CRISPRs (clustered regularly interspaced short palindromic repeats) 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. It is pronounced "crisper".
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 RNAi in eukaryotic organisms. CRISPRs are found in approximately 40% of sequenced bacteria genomes and 90% of sequenced archaea.[note 1]
The CRISPR/Cas system has been used for gene editing (adding, disrupting or changing the sequence of specific genes) and gene regulation in species throughout the tree of life. By delivering the Cas9 protein and appropriate guide RNAs into a cell, the organism's genome can be relatively cheaply cut at any desired location. CRISPR has a number of potential applications including treating genetic diseases, fighting infections, and increasing food crop yields, but the application of this method is accompanied by ethical concerns.
- 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, though it has only recently been studied, as bacteria may incorporate foreign DNA in other circumstances and even scavenge damaged DNA from their environment.
Clustered repeats were first described in 1987 for the bacterium Escherichia coli by Yoshizumi Ishino, but at that time their function was not known. In 2000, similar repeats were identified in other bacteria and archaea, and were termed Short Regularly Spaced Repeats (SRSR). SRSR were renamed CRISPR in 2002. 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 DNA.
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.
Doudna and 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. Jinek combined tracrRNA and spacer RNA into a "single-guide RNA" molecule that, mixed with Cas9, could find and cut the correct DNA targets. Jinek et al proposed that such synthetic guide RNAs could be used for gene editing.
CRISPR was first shown to work as a genome engineering/editing tool in human cell culture by 2012. It has since been used in a wide range of organisms including baker's yeast (S. cerevisiae), zebrafish (D. rerio), flies (D. melanogaster), axolotl (A. mexicanum), nematodes (C. elegans), plants, mice, monkeys, nonviable human embryos and other organisms.
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 they make a short RNA sequence that is paired to the targeted DNA sequence, rather than engineering an entire custom protein.
Repeats and spacers
CRISPR loci 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
CRISPR-associated (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, 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.|||
|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 was the first clue that they were involved in spacer acquisition. Mutation studies confirmed this hypothesis, showing that removal of cas1 or cas2 stopped spacer acquisition, without affecting CRISPR immune response.
The exact function of Cas1 and Cas2 is unknown, however a number of Cas1 proteins were biochemically characterised and their structures resolved. Cas1 proteins have very diverse amino acid sequences, however their crystal structures are similar and all purified Cas1 proteins are metal-dependent nucleases that bind to DNA in a sequence-independent manner.
Representative Cas2 proteins were characterised and possess either ssRNA- or dsDNA- specific endoribonuclease activity. Functional data and genetic mutation studies suggests that Cas1 and Cas2 excise fragments of invading DNA and insert them into CRISPR arrays.
Bioinformatic analysis of regions of phage genomes that were excised as spacers (termed protospacers) revealed that they were not randomly 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, 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 inherent to the Cas1 protein, 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 have demonstrated that the first direct repeat, adjacent to the leader sequence is copied, with the newly acquired spacer inserted between the first and second direct repeats.
The PAM sequence also appears to be important during spacer insertion in type I-E systems. That sequence contains a strongly conserved final nucleotide (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.
A number of 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 both the Cas proteins involved in acquisition and interference to interact with each other. Newly acquired spacers that result from the priming mechanism are always found on the same strand as the 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 an 8 nt 5ʹ-handle originating from the repeat on mature crRNAs along with a single spacer sequence. Type III systems also use Cas6, however the repeats found in type III systems do not produce stem-loops, instead cleavage occurs by the primary transcript wrapping around the Cas6 to allow cleavage 8 nt upstream of the repeat spacer junction. 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. A number of phage and plasmid challenge experiments have shown that crRNAs show no preference between coding and non-coding strand, which is indicative of an RNA-guided DNA-targeting system. 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 have 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 inferred to be common to all three systems. Even through the distinctive maturation process of each major type, all crRNAs contain a spacer sequence and some portion of the repeat at one or both ends. It is the partial repeat sequence that prevents the CRISPR-Cas system from targeting the chromosome as base pairing beyond the spacer sequence signals self and prevents DNA cleavage of the chromosome. 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|
Evolution and diversity
Studies of Streptococcus thermophilus first showed how CRISPRs drive phage and bacterial evolution. 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 where there are point mutations in the spacer. Similar stringency is required in PAM or the bacterial strain will remain phage sensitive. 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.
CRISPR evolution has been studied using comparative genomics of many strains of S. thermophilus, Escherichia coli and Salmonella enterica. 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. The results showed that particular CRISPR loci evolve more rapidly than others, which allowed the strains' phylogenetic relationships to be determined. A similar analysis of E. coli and S. enterica strains revealed that they evolved much slower than S. thermophilus. The latter's strains that had diverged 250 thousand years ago still contained the same spacer complement.
CRISPR diversity was studied in multiple environmental communities using metagenomics. Analysis of two acid mine drainage biofilms showed that one of the analyzed CRISPRs contained extensive deletions and spacer additions in comparison to the other biofilm, suggesting a higher phage activity/prevalence in one community compared to the other. In the oral cavity, a temporal study determined that 7-22% of spacers were shared between timepoints over 17 months within an individual and less than 2% of spacers were shared between individuals at any timepoint. From the same environment a single strain was tracked using PCR primers specific to its CRISPR. Unlike the broad-level results of spacer presence/absence, which showed significant diversity, this CRISPR added 3 spacers over 17 months, 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 between timepoints.
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 the outcome of CRISPR and phage 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 there are many reference genomes available, PCR can be used to amplify CRISPR arrays and analyse spacer content. However, this approach will only yield information for CRISPRs specifically targeted and for organisms with sufficient representation in public databases to design reliable PCR primers.
The alternative approach is to extract and reconstruct CRISPR arrays from shotgun metagenomic data. Identification of CRISPR arrays from metagenomic reads is computationally more difficult, particularly with second generation sequencing technologies (e.g. 454, Illumina), as the short read lengths prevent more than two or three repeat units being present in a single read. CRISPR identification in raw reads has been achieved using purely denovo identification or by using direct repeat sequences in partially assembled CRISPR arrays from contigs 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, CRISPR/Cas can be described as a Lamarckian inheritance mechanism. 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.
CRISPRs can add and delete base pairs at specifically targeted DNA loci and have been used to cut as many as five genes at once. Or up to 62 genes at once—Pig cells have been engineered to inactivate all 62 Porcine Endogenous Retrovirus in the pig genome using CRISPR Cas9 genome editing technology, and 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
"CRISPRi" like RNAi, turns off genes in a reversible fashion by targeting but not cutting a site. The targeted site is methylated so the gene is epigentically modified. This modification inhibits transcription of the gene. RNA-guided CRISPR associated nuclease 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, but for mammalian applications, a section of protein is added. Its guide RNA targets regulatory DNA, called promoters that immediately precede the gene target.
Cas9 was used to carry synthetic transcription factors (protein fragments that turn on genes) that activated specific human genes. The technique achieved a strong effect by targeting multiple CRISPR constructs to slightly different spots on the gene's promoter.
Some of the affected genes tied to human diseases, including those involved in muscle differentiation, cancer, inflammation and producing fetal hemoglobin.
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.
Patents and commercialization
As of December 2014, patent rights to CRISPR were still developing. Several companies had been formed to develop related drugs and research tools.
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
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, writing that "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 noted that CRISPR is not ready for clinical application in reproductive medicine, and 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.”
- CRISPR/Cas Tools
- CRISPR interference
- Gene drive
- Transcription activator-like effector nuclease (TALEN)
- Zinc finger nuclease
- Gene knockout
- Gene silencing
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