CRISPR

From Wikipedia, the free encyclopedia

This is an old revision of this page, as edited by Jytdog (talk | contribs) at 00:40, 2 March 2015 (Undid revision 649457980 by NeMewSys (talk) not in source). The present address (URL) is a permanent link to this revision, which may differ significantly from the current revision.

Diagram of the possible mechanism for CRISPR.[1]

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

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

CRISPRs are often associated with cas genes that code for proteins related to CRISPRs. The CRISPR/Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and phages[5][6] and provides a form of acquired immunity. CRISPR spacers recognize and cut these exogenous genetic elements in a manner analogous to RNAi in eukaryotic organisms.[2]

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

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

History

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

Repeats were first described in 1987 for the bacterium Escherichia coli.[10] In 2000, similar clustered repeats were identified in additional bacteria and archaea and were termed Short Regularly Spaced Repeats (SRSR).[11] SRSR were renamed CRISPR in 2002.[12] A set of genes, some encoding putative nuclease or helicase proteins, were found to be associated with CRISPR repeats (the cas, or CRISPR-associated genes).[12]

Simplified diagram of a CRISPR locus. The three major components of a CRISPR locus are shown: cas genes, a leader sequence, and a repeat-spacer array. Repeats are shown as grey boxes and spacers are colored bars. The arrangement of the three components is not always as shown.[1][2] In addition, several CRISPRs with a similar DR can be present in a single genome, only one of which being associated with cas genes.[4]

In 2005, three independent research groups showed that some CRISPR spacers are derived from several phage DNA and extrachromosomal DNA such as plasmids.[13][14][15] These key observations were an indication that the CRISPR/cas system could have a role in adaptive immunity in bacteria.[1] Koonin and colleagues[16] proposed that spacers serve as a template for RNA molecules, analogous to eukaryotic cells that use a system called RNA interference.

In 2007 Barrangou, Horvath (food industry scientists at Danisco) and Moineau's group at Université Laval (Canada) showed that they could alter the resistance of Streptococcus thermophilus to phage attack with spacer DNA.[16]

Doudna and Charpentier had independently been exploring CRISPR-associated proteins to learn how bacteria deploy spacers in their immune defenses. They jointly studied a simpler CRISPR system that relies on a protein called Cas9. They found that bacteria respond to an invading phage by transcribing spacers and palindromic DNA into a long RNA molecule that the cell then uses tracrRNA and Cas9 to cut it into pieces called crRNAs.[16]

Cas9 is a nuclease, an enzyme specialized for cutting DNA, with two active cutting sites (HNH and RuvC), one for each strand of the double helix. The team demonstrated that they could disable one or both sites while preserving Cas9's ability to home in on its target DNA. Jinek combined tracrRNA and spacer RNA into a "single-guide RNA" molecule that, mixed with Cas9, could find and cut the correct DNA targets. Jinek et al proposed that such synthetic guide RNAs might be able to be used for gene editing.[16]

CRISPR was first shown to work as a genome engineering/editing tool in human cell culture by 2012[17][18] It has since been used in a wide range of organisms including baker's yeast (S. cerevisiae),[19] zebra fish (D. rerio),[20] flies (D. melanogaster),[21] nematodes (C. elegans),[22] plants,[23] mice,[24] and several other organisms.

Additionally CRISPR has been modified to make programmable transcription factors that allow scientists to target and activate or silence specific genes.[25]

Libraries of tens of thousands of guide RNAs are now available.[16]

The first evidence that CRISPR can reverse disease symptoms in living animals was demonstrated in March 2014, when MIT researchers cured mice of a rare genetic liver disorder.[26]

Gene-editing predecessors

In the early 2000s, researchers developed zinc finger nucleases, synthetic proteins whose DNA-binding domains enable them to cut DNA at specific spots. Later, synthetic nucleases called TALENs provided an easier way to target specific DNA and were predicted to surpass zinc fingers. They both depend on making custom proteins for each DNA target, a more cumbersome procedure than guide RNAs. CRISPRs are more efficient and can target more genes than these earlier techniques.[27]

Locus structure

Repeats and spacers

CRISPR loci range in size from 24 to 48 base pairs.[28] They usually show some dyad symmetry, implying the formation of a secondary structure such as a hairpin, but are not truly palindromic.[29] Repeats are separated by spacers of similar length.[28] Some CRISPR spacer sequences exactly match sequences from plasmids and phages,[13][14][15] although some spacers match the prokaryote's genome (self-targeting spacers).[13][30] New spacers can be added rapidly in response to phage infection.[31]

Cas genes and CRISPR subtypes

CRISPR-associated (cas) genes are often associated with CRISPR repeat-spacer arrays. Extensive comparative genomics have identified many different cas genes; an initial analysis of 40 bacterial and archaeal genomes suggested that there may be 45 cas gene families, with only two genes, cas1 and cas2, universally present.[28] The current CRISPR classification groups cas operons into three major divisions, each with multiple subdivisions based on cas1 phylogeny and cas operon gene complement.[32] Aside from cas1 and cas2, the three major divisions have vastly different sets of constituent genes, with each of the subdivisions characterised by a ‘signature gene’ found exclusively in that subdivision. Many organisms contain multiple CRISPR-Cas systems suggesting that they are compatible and may even share components.[33][34] The sporadic distribution of the CRISPR/Cas subtypes suggests that the system is subject to horizontal gene transfer during microbial evolution.

Signature genes and their putative functions for the major and minor CRISPR-cas types.
Cas type Signature gene Function Reference
I Cas3 Single-stranded DNA nuclease (HD domain) and ATP-dependent helicase [35]
IA Cas8a Subunit of the interference module [36]
IB Cas8b
IC Cas8c
ID Cas10d contains a domain homologous to the palm domain of nucleic acid polymerases and nucleotide cyclases [32][37]
IE Cse1
IF Csy1 Not Determined
II Cas9 Nucleases RuvC and HNH together produce DSBs, and separately can produce single-strand breaks [38]
IIA Csn2 Not Determined
IIB Cas4 Not Determined
IIC Characterized by the absence of either Csn2 or Cas4 [39]
III Cas10 Homolog of Cas10d and Cse1 [37]
IIIA Csm2 Not Determined
IIIB Cmr5 Not Determined

Mechanism

The stages of CRISPR immunity for each of the three major types of adaptive immunity. (1) Acquisition begins by recognition of invading DNA by Cas1 and Cas2 and cleavage of a protospacer. (2) The protospacer is ligated to the direct repeat adjacent to the leader sequence and (3) single strand extension repairs the CRISPR and duplicates the direct repeat. The crRNA processing and interference stages occur differently in each of the three major CRISPR systems. (4) The primary CRISPR transcript is cleaved by cas genes to produce crRNAs. (5) In type I systems Cas6e/Cas6f cleave at the junction of ssRNA and dsRNA formed by hairpin loops in the direct repeat. Type II systems use a trans-activating (tracr) RNA to form dsRNA, which is cleaved by Cas9 and RNaseIII. Type III systems use a Cas6 homolog that does not require hairpin loops in the direct repeat for cleavage. (6) In type II and type III systems secondary trimming is performed at either the 5’ or 3’ end to produce mature crRNAs. (7) Mature crRNAs associate with Cas proteins to form interference complexes. (8) In type I and type II systems, basepairing between the crRNA and the PAM causes degradation of invading DNA. Type III systems do not require a PAM for successful degradation and inn type III-A systems basepairing occurs between the crRNA and mRNA rather than the DNA, targeted by type III-B systems.

Acquisition of spacers into CRISPR loci

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

Bioinformatic analysis of regions of phage genomes that were excised as spacers (termed protospacers) revealed that they were not randomly distributed in but instead were found adjacent to short (3 – 5 bp) DNA sequences termed PAMs (protospacer adjacent motifs). Analysis of CRISPR-Cas systems from the three major divisions have shown PAMs to be important for type I, type II but not type III systems during the spacer acquisition process.[15][50][51][52][53][54] In type I and type II systems, protospacers are excised at positions adjacent to a PAM sequence, with the other end of the spacer cut using a ruler mechanism inherent to the Cas1 protein, thus maintaining the regularity of the spacer size in the CRISPR array.[55][56] The conservation of the PAM sequence differs between CRISPR-Cas systems and appears to be evolutionarily linked to cas1 and the leader sequence.[54][57]

New spacers are added to a CRISPR array in a directional manner,[13] occurring preferentially [50][51][58][59][60] but not exclusively adjacent [53][56] to the leader sequence. Analysis of the type I-E system from E. coli have demonstrated that the first direct repeat, adjacent to the leader sequence is copied, with the newly acquired spacer inserted between the first and second direct repeats.[42][55] The PAM sequence also appears to be important during spacer insertion in type I-E systems. The PAM sequence of the I-E system contains a strongly conserved final nucleotide (adjacent to the first nucleotide of the protospacer) and it has been shown that this nucleotide becomes the final base in the first direct repeat.[43][61][62] This suggests that the spacer acquisition machinery generates single stranded overhangs in the second-to-last position of the direct repeat and in the PAM during spacer insertion. However, not all CRISPR-Cas systems appear to share this mechanism as PAMs characterised in other organisms do not show the same level of conservation in the final position.[57] It is likely that in those systems, a blunt end is generated at the very end of the direct repeat and the protospacer during acquisition. Recent analysis of Sulfolobus solfataricus CRISPRs revealed further complexities to the canonical model of spacer insertion as one of its six CRISPR loci inserted new spacers randomly throughout its CRISPR array, as opposed to inserting closest to the leader sequence.[56]

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

Interference stage

The CRISPR immune response occurs through two steps: CRISPR-RNA (crRNA) biogenesis and crRNA-guided interference. A CRISPR array is transcribed from a promoter in the leader into a single long transcript.[36][63][64] This transcript is processed by cleavage inside the repeat sequence to form crRNAs. The mechanisms to produce mature crRNAs differ greatly between the three main CRISPR-Cas systems. In both type I-E and type I-F systems, the proteins Cas6e and Cas6f respectively, recognise stem-loops [65][66][67] created by the palindromic nature of the direct repeats.[29] These proteins cleave the primary transcript at the junction between double-stranded and single-stranded RNA, leaving an 8 nt 5ʹ-handle originating from the repeat on mature crRNAs along with a single spacer sequence. Type III systems also use Cas6, however the repeats found in type III systems do not produce stem-loops, instead cleavage occurs by the primary transcript wrapping around the Cas6 to allow cleavage 8 nt upstream of the repeat spacer junction.[68][69][70] Type II systems lack the Cas6 gene and instead utilize RNaseIII for cleavage. Functional type II systems encode an extra small RNA that is complementary to the repeat sequence, known as a trans-activating RNA (tracrRNA).[40] Transcription of the tracrRNA and the primary CRISPR transcript results in base pairing and the formation of dsRNA at the repeat sequence, which is subsequently targeted by RNaseIII to produce crRNAs. Unlike the other two systems the crRNA does not contain the full spacer but instead is truncated at one end by 10 nt.[38]

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

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

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

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

CRISPR associated protein
crystal structure of a crispr-associated protein from thermus thermophilus
Identifiers
SymbolCRISPR_assoc
PfamPF08798
Pfam clanCL0362
InterProIPR010179
CDDcd09727
Available protein structures:
Pfam  structures / ECOD  
PDBRCSB PDB; PDBe; PDBj
PDBsumstructure summary
CRISPR associated protein Cas2
crystal structure of a hypothetical protein tt1823 from thermus thermophilus
Identifiers
SymbolCRISPR_Cas2
PfamPF09827
InterProIPR019199
CDDcd09638
Available protein structures:
Pfam  structures / ECOD  
PDBRCSB PDB; PDBe; PDBj
PDBsumstructure summary
CRISPR-associated protein Cse1
Identifiers
SymbolCRISPR_Cse1
PfamPF09481
InterProIPR013381
CDDcd09729
Available protein structures:
Pfam  structures / ECOD  
PDBRCSB PDB; PDBe; PDBj
PDBsumstructure summary
CRISPR-associated protein Cse2
Identifiers
SymbolCRISPR_Cse2
PfamPF09485
InterProIPR013382
CDDcd09670
Available protein structures:
Pfam  structures / ECOD  
PDBRCSB PDB; PDBe; PDBj
PDBsumstructure summary

Evolution and diversity

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

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

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

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

Bioinformatic identification of CRISPRs in genomes and metagenomes

CRISPRs are widely distributed amongst the bacteria and archaea [32] and show some sequence similarities,[29] however their most notable characteristic is their repeating spacers and direct repeats. This characteristic makes CRISPRs easily identifiable in long sequences of DNA, since the number of repeat copies decreases the likelihood of a false positive match. There are currently three programs used for CRISPR repeat identification that search for regularly interspaced repeats in long sequences: CRT,[84] PILER-CR [85] and CRISPRfinder.[86]

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

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

Evolutionary significance

A bioinformatic study showed that CRISPRs are evolutionarily conserved and cluster into related types. Many show signs of a conserved secondary structure.[29]

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

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

Applications

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

  • Artificial immunization against phage by introduction of engineered CRISPR loci in industrially important bacteria, including those used in food production and large-scale fermentation
  • Genome engineering at cellular or organismic level by reprogramming a CRISPR/Cas system to achieve RNA-guided genome engineering. Proof of concept studies demonstrated examples both in vitro[17][97] and in vivo[24][98][99]
  • Discrimination of bacterial strains by comparison of spacer sequences

Therapeutics

Editas Medicine, a $43 million startup, aims to develop treatments that employ CRISPR/Cas to make edits to single base pairs and larger stretches of DNA. Inherited diseases such as cystic fibrosis and sickle-cell anemia are caused by single base pair mutations; CRISPR/Cas technology has the potential to correct these errors. The "corrected" gene remains in its normal location on its chromosome, which preserves the way the cell normally activates and/or inhibits its expression.[100]

After harvesting blood cell precursors called hematopoietic stem cells from a patient's bone marrow, CRISPR gene surgery would correct the defective gene. Then the gene-corrected stem cells would be returned to the patient's marrow, which would then produce healthy red blood cells. Replacing 70% of the sickle cells would produce a cure.[27]

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

Other pathologies potentially treatable by CRISPR include Huntington’s disease, aging, schizophrenia and autism, not to mention modifying DNA in living embryos.[27]

Improved targeting is required before CRISPR can be used in medical applications. Current guide RNAs may target sequences that differ by multiple base pairs from the intended sequence.[16]

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

Mouse models

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

Agriculture

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

Functions

Editing

CRISPRs can add and delete base pairs at specifically targeted DNA loci. CRISPRs have been used to cut as many as five genes at once.[16]

Reversible knockdown

Main article: CRISPR interference

"CRISPRi" like RNAi, turns off genes in a reversible fashion by targeting but not cutting a site. In bacteria, the presence of Cas9 alone is enough to block transcription, but for mammalian applications, a section of protein is added. Its guide RNA targets regulatory DNA, called promoters that immediately precede the gene target.[16]

Activation

Main article: CRISPR interference

Cas9 was used to carry synthetic transcription factors (protein fragments that turn on genes) that activated specific human genes. The technique achieved a strong effect by targeting multiple CRISPR constructs to slightly different spots on the gene's promoter.[16]

The genes included some tied to human diseases, including those involved in muscle differentiation, cancer, inflammation and producing fetal hemoglobin.[16]

Use by phages

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

Automation and library support

Free software is available to design RNA to target any desired gene. The Addgene repository offers academics the DNA to make their own CRISPR system for $65. In 2013 Addgene distributed more than 10,000 CRISPR constructs. The facility has received CRISPR-enabling DNA sequences from 11 independent research teams.[16]

Patents and commercialization

As of December 2014, patent rights to CRISPR were still developing, and several companies had been formed to develop drugs and research tools based on the technology.[106]

See also

References

  1. ^ a b c Attention: This template ({{cite pmid}}) is deprecated. To cite the publication identified by PMID 20056882, please use {{cite journal}} with |pmid=20056882 instead.
  2. ^ a b c Marraffini LA, Sontheimer EJ (Mar 2010). "CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea". Nature Reviews. Genetics. 11 (3): 181–190. doi:10.1038/nrg2749. PMC 2928866. PMID 20125085.
  3. ^ 71/79 Archaea, 463/1008 Bacteria CRISPRdb, Date: 19.6.2010
  4. ^ a b Grissa I, Vergnaud G, Pourcel C (2007). "The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats". BMC Bioinformatics. 8: 172. doi:10.1186/1471-2105-8-172. PMC 1892036. PMID 17521438.{{cite journal}}: CS1 maint: unflagged free DOI (link)
  5. ^ Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, Romero DA, Horvath P (Mar 2007). "CRISPR provides acquired resistance against viruses in prokaryotes". Science. 315 (5819): 1709–1712. doi:10.1126/science.1138140. PMID 17379808.
  6. ^ a b Marraffini LA, Sontheimer EJ (Dec 2008). "CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA". Science. 322 (5909): 1843–1845. doi:10.1126/science.1165771. PMC 2695655. PMID 19095942.
  7. ^ Mali P, Esvelt KM, Church GM (Oct 2013). "Cas9 as a versatile tool for engineering biology". Nature Methods. 10 (10): 957–63. doi:10.1038/nmeth.2649. PMC 4051438. PMID 24076990.
  8. ^ Esvelt KM, Smidler AL, Catteruccia F, Church GM (Jul 2014). "Concerning RNA-guided gene drives for the alteration of wild populations". eLife. doi:10.7554/eLife.03401. PMID 25035423.{{cite journal}}: CS1 maint: unflagged free DOI (link)
  9. ^ Overballe-Petersen S, Harms K, Orlando LA, Mayar JV, Rasmussen S, Dahl TW, Rosing MT, Poole AM, Sicheritz-Ponten T, Brunak S, Inselmann S, de Vries J, Wackernagel W, Pybus OG, Nielsen R, Johnsen PJ, Nielsen KM, Willerslev E (Dec 2013). "Bacterial natural transformation by highly fragmented and damaged DNA". Proceedings of the National Academy of Sciences of the United States of America. 110 (49): 19860–5. doi:10.1073/pnas.1315278110. PMID 24248361.
    "Bacteria incorporate pieces of old DNA in their own genome, scientists discover". KurzweilAI. doi:10.1073/pnas.1315278110. Retrieved 2013-11-26.
  10. ^ Ishino Y, Shinagawa H, Makino K, Amemura M, Nakata A (Dec 1987). "Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product". Journal of Bacteriology. 169 (12): 5429–5433. PMC 213968. PMID 3316184.
  11. ^ Mojica FJ, Díez-Villaseñor C, Soria E, Juez G (Apr 2000). "Biological significance of a family of regularly spaced repeats in the genomes of Archaea, Bacteria and mitochondria". Molecular Microbiology. 36 (1): 244–246. doi:10.1046/j.1365-2958.2000.01838.x. PMID 10760181.
  12. ^ a b Jansen R, Embden JD, Gaastra W, Schouls LM (Mar 2002). "Identification of genes that are associated with DNA repeats in prokaryotes". Molecular Microbiology. 43 (6): 1565–1575. doi:10.1046/j.1365-2958.2002.02839.x. PMID 11952905.
  13. ^ a b c d Pourcel C, Salvignol G, Vergnaud G (Mar 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): 653–663. doi:10.1099/mic.0.27437-0. PMID 15758212.
  14. ^ a b Mojica FJ, Díez-Villaseñor C, García-Martínez J, Soria E (Feb 2005). "Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements". Journal of Molecular Evolution. 60 (2): 174–182. doi:10.1007/s00239-004-0046-3. PMID 15791728.
  15. ^ a b c Bolotin A, Quinquis B, Sorokin A, Ehrlich SD (Aug 2005). "Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin". Microbiology. 151 (Pt 8): 2551–2561. doi:10.1099/mic.0.28048-0. PMID 16079334.
  16. ^ a b c d e f g h i j k Pennisi E (Aug 2013). "The CRISPR craze". Science. 341 (6148): 833–836. doi:10.1126/science.341.6148.833. PMID 23970676.
  17. ^ a b Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (Aug 2012). "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity". Science. 337 (6096): 816–821. doi:10.1126/science.1225829. PMID 22745249.
  18. ^ "CRISPR gene therapy: Scientists call for more public debate around breakthrough technique - Science - News". The Independent. 2013-11-07. Retrieved 2013-11-25.
  19. ^ DiCarlo JE, Norville JE, Mali P, Rios X, Aach J, Church GM (Apr 2013). "Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems". Nucleic Acids Research. 41 (7): 4336–43. doi:10.1093/nar/gkt135. PMC 3627607. PMID 23460208.
  20. ^ Hwang WY, Fu Y, Reyon D, Maeder ML, Tsai SQ, Sander JD, Peterson RT, Yeh JR, Joung JK (Mar 2013). "Efficient genome editing in zebrafish using a CRISPR-Cas system". Nature Biotechnology. 31 (3): 227–9. doi:10.1038/nbt.2501. PMC 3686313. PMID 23360964.
  21. ^ Gratz SJ, Cummings AM, Nguyen JN, Hamm DC, Donohue LK, Harrison MM, Wildonger J, O'Connor-Giles KM (Aug 2013). "Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease". Genetics. 194 (4): 1029–35. doi:10.1534/genetics.113.152710. PMC 3730909. PMID 23709638.
  22. ^ Friedland AE, Tzur YB, Esvelt KM, Colaiácovo MP, Church GM, Calarco JA (Aug 2013). "Heritable genome editing in C. elegans via a CRISPR-Cas9 system". Nature Methods. 10 (8): 741–3. doi:10.1038/nmeth.2532. PMC 3822328. PMID 23817069.
  23. ^ Jiang W, Zhou H, Bi H, Fromm M, Yang B, Weeks DP (Nov 2013). "Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice". Nucleic Acids Research. 41 (20): e188. doi:10.1093/nar/gkt780. PMC 3814374. PMID 23999092.
  24. ^ a b 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): 910–918. doi:10.1016/j.cell.2013.04.025. PMID 23643243.
  25. ^ Larson MH, Gilbert LA, Wang X, Lim WA, Weissman JS, Qi LS (Nov 2013). "CRISPR interference (CRISPRi) for sequence-specific control of gene expression". Nature Protocols. 8 (11): 2180–96. doi:10.1038/nprot.2013.132. PMID 24136345.
  26. ^ "Researchers reverse a liver disorder in mice by correcting a mutated gene". PhysOrg. 30 March 2014. Retrieved 31 March 2014.
  27. ^ a b c Young, Susan (2014-02-11). "CRISPR and Other Genome Editing Tools Boost Medical Research and Gene Therapy's Reach | MIT Technology Review". Technologyreview.com. Retrieved 2014-04-13.
  28. ^ a b c Haft DH, Selengut J, Mongodin EF, Nelson KE (Nov 2005). "A guild of 45 CRISPR-associated (Cas) protein families and multiple CRISPR/Cas subtypes exist in prokaryotic genomes". PLoS Computational Biology. 1 (6): e60. doi:10.1371/journal.pcbi.0010060. PMC 1282333. PMID 16292354.{{cite journal}}: CS1 maint: unflagged free DOI (link)
  29. ^ a b c d Kunin V, Sorek R, Hugenholtz P (2007). "Evolutionary conservation of sequence and secondary structures in CRISPR repeats". Genome Biology. 8 (4): R61. doi:10.1186/gb-2007-8-4-r61. PMC 1896005. PMID 17442114.{{cite journal}}: CS1 maint: unflagged free DOI (link)
  30. ^ Stern A, Keren L, Wurtzel O, Amitai G, Sorek R (Aug 2010). "Self-targeting by CRISPR: gene regulation or autoimmunity?". Trends in Genetics. 26 (8): 335–340. doi:10.1016/j.tig.2010.05.008. PMC 2910793. PMID 20598393.
  31. ^ Tyson GW, Banfield JF (Jan 2008). "Rapidly evolving CRISPRs implicated in acquired resistance of microorganisms to viruses". Environmental Microbiology. 10 (1): 200–207. doi:10.1111/j.1462-2920.2007.01444.x. PMID 17894817.
  32. ^ a b c Chylinski K, Makarova KS, Charpentier E, Koonin EV (Jun 2014). "Classification and evolution of type II CRISPR-Cas systems". Nucleic Acids Research. 42 (10): 6091–105. doi:10.1093/nar/gku241. PMC 4041416. PMID 24728998.
  33. ^ a b c Wiedenheft B, Sternberg SH, Doudna JA (Feb 2012). "RNA-guided genetic silencing systems in bacteria and archaea". Nature. 482 (7385): 331–8. doi:10.1038/nature10886. PMID 22337052.
  34. ^ a b Deng L, Garrett RA, Shah SA, Peng X, She Q (Mar 2013). "A novel interference mechanism by a type IIIB CRISPR-Cmr module in Sulfolobus". Molecular Microbiology. 87 (5): 1088–99. doi:10.1111/mmi.12152. PMID 23320564.
  35. ^ Sinkunas T, Gasiunas G, Fremaux C, Barrangou R, Horvath P, Siksnys V (Apr 2011). "Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system". The EMBO Journal. 30 (7): 1335–42. doi:10.1038/emboj.2011.41. PMC 3094125. PMID 21343909.
  36. ^ a b c d Aliyari R, Ding SW (Jan 2009). "RNA-based viral immunity initiated by the Dicer family of host immune receptors". Immunological Reviews. 227 (1): 176–88. doi:10.1111/j.1600-065X.2008.00722.x. PMC 2676720. PMID 19120484.
  37. ^ a b Makarova KS, Aravind L, Wolf YI, Koonin EV (2011). "Unification of Cas protein families and a simple scenario for the origin and evolution of CRISPR-Cas systems". Biology Direct. 6: 38. doi:10.1186/1745-6150-6-38. PMC 3150331. PMID 21756346.{{cite journal}}: CS1 maint: unflagged free DOI (link)
  38. ^ a b c Gasiunas G, Barrangou R, Horvath P, Siksnys V (Sep 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–E2586. doi:10.1073/pnas.1208507109. PMC 3465414. PMID 22949671.
  39. ^ 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. doi:10.4161/rna.24321. PMC 3737331. PMID 23563642.
  40. ^ a b 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. doi:10.1371/journal.pgen.1003495. PMC 3656092. PMID 23696746.{{cite journal}}: CS1 maint: unflagged free DOI (link)
  41. ^ Hatoum-Aslan A, Maniv I, Marraffini LA (Dec 2011). "Mature clustered, regularly interspaced, short palindromic repeats RNA (crRNA) length is measured by a ruler mechanism anchored at the precursor processing site". Proceedings of the National Academy of Sciences of the United States of America. 108 (52): 21218–22. doi:10.1073/pnas.1112832108. PMC 3248500. PMID 22160698.
  42. ^ a b Yosef I, Goren MG, Qimron U (Jul 2012). "Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli". Nucleic Acids Research. 40 (12): 5569–76. doi:10.1093/nar/gks216. PMC 3384332. PMID 22402487.
  43. ^ a b c d Swarts DC, Mosterd C, van Passel MW, Brouns SJ (2012). "CRISPR interference directs strand specific spacer acquisition". PloS One. 7 (4): e35888. doi:10.1371/journal.pone.0035888. PMC 3338789. PMID 22558257.{{cite journal}}: CS1 maint: unflagged free DOI (link)
  44. ^ Babu M, Beloglazova N, Flick R, Graham C, Skarina T, Nocek B, Gagarinova A, Pogoutse O, Brown G, Binkowski A, Phanse S, Joachimiak A, Koonin EV, Savchenko A, Emili A, Greenblatt J, Edwards AM, Yakunin AF (Jan 2011). "A dual function of the CRISPR-Cas system in bacterial antivirus immunity and DNA repair". Molecular Microbiology. 79 (2): 484–502. doi:10.1111/j.1365-2958.2010.07465.x. PMC 3071548. PMID 21219465.
  45. ^ Han D, Lehmann K, Krauss G (Jun 2009). "SSO1450--a CAS1 protein from Sulfolobus solfataricus P2 with high affinity for RNA and DNA". FEBS Letters. 583 (12): 1928–32. doi:10.1016/j.febslet.2009.04.047. PMID 19427858.
  46. ^ Wiedenheft B, Zhou K, Jinek M, Coyle SM, Ma W, Doudna JA (Jun 2009). "Structural basis for DNase activity of a conserved protein implicated in CRISPR-mediated genome defense". Structure. 17 (6): 904–12. doi:10.1016/j.str.2009.03.019. PMID 19523907.
  47. ^ 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 (Jul 2008). "A novel family of sequence-specific endoribonucleases associated with the clustered regularly interspaced short palindromic repeats". The Journal of Biological Chemistry. 283 (29): 20361–71. doi:10.1074/jbc.M803225200. PMC 2459268. PMID 18482976.{{cite journal}}: CS1 maint: unflagged free DOI (link)
  48. ^ Samai P, Smith P, Shuman S (Dec 2010). "Structure of a CRISPR-associated protein Cas2 from Desulfovibrio vulgaris". Acta Crystallographica. Section F, Structural Biology and Crystallization Communications. 66 (Pt 12): 1552–6. doi:10.1107/S1744309110039801. PMC 2998353. PMID 21139194.
  49. ^ Nam KH, Ding F, Haitjema C, Huang Q, DeLisa MP, Ke A (Oct 2012). "Double-stranded endonuclease activity in Bacillus halodurans clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas2 protein". The Journal of Biological Chemistry. 287 (43): 35943–52. doi:10.1074/jbc.M112.382598. PMC 3476262. PMID 22942283.{{cite journal}}: CS1 maint: unflagged free DOI (link)
  50. ^ a b c d Horvath P, Romero DA, Coûté-Monvoisin AC, Richards M, Deveau H, Moineau S, Boyaval P, Fremaux C, Barrangou R (Feb 2008). "Diversity, activity, and evolution of CRISPR loci in Streptococcus thermophilus". Journal of Bacteriology. 190 (4): 1401–1412. doi:10.1128/JB.01415-07. PMC 2238196. PMID 18065539.
  51. ^ a b c Deveau H, Barrangou R, Garneau JE, Labonté J, Fremaux C, Boyaval P, Romero DA, Horvath P, Moineau S (Feb 2008). "Phage response to CRISPR-encoded resistance in Streptococcus thermophilus". Journal of Bacteriology. 190 (4): 1390–1400. doi:10.1128/JB.01412-07. PMC 2238228. PMID 18065545.
  52. ^ Mojica FJ, Díez-Villaseñor C, García-Martínez J, Almendros C (Mar 2009). "Short motif sequences determine the targets of the prokaryotic CRISPR defence system". Microbiology. 155 (Pt 3): 733–740. doi:10.1099/mic.0.023960-0. PMID 19246744.
  53. ^ a b Lillestøl RK, Shah SA, Brügger K, Redder P, Phan H, Christiansen J, Garrett RA (Apr 2009). "CRISPR families of the crenarchaeal genus Sulfolobus: bidirectional transcription and dynamic properties". Molecular Microbiology. 72 (1): 259–272. doi:10.1111/j.1365-2958.2009.06641.x. PMID 19239620.
  54. ^ a b Shah SA, Hansen NR, Garrett RA (Feb 2009). "Distribution of CRISPR spacer matches in viruses and plasmids of crenarchaeal acidothermophiles and implications for their inhibitory mechanism". Biochemical Society Transactions. 37 (Pt 1): 23–28. doi:10.1042/BST0370023. PMID 19143596.
  55. ^ a b 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. doi:10.4161/rna.24023. PMC 3737337. PMID 23445770.
  56. ^ a b c Erdmann S, Garrett RA (Sep 2012). "Selective and hyperactive uptake of foreign DNA by adaptive immune systems of an archaeon via two distinct mechanisms". Molecular Microbiology. 85 (6): 1044–56. doi:10.1111/j.1365-2958.2012.08171.x. PMC 3468723. PMID 22834906.
  57. ^ a b Shah SA, Erdmann S, Mojica FJ, Garrett RA (May 2013). "Protospacer recognition motifs: mixed identities and functional diversity". RNA Biology. 10 (5): 891–9. doi:10.4161/rna.23764. PMC 3737346. PMID 23403393.
  58. ^ Andersson AF, Banfield JF (May 2008). "Virus population dynamics and acquired virus resistance in natural microbial communities". Science. 320 (5879): 1047–1050. doi:10.1126/science.1157358. PMID 18497291.
  59. ^ a b Tyson GW, Banfield JF (Jan 2008). "Rapidly evolving CRISPRs implicated in acquired resistance of microorganisms to viruses". Environmental Microbiology. 10 (1): 200–207. doi:10.1111/j.1462-2920.2007.01444.x. PMID 17894817.
  60. ^ a b c d Pride DT, Sun CL, Salzman J, Rao N, Loomer P, Armitage GC, Banfield JF, Relman DA (Jan 2011). "Analysis of streptococcal CRISPRs from human saliva reveals substantial sequence diversity within and between subjects over time". Genome Research. 21 (1): 126–36. doi:10.1101/gr.111732.110. PMC 3012920. PMID 21149389.
  61. ^ a b Goren MG, Yosef I, Auster O, Qimron U (Oct 2012). "Experimental definition of a clustered regularly interspaced short palindromic duplicon in Escherichia coli". Journal of Molecular Biology. 423 (1): 14–6. doi:10.1016/j.jmb.2012.06.037. PMID 22771574.
  62. ^ a b c Datsenko KA, Pougach K, Tikhonov A, Wanner BL, Severinov K, Semenova E (2012). "Molecular memory of prior infections activates the CRISPR/Cas adaptive bacterial immunity system". Nature Communications. 3: 945. doi:10.1038/ncomms1937. PMID 22781758.
  63. ^ Tang TH, Bachellerie JP, Rozhdestvensky T, Bortolin ML, Huber H, Drungowski M, Elge T, Brosius J, Hüttenhofer A (May 2002). "Identification of 86 candidates for small non-messenger RNAs from the archaeon Archaeoglobus fulgidus". Proceedings of the National Academy of Sciences of the United States of America. 99 (11): 7536–41. doi:10.1073/pnas.112047299. PMC 124276. PMID 12032318.
  64. ^ Tang TH, Polacek N, Zywicki M, Huber H, Brugger K, Garrett R, Bachellerie JP, Hüttenhofer A (Jan 2005). "Identification of novel non-coding RNAs as potential antisense regulators in the archaeon Sulfolobus solfataricus". Molecular Microbiology. 55 (2): 469–81. doi:10.1111/j.1365-2958.2004.04428.x. PMID 15659164.
  65. ^ Gesner EM, Schellenberg MJ, Garside EL, George MM, Macmillan AM (Jun 2011). "Recognition and maturation of effector RNAs in a CRISPR interference pathway". Nature Structural & Molecular Biology. 18 (6): 688–92. doi:10.1038/nsmb.2042. PMID 21572444.
  66. ^ Sashital DG, Jinek M, Doudna JA (Jun 2011). "An RNA-induced conformational change required for CRISPR RNA cleavage by the endoribonuclease Cse3". Nature Structural & Molecular Biology. 18 (6): 680–7. doi:10.1038/nsmb.2043. PMID 21572442.
  67. ^ Haurwitz RE, Jinek M, Wiedenheft B, Zhou K, Doudna JA (Sep 2010). "Sequence- and structure-specific RNA processing by a CRISPR endonuclease". Science. 329 (5997): 1355–8. doi:10.1126/science.1192272. PMC 3133607. PMID 20829488.
  68. ^ Carte J, Wang R, Li H, Terns RM, Terns MP (Dec 2008). "Cas6 is an endoribonuclease that generates guide RNAs for invader defense in prokaryotes". Genes & Development. 22 (24): 3489–3496. doi:10.1101/gad.1742908. PMC 2607076. PMID 19141480.
  69. ^ Wang R, Preamplume G, Terns MP, Terns RM, Li H (Feb 2011). "Interaction of the Cas6 riboendonuclease with CRISPR RNAs: recognition and cleavage". Structure. 19 (2): 257–64. doi:10.1016/j.str.2010.11.014. PMC 3154685. PMID 21300293.
  70. ^ Niewoehner O, Jinek M, Doudna JA (Jan 2014). "Evolution of CRISPR RNA recognition and processing by Cas6 endonucleases". Nucleic Acids Research. 42 (2): 1341–53. doi:10.1093/nar/gkt922. PMC 3902920. PMID 24150936.
  71. ^ Garneau JE, Dupuis MÈ, Villion M, Romero DA, Barrangou R, Boyaval P, Fremaux C, Horvath P, Magadán AH, Moineau S (Nov 2010). "The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA". Nature. 468 (7320): 67–71. Bibcode:2010Natur.468...67G. doi:10.1038/nature09523. PMID 21048762.
  72. ^ Semenova E, Jore MM, Datsenko KA, Semenova A, Westra ER, Wanner B, van der Oost J, Brouns SJ, Severinov K (Jun 2011). "Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence". Proceedings of the National Academy of Sciences of the United States of America. 108 (25): 10098–103. doi:10.1073/pnas.1104144108. PMC 3121866. PMID 21646539.
  73. ^ Gudbergsdottir S, Deng L, Chen Z, Jensen JV, Jensen LR, She Q, Garrett RA (Jan 2011). "Dynamic properties of the Sulfolobus CRISPR/Cas and CRISPR/Cmr systems when challenged with vector-borne viral and plasmid genes and protospacers". Molecular Microbiology. 79 (1): 35–49. doi:10.1111/j.1365-2958.2010.07452.x. PMC 3025118. PMID 21166892.
  74. ^ Manica A, Zebec Z, Teichmann D, Schleper C (Apr 2011). "In vivo activity of CRISPR-mediated virus defence in a hyperthermophilic archaeon". Molecular Microbiology. 80 (2): 481–91. doi:10.1111/j.1365-2958.2011.07586.x. PMID 21385233.
  75. ^ 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. doi:10.1038/nsmb.2019. PMID 21460843.
  76. ^ Wiedenheft B, Lander GC, Zhou K, Jore MM, Brouns SJ, van der Oost J, Doudna JA, Nogales E (Sep 2011). "Structures of the RNA-guided surveillance complex from a bacterial immune system". Nature. 477 (7365): 486–9. doi:10.1038/nature10402. PMID 21938068.
  77. ^ 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 (Feb 2012). "Structure and mechanism of the CMR complex for CRISPR-mediated antiviral immunity". Molecular Cell. 45 (3): 303–13. doi:10.1016/j.molcel.2011.12.013. PMC 3381847. PMID 22227115.
  78. ^ a b Hale CR, Zhao P, Olson S, Duff MO, Graveley BR, Wells L, Terns RM, Terns MP (Nov 2009). "RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex". Cell. 139 (5): 945–956. doi:10.1016/j.cell.2009.07.040. PMC 2951265. PMID 19945378.
  79. ^ a b c Marraffini LA, Sontheimer EJ (Jan 2010). "Self versus non-self discrimination during CRISPR RNA-directed immunity". Nature. 463 (7280): 568–571. doi:10.1038/nature08703. PMC 2813891. PMID 20072129.
  80. ^ Touchon M, Rocha EP (2010). Randau L (ed.). "The small, slow and specialized CRISPR and anti-CRISPR of Escherichia and Salmonella". PloS One. 5 (6): e11126. doi:10.1371/journal.pone.0011126. PMC 2886076. PMID 20559554.{{cite journal}}: CS1 maint: unflagged free DOI (link)
  81. ^ a b c Rho M, Wu YW, Tang H, Doak TG, Ye Y (2012). "Diverse CRISPRs evolving in human microbiomes". PLoS Genetics. 8 (6): e1002441. doi:10.1371/journal.pgen.1002441. PMC 3374615. PMID 22719260.{{cite journal}}: CS1 maint: unflagged free DOI (link)
  82. ^ a b Sun CL, Barrangou R, Thomas BC, Horvath P, Fremaux C, Banfield JF (Feb 2013). "Phage mutations in response to CRISPR diversification in a bacterial population". Environmental Microbiology. 15 (2): 463–70. doi:10.1111/j.1462-2920.2012.02879.x. PMID 23057534.
  83. ^ 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. doi:10.1099/mic.0.073494-0. PMID 24586036.
  84. ^ Bland C, Ramsey TL, Sabree F, Lowe M, Brown K, Kyrpides NC, Hugenholtz P (2007). "CRISPR recognition tool (CRT): a tool for automatic detection of clustered regularly interspaced palindromic repeats". BMC Bioinformatics. 8: 209. doi:10.1186/1471-2105-8-209. PMC 1924867. PMID 17577412.{{cite journal}}: CS1 maint: unflagged free DOI (link)
  85. ^ Edgar RC (2007). "PILER-CR: fast and accurate identification of CRISPR repeats". BMC Bioinformatics. 8: 18. doi:10.1186/1471-2105-8-18. PMC 1790904. PMID 17239253.{{cite journal}}: CS1 maint: unflagged free DOI (link)
  86. ^ Grissa I, Vergnaud G, Pourcel C (Jul 2007). "CRISPRFinder: a web tool to identify clustered regularly interspaced short palindromic repeats". Nucleic Acids Research. 35 (Web Server issue): W52–7. doi:10.1093/nar/gkm360. PMC 1933234. PMID 17537822.
  87. ^ Pride DT, Salzman J, Relman DA (Sep 2012). "Comparisons of clustered regularly interspaced short palindromic repeats and viromes in human saliva reveal bacterial adaptations to salivary viruses". Environmental Microbiology. 14 (9): 2564–76. doi:10.1111/j.1462-2920.2012.02775.x. PMC 3424356. PMID 22583485.
  88. ^ Held NL, Herrera A, Whitaker RJ (Apr 2013). "Reassortment of CRISPR repeat-spacer loci in Sulfolobus islandicus". Environmental Microbiology. doi:10.1111/1462-2920.12146. PMID 23701169.
  89. ^ Held NL, Herrera A, Cadillo-Quiroz H, Whitaker RJ (2010). "CRISPR associated diversity within a population of Sulfolobus islandicus". PloS One. 5 (9). doi:10.1371/journal.pone.0012988. PMC 2946923. PMID 20927396.{{cite journal}}: CS1 maint: unflagged free DOI (link)
  90. ^ Skennerton CT, Imelfort M, Tyson GW (May 2013). "Crass: identification and reconstruction of CRISPR from unassembled metagenomic data". Nucleic Acids Research. 41 (10): e105. doi:10.1093/nar/gkt183. PMC 3664793. PMID 23511966.
  91. ^ Stern A, Mick E, Tirosh I, Sagy O, Sorek R (Oct 2012). "CRISPR targeting reveals a reservoir of common phages associated with the human gut microbiome". Genome Research. 22 (10): 1985–94. doi:10.1101/gr.138297.112. PMC 3460193. PMID 22732228.
  92. ^ Koonin EV, Wolf YI (2009). "Is evolution Darwinian or/and Lamarckian?". Biology Direct. 4: 42. doi:10.1186/1745-6150-4-42. PMC 2781790. PMID 19906303.{{cite journal}}: CS1 maint: unflagged free DOI (link)
  93. ^ 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. doi:10.1371/journal.pone.0004169. PMC 2612747. PMID 19132092.{{cite journal}}: CS1 maint: unflagged free DOI (link)
  94. ^ 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. doi:10.1038/nature12048. PMC 3651764. PMID 23584588.
  95. ^ Hale CR, Majumdar S, Elmore J, Pfister N, Compton M, Olson S, Resch AM, Glover CV, Graveley BR, Terns RM, Terns MP (Feb 2012). "Essential features and rational design of CRISPR RNAs that function with the Cas RAMP module complex to cleave RNAs". Molecular Cell. 45 (3): 292–302. doi:10.1016/j.molcel.2011.10.023. PMC 3278580. PMID 22227116.
  96. ^ Sorek R, Kunin V, Hugenholtz P (Mar 2008). "CRISPR--a widespread system that provides acquired resistance against phages in bacteria and archaea". Nature Reviews. Microbiology. 6 (3): 181–186. doi:10.1038/nrmicro1793. PMID 18157154.
  97. ^ Gasiunas G, Barrangou R, Horvath P, Siksnys V (Sep 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–E2586. doi:10.1073/pnas.1208507109. PMC 3465414. PMID 22949671.
  98. ^ Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F (Feb 2013). "Multiplex genome engineering using CRISPR/Cas systems". Science. 339 (6121): 819–823. doi:10.1126/science.1231143. PMC 3795411. PMID 23287718.
  99. ^ Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM (Feb 2013). "RNA-guided human genome engineering via Cas9". Science. 339 (6121): 823–826. doi:10.1126/science.1232033. PMC 3712628. PMID 23287722.
    Hou Z, Zhang Y, Propson NE, Howden SE, Chu LF, Sontheimer EJ, Thomson JA (Sep 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. doi:10.1073/pnas.1313587110.
  100. ^ a b Young, Susan. "Biotech Startup Editas Medicine Wants to Cure Grievous Genetic Diseases with New Genome Editing Technology | MIT Technology Review". Technologyreview.com. Retrieved 2013-11-30.
  101. ^ "Seamless gene correction of β-thalassemia mutations in patient-specific iPSCs using CRISPR/Cas9 and piggyBac".
  102. ^ Talbot, David (2014-07-19). "Beijing Researchers Use Gene Editing to Create Disease-Resistant Wheat | MIT Technology Review". Technologyreview.com. Retrieved 2014-07-23.
  103. ^ Wang Y, Cheng X, Shan Q, Zhang Y, Liu J, Gao C, Qiu JL (Sep 2014). "Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew". Nature Biotechnology. 32 (9). doi:10.1038/nbt.2969. PMID 25038773.
  104. ^ Novick RP, Christie GE, Penadés JR (Aug 2010). "The phage-related chromosomal islands of Gram-positive bacteria". Nature Reviews. Microbiology. 8 (8): 541–551. doi:10.1038/nrmicro2393. PMC 3522866. PMID 20634809.
  105. ^ Seed KD, Lazinski DW, Calderwood SB, Camilli A (Feb 2013). "A bacteriophage encodes its own CRISPR/Cas adaptive response to evade host innate immunity". Nature. 494 (7438): 489–491. doi:10.1038/nature11927. PMC 3587790. PMID 23446421.
  106. ^ Regalado A. "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 {{cite web}}: line feed character in |title= at position 55 (help)

Further reading

Retrieved from "https://en.wikipedia.org/w/index.php?title=CRISPR&oldid=649460399"