CRISPR

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Diagram of the possible mechanism for CRISPR.[1]

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.[2] It is pronounced "crisper".[3]

The CRISPR/Cas system is a prokaryotic immune system that confer resistance to foreign genetic elements such as plasmids and phages,[4][5] 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] CRISPRs are found in approximately 40% of sequenced bacteria genomes and 90% of sequenced archaea.[6][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.[7] 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.[8][9][10]

History[edit]

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.[11]

Clustered repeats were first described in 1987 for the bacterium Escherichia coli, but at that time their function was not known.[12] In 2000, similar repeats were identified in other bacteria and archaea, and were termed Short Regularly Spaced Repeats (SRSR).[13] SRSR were renamed CRISPR in 2002.[14] A set of genes were found to be associated with CRISPR repeats, and were named the cas, or CRISPR-associated genes. These genes encoded putative nuclease or helicase proteins, which are enzymes that can cut DNA.[14]

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.[6]

In 2005, three independent research groups showed that some CRISPR spacers are derived from phage DNA and extrachromosomal DNA such as plasmids.[15][16][17] 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.[1][18] Koonin and colleagues proposed that spacers serve as a template for RNA molecules, analogous to a system called RNA interference used by eukaryotic cells.[19]

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.[19]

Doudna and Charpentier had independently been exploring CRISPR-associated proteins to learn how bacteria use spacers in their immune defenses.[20] 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.[19]

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.[19]

CRISPR was first shown to work as a genome engineering/editing tool in human cell culture by 2012.[21][22] It has since been used in a wide range of organisms including baker's yeast (S. cerevisiae),[23] zebrafish (D. rerio),[24] flies (D. melanogaster),[25] axolotl (A. mexicanum),[26] nematodes (C. elegans),[27] plants,[28] mice,[29] monkeys,[30] nonviable human embryos,[31] and other organisms.

Additionally CRISPR has been modified to make programmable transcription factors that allow scientists to target and activate or silence specific genes.[32]

Libraries of tens of thousands of guide RNAs are now available.[19]

Gene-editing predecessors[edit]

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.[33]

Locus structure[edit]

Repeats and spacers[edit]

CRISPR loci range in size from 24 to 48 base pairs.[34] They usually show some dyad symmetry, implying the formation of a secondary structure such as a hairpin, but are not truly palindromic.[35] Repeats are separated by spacers of similar length.[34] Some CRISPR spacer sequences exactly match sequences from plasmids and phages,[15][16][17] although some spacers match the prokaryote's genome (self-targeting spacers).[15][36] New spacers can be added rapidly as part of the immune response to phage infection.[37]

Cas genes and CRISPR subtypes[edit]

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, and that only the cas1 and cas2 genes are universally present in the 45 families.[34] The current CRISPR classification groups cas operons into three major divisions, each with multiple subdivisions based on cas1 phylogeny and cas operon gene complement.[38] 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.[39][40] The sporadic distribution of the CRISPR/Cas subtypes suggests that the CRISPR/Cas 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 [41]
IA Cas8a Subunit of the interference module. Important in targeting of invading DNA by recognizing the PAM sequence [42][43]
IB Cas8b
IC Cas8c
ID Cas10d contains a domain homologous to the palm domain of nucleic acid polymerases and nucleotide cyclases [38][44]
IE Cse1
IF Csy1 Not Determined
II Cas9 Nucleases RuvC and HNH together produce DSBs, and separately can produce single-strand breaks. Ensures the acquisition of functional spacers during adaptation. [45][46]
IIA Csn2 Not Determined
IIB Cas4 Not Determined
IIC Characterized by the absence of either Csn2 or Cas4 [47]
III Cas10 Homolog of Cas10d and Cse1 [44]
IIIA Csm2 Not Determined
IIIB Cmr5 Not Determined

Mechanism[edit]

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[edit]

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.[42][48][49][50][51] The exact function of Cas1 and Cas2 is unknown, however a number of Cas1 proteins have been biochemically characterised and their structures resolved.[52][53][54] 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.[39] Representative Cas2 proteins have also been characterised and possess either ssRNA [55] or dsDNA [56][57] 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 selected 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.[17][58][59][60][61][62] 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.[63][64] The conservation of the PAM sequence differs between CRISPR-Cas systems and appears to be evolutionarily linked to cas1 and the leader sequence.[62][65]

New spacers are added to a CRISPR array in a directional manner,[15] occurring preferentially [37][58][59][66][67] but not exclusively adjacent [61][64] 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.[50][63] 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.[51][68][69] 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.[65] 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.[64]

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.[51][68][69] This observation has led to the hypothesis that the acquisition machinery slides along the foreign DNA after priming to find a new protospacer.[69]

Interference stage[edit]

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.[42][70][71] 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 [72][73][74] created by the palindromic nature of the direct repeats.[35] 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.[75][76][77] 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).[48] 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.[45]

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.[5][42][51][78][79][80][81] 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.[82][83] 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.[45] 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.[84][85] Surprisingly, the type III systems analysed from S. solfataricus and P. furiosus have both target the mRNA of phage/plasmids,[40][85] which may make these systems uniquely capable of targeting RNA based phage genomes.[39]

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.[86] RNA-guided CRISPR enzymes are classified as type V restriction enzymes.

CRISPR associated protein
PDB 1wj9 EBI.jpg
crystal structure of a crispr-associated protein from thermus thermophilus
Identifiers
Symbol CRISPR_assoc
Pfam PF08798
Pfam clan CL0362
InterPro IPR010179
CDD cd09727
CRISPR associated protein Cas2
PDB 1zpw EBI.jpg
crystal structure of a hypothetical protein tt1823 from thermus thermophilus
Identifiers
Symbol CRISPR_Cas2
Pfam PF09827
InterPro IPR019199
CDD cd09638
CRISPR-associated protein Cse1
Identifiers
Symbol CRISPR_Cse1
Pfam PF09481
InterPro IPR013381
CDD cd09729
CRISPR-associated protein Cse2
Identifiers
Symbol CRISPR_Cse2
Pfam PF09485
InterPro IPR013382
CDD cd09670

Evolution and diversity[edit]

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.[86] Similar stringency is required in PAM or the bacterial strain will remain phage sensitive.[59][86] 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 strains of S. thermophilus showed that 26% of all spacers were unique and that different CRISPR loci showed different rates of new spacer acquisition.[58] 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.[87]

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.[37] 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.[67] 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,[67] 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.[88] 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.[88]

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.[89] 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.[89] 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.[90] 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[edit]

CRISPRs are widely distributed amongst the bacteria and archaea [38] and show some sequence similarities,[35] 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,[91] PILER-CR [92] and CRISPRfinder.[93]

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.[58][67][94][95][96] 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 [97] or by using direct repeat sequences in partially assembled CRISPR arrays from contigs [88] and direct repeat sequences from published genomes [98] as a hook for identifying direct repeats in individual reads.

Evolutionary significance[edit]

A bioinformatic study showed that CRISPRs are evolutionarily conserved and cluster into related types. Many show signs of a conserved secondary structure.[35]

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.[99] Others investigated the coevolution of host and viral genomes by analysis of CRISPR sequences.[100]

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.[101]

Applications[edit]

The proof-of-principle demonstration of selective engineered redirection of the CRISPR/Cas system in 2012[102] provided a first step toward realization of proposals for CRISPR-derived biotechnology:[103]

  • 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[10][21][45] and in vivo[29][104][105]
  • Discrimination of bacterial strains by comparison of spacer sequences

In particular the low cost of the CRISPR approach compared to alternatives is widely seen as revolutionary.[8][9]

Therapeutics[edit]

Editas Medicine was founded in 2013 with $43 million in venture capital financing, with the goal of developing treatments that employ CRISPR/Cas to make edits to single base pairs and larger stretches of DNA. The company plans to use CRISPR by harvesting hematopoietic stem cells from a patient's bone marrow, correcting one or more defective genes with CRISPR, and then returning the cells to the patient's marrow, which would then produce healthy red blood cells.[33] It aims to treat cystic fibrosis and sickle-cell anemia, each of which are caused by single base pair mutations.[106]

Animal models[edit]

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.[107][108][109]

Non-animal research[edit]

A major current application of CRISPR technology is in research, and by the end of 2014 about 600 research papers had been published mentioning CRISPR.[110] The technology has been used to functionally inactivate genes in cell lines and cells taken directly from humans, study Candida albicans, yeasts used to make biofuels, and make genetically modified crops.[110]

Agriculture[edit]

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.[111][112]

Populations[edit]

It may be possible to use CRISPR to build RNA-guided gene drives capable of altering the genomes of entire populations.[113]

Functions[edit]

Editing[edit]

CRISPRs can add and delete base pairs at specifically targeted DNA loci.[10] CRISPRs have been used to cut as many as five genes at once.[19]

Reversible knockdown[edit]

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.[19]

Activation[edit]

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.[19]

The genes included some tied to human diseases, including those involved in muscle differentiation, cancer, inflammation and producing fetal hemoglobin.[19]

Use by phages[edit]

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.[114] 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.[115]

Automation and library support[edit]

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.[19]

Patents and commercialization[edit]

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.[116]

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.[117]

Society and culture[edit]

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,[118] 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".[31][119] 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.[120]

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.[121][122] 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.[123] 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.”[123]

See also[edit]

Notes[edit]

  1. ^ 71/79 Archaea, 463/1008 Bacteria CRISPRdb, Date: 19.6.2010

References[edit]

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