complementary to the repeat sequence, known as a trans-activating RNA (tracrRNA). Transcription of the tracrRNA and the primary CRISPR transcript results in base pairing and the formation of dsRNA at the repeat sequence, which is subsequently targeted by RNaseIII to produce crRNAs. Unlike the other two systems the crRNA does not contain the full spacer but instead is truncated at one end by 10 nt.
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 arranged in a ‘seahorse’ conformation, bound to a single crRNA that runs down the spine.
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 also required in type II systems, however the PAM is recognized on the same strand as the crRNA (the opposite strand to type I systems).
Type III systems, like type I require a multi-protein complex to associate with the crRNA. Biochemical and structural analyses of complexes from S. solfataricus and Pyrococcus furiosus have elucidated that six or seven cas proteins bind to crRNAs, respectively. Surprisingly, the type III systems analysed from S. solfataricus and P. furiosus both target the mRNA of phage/plasmids, 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
Scientists can use viral or non-viral system for delivery of the Cas9 and gRNA 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 adenoassociated (AAV).
|CRISPR-associated protein Cse1|
|CRISPR-associated protein Cse2|
Evolution and diversity
The basic model of CRISPR evolution is one where newly incorporated spacers drive phages to mutate their genomes to avoid the bacteria 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 will remain 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 new spacer acquisition. Particular 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 had 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 in comparison to the other biofilm, suggesting a higher phage activity/prevalence in one community compared to 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% of spacers were shared between individuals.
From the same environment a single strain was tracked using PCR primers specific to its CRISPR system. Unlike the broad-level results of spacer presence/absence, which showed significant diversity, this CRISPR added 3 spacers over 17 months, suggesting that even in an environment with significant CRISPR diversity some loci evolve slowly. CRISPRs have also been analysed from the metagenomes produced for the human microbiome project. Although most CRISPRs were body-site specific, some CRISPRs within a body site are widely shared among individuals. One of these CRISPR loci originated from streptococcal species and contained ~15,000 spacers, 50% of which were unique. Similar to the targeted studies of the oral cavity, some of the CRISPRs showed little evolution over time.
CRISPR evolution has been studied in chemostats using S. thermophilus to explicitly 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 CRISPR targeting had prevented phage replication absent these mutations. Other S. thermophilus experiments showed that phages can still infect and replicate in hosts that have only one targeting spacer and that sensitive hosts can exist in environments with high phage titres. 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. However their most notable characteristic is their repeating spacers and direct repeats. This characteristic makes CRISPRs easily identifiable in long sequences of DNA, since the number of repeat copies decreases the likelihood of a false positive match. Three programs are used for CRISPR repeat identification that 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 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 denovo 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.
Through the CRISPR/Cas mechanism, bacteria can acquire immunity to certain phages and thus halt further transmission of targeted phages. For this reason, Eugene Koonin has described CRISPR/Cas as a Lamarckian inheritance mechanism. However, this has been disputed by a recent critic noting "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.
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 has 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/Cas9 genome editing is carried out with a Type II CRISPR system. When utilized for genome editing, this system includes Cas9, CRISPR RNA (crRNA), trans-activating 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 RNA used by Cas9 to guide it to the correct section of host DNA along with a region that binds to tracrRNA (generally in a hairpin loop form) forming an active complex with Cas9|
|tracrRNA||Binds to crRNA and forms an active complex with Cas9|
|sgRNA||Single guide RNAs are a combined RNA consisting of a tracrRNA and at least one crRNA|
|Cas9||Protein that in its active form is able to modify DNA utilizing crRNA as its guide. Many variants exist with differing functions (i.e. single strand nicking, double strand break, DNA binding) due to Cas9's DNA site recognition function that is independent of its two DNA cleaving domains (one for each strand).|
|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 must also be designed for each application, as it must overlap with the sequences on either side of the cut and code for the insertion sequence.
Multiple crRNA's 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 (see image for overview).
CRISPR/Cas9 is a widely used system for genome editing due to its high degree of fidelity and relatively simple construction. CRISPR/Cas9 depends on two factors for its specificity – the CRISPR target sequence and the PAM. The CRISPR 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 for base pair bonding with 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 the protein structure of Cas9 and generally cannot be easily modified to recognize a difference sequence. However this is not limiting as it is a short sequence and not very specific (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 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 non-homologous end joining that typically follows a double strand break. Providing a section of 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.
CRISPRs have been used to cut five to 62 genes at once: pig cells have been engineered to inactivate all 62 Porcine Endogenous Retrovirus in the pig genome, which eliminated infection 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
Like RNAi, CRISPR interference (CRISPRi) turns off genes in a reversible fashion by targeting, but not cutting a site. The targeted site is methylated so the gene is epigenetically modified. 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 spots on the gene's promoter.
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 locally targeted.
CRISPR can also be utilized to create human cellular models of disease. For instance, CRISPR was applied to human pluripotent stem cells to introduce targeted mutations in genes relevant to two different kidney diseases, polycystic kidney disease and focal segmental glomerulosclerosis. These CRISPR-modified pluripotent stem cells were subsequently grown into human kidney organoids, which exhibited disease-specific phenotypes. Kidney organoids from stem cells with polycystic kidney disease mutations formed large, translucent cyst structures from kidney tubules. Kidney organoids with mutations in a gene linked to focal segmental glomerulosclerosis 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 mutations. A similar approach has been 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 diseases and test drugs that might work in human patients.
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 spot 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. Only weeks later, the team reported a second drive with genes that rendered female mosquitoes infertile and could quickly wipe out a population. The work was done in the lab, leading to debates over the desirability of field testing.
Using “dead” versions of Cas9 eliminates CRISPR’s DNA-cutting ability, while preserving its ability to target desirable sequences. Multiple groups added various regulatory factors to dead Cas9s, enabling them to turn almost any gene on or off or subtly adjust its level of activity.
In another 2015 experiment, the 20,000 or so known human genes were separately targeted, turning them on one by one in groups of cells 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.
Clinical researchers are applying it 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.
It may also 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 every individual human gene, in an attempt to identify which genes were essential. Between 1,600 and 1,800 genes passed their test—around one in ten. 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 specific functions of some 18 percent of the essential genes are unidentified. 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.
Patents and commercialization
As of December 2014, patent rights to CRISPR were contested. Several companies had been formed to develop related drugs and research tools. As companies ramp up financing, doubts as to whether or not CRISPR can be successfully monetized in the short-term have been raised.
As of November 2013 SAGE Labs had exclusive rights from one of those companies to produce and sell genetically engineered rats and nonexclusive rights for mouse and rabbit models.
Society and culture
Human germline modification
In light of plans or ongoing research to apply CRISPR to human embryos in at least four labs in the US, labs in China and the UK, and by a US biotechnology company called Ovascience, scientists, including an inventor of CRISPR, 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 inheritable changes to people.
In April 2015, scientists from China published a paper in the journal Protein & Cell reporting 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. According to the paper's lead author, the study had previously been rejected by both Nature and Science in part because of ethical concerns; the journals did not comment to reporters. The experiments resulted in changing only some of the genes, and had off-target effects on other genes. The scientists who conducted the research stated that CRISPR is not ready for clinical application in reproductive medicine. One said to a reporter at Nature: "If you want to do it in normal embryos, you need to be close to 100%.... That’s why we stopped. We still think it’s too immature."
In December 2015, the 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. In conclusion, they agreed to proceed further with basic and clinical research under appropriate legal and ethical guidelines. A specific distinction was made between clinical use in somatic cells, where the effects of edits are limited to a single individual, versus germline cells, where genome changes would be inherited by future generations. This could have unintended and far-reaching consequences for human evolution, genetically (e.g. gene/environment interactions) and culturally (e.g. Social Darwinism), hence altering of gametocytes and embryos to generate inheritable changes in humans was claimed irresponsible. In addition, they agreed to initiate an international forum where these concerns will be continuously addressed, and regulations in research harmonised across countries.
In February 2016, British scientists were given permission by regulators to genetically modify human embryos by using CRISPR-Cas9 and related techniques. The embryos were to be destroyed after seven days.
American Jennifer Doudna and French-born Emmanuelle Charpentier co-authored a key study published in August 2012 that demonstrated the technical power of Crispr-Cas9 to cut and splice genes with extreme efficiency at the highest resolution possible. 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 also named as one of MIT Technology Review's 10 breakthroughs technologies in 2014 and 2016.
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- "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
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- Antonio Regalado for MIT Technology Review, March 5, 2015 Engineering the Perfect Baby
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- Wade, Nicholas (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.
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This tab holds the annotation information that is stored in the Pfam database. As we move to using Wikipedia as our main source of annotation, the contents of this tab will be gradually replaced by the Wikipedia tab.
CRISPR associated protein Provide feedback
This domain forms an anti-parallel beta strand structure with flanking alpha helical regions.
This tab holds annotation information from the InterPro database.
InterPro entry IPR010179
The CRISPR-Cas system is a prokaryotic defense mechanism against foreign genetic elements. The key elements of this defense system are the Cas proteins and the CRISPR RNA.
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) are a family of DNA direct repeats separated by regularly sized non-repetitive spacer sequences that are found in most bacterial and archaeal genomes [PUBMED:17442114]. CRISPRs appear to provide acquired resistance against mobile genetic elements (viruses, transposable elements and conjugative plasmids). CRISPR clusters contain sequences complementary to antecedent mobile elements and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA).
The defense reaction is divided into three stages. In the adaptation stage, the invader DNA is cleaved, and a piece of it is selected to be integrated as a new spacer into the CRISPR locus, where it is stored as an identity tag for future attacks by this invader. During the second stage (the expression stage), the CRISPR RNA (pre-crRNA) is transcribed and subsequently processed into the mature crRNAs. In the third stage (the interference stage), Cas proteins, together with crRNAs, identify and degrade the invader [PUBMED:17379808, PUBMED:16545108, PUBMED:21699496].
The CRISPR-Cas systems have been sorted into three major classes. In CRISPR-Cas types I and III, the mature crRNA is generally generated by a member of the Cas6 protein family. Whereas in system III the Cas6 protein acts alone, in some class I systems it is part of a complex of Cas proteins known as Cascade (CRISPR-associated complex for antiviral defense). The Cas6 protein is necessary for crRNA production whereas the additional Cas proteins that form the Cascade complex are needed for crRNA stability [PUBMED:24459147].
This entry represents the Cse3 (CRISPR/Cas Subtype Ecoli protein 3) family of Cas proteins. The Thermus thermophilus HB8 family member has been crystallised and found to have a structure consisting of two domains with opposing parallel beta-sheets, known as a beta-sheet platform [PUBMED:16672237]. This structure is similar to those found in the sex-lethal protein and poly(A)-binding protein and is consistent with an RNA-binding function.
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...
This group of families is one of several protein families that are always found associated with prokaryotic CRISPRs, themselves a family of clustered regularly interspaced short palindromic repeats, DNA repeats found in nearly half of all bacterial and archaeal genomes. These DNA repeat regions have a remarkably regular structure: unique sequences of constant size, called spacers, sit between each pair of repeats . It has been shown that the CRISPRs are virus-derived sequences acquired by the host to enable them to resist viral infection. The Cas proteins from the host use the CRISPRs to mediate an antiviral response. After transcription of the CRISPR, a complex of Cas proteins termed Cascade cleaves a CRISPR RNA precursor in each repeat and retains the cleavage products containing the virus-derived sequence. Assisted by the helicase Cas3, these mature CRISPR RNAs then serve as small guide RNAs that enable Cascade to interfere with virus proliferation . Cas5 contains an endonuclease motif, whose inactivation leads to loss of resistance, even in the presence of phage-derived spacers .
The clan contains the following 7 members:Cas6 Cas_Cas5d Cas_Cas6 Cas_Cmr3 CRISPR_assoc DUF2276 RAMPs
We store a range of different sequence alignments for families. As well as the seed alignment from which the family is built, we provide the full alignment, generated by searching the sequence database (reference proteomes) using the family HMM. We also generate alignments using four representative proteomes (RP) sets, the UniProtKB sequence database, the NCBI sequence database, and our metagenomics sequence database. More...
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.
|Number in seed:||114|
|Number in full:||180|
|Average length of the domain:||218.60 aa|
|Average identity of full alignment:||24 %|
|Average coverage of the sequence by the domain:||97.26 %|
|HMM build commands:||
build method: hmmbuild -o /dev/null HMM SEED
search method: hmmsearch -Z 11927849 -E 1000 --cpu 4 HMM pfamseq
|Family (HMM) version:||8|
|Download:||download the raw HMM for this family|
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This visualisation provides a simple graphical representation of the distribution of this family across species. You can find the original interactive tree in the 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_assoc domain has been found. There are 20 instances of this domain found in the PDB. Note that there may be multiple copies of the domain in a single PDB structure, since many structures contain multiple copies of the same protein seqence.
Loading structure mapping...