CRISPR

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CRISPR associated protein
PDB 1wj9 EBI.jpg
crystal structure of a crispr-associated protein from thermus thermophilus
Identifiers
SymbolCRISPR_assoc
PfamPF08798
Pfam clanCL0362
InterProIPR010179
CDDcd09727
 
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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 eubacteria 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 silence these exogenous genetic elements like RNAi in eukaryotic organisms.[2]

Since 2012, the CRISPR/Cas system has been used for gene editing (silencing, enhancing or changing specific genes) that even works in eukaryotes like mice and primates.[7] By inserting a plasmid containing cas genes and specifically designed CRISPRs, an organism's genome can be cut at any desired location.

History[edit]

Bacteria may incorporate foreign DNA in other circumstances and even to scavenge damaged DNA from their environment.[8]

Repeats were first described in 1987 for the bacterium Escherichia coli.[9] In 2000, similar clustered repeats were identified in additional bacteria and archaea and were termed Short Regularly Spaced Repeats (SRSR).[10] SRSR were renamed CRISPR in 2002.[11] 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).[11]

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. While most CRISPR loci contain each of the three components, the arrangement is not always as shown.[1][2]

In 2005, three independent researchers showed that CRISPR spacers showed homology to several phage DNA and extrachromosomal DNA such as plasmids. This was an indication that the CRISPR/cas system could have a role in adaptive immunity in bacteria.[1] Koonin and colleagues proposed that spacers serve as a template for RNA molecules, analogously to eukaryotic cells that use a system called RNA interference.[12]

In 2007 Barrangou, Horvath (food industry scientists at Danisco) and others showed that they could alter the resistance of Streptococcus thermophilus to phage attack with spacer DNA.[12]

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.[12]

Cas9 is a nuclease, an enzyme specialized for cutting DNA, with two active cutting sites, 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 located 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.[12]

CRISPR was first shown to work as a genome engineering/editing tool in human cell culture by 2012[13][14] It has since been used in a wide range of organisms including bakers yeast (S. cerevisiae),[15] zebra fish,[16] nematodes (C. elegans),[17] plants,[18] mice,[19] 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.[20]

Libraries of tens of thousands of guide RNAs are now available.[12]

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 liver disorder.[21]

Gene-editing predecessors[edit]

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.[22]

Locus structure[edit]

Repeats and spacers[edit]

CRISPR repeats range in size from 24 to 48 base pairs.[23] They usually show some dyad symmetry, implying the formation of a secondary structure such as a hairpin, but are not truly palindromic.[24] Repeats are separated by spacers of similar length.[23] Some CRISPR spacer sequences exactly match sequences from plasmids and phages,[25][26][27] although some spacers match the prokaryote's genome (self-targeting spacers).[28] New spacers can be added rapidly in response to phage infection.[29]

Cas genes and CRISPR subtypes[edit]

CRISPR-associated (cas) genes are often associated with CRISPR repeat-spacer arrays. As of 2013, more than forty different Cas protein families had been described.[23] Of these protein families, Cas1 appears to be ubiquitous among different CRISPR/Cas systems. Particular combinations of cas genes and repeat structures have been used to define 8 CRISPR subtypes (Ecoli, Ypest, Nmeni, Dvulg, Tneap, Hmari, Apern, and Mtube), some of which are associated with an additional gene module encoding repeat-associated mysterious proteins (RAMPs).[23] More than one CRISPR subtype may occur in a single genome. The sporadic distribution of the CRISPR/Cas subtypes suggests that the system is subject to horizontal gene transfer during microbial evolution.

CRISPR associated protein
PDB 1wj9 EBI.jpg
crystal structure of a crispr-associated protein from thermus thermophilus
Identifiers
SymbolCRISPR_assoc
PfamPF08798
Pfam clanCL0362
InterProIPR010179
CDDcd09727
CRISPR associated protein Cas2
PDB 1zpw EBI.jpg
crystal structure of a hypothetical protein tt1823 from thermus thermophilus
Identifiers
SymbolCRISPR_Cas2
PfamPF09827
InterProIPR019199
CDDcd09638
CRISPR-associated protein Cse1
Identifiers
SymbolCRISPR_Cse1
PfamPF09481
InterProIPR013381
CDDcd09729
CRISPR-associated protein Cse2
Identifiers
SymbolCRISPR_Cse2
PfamPF09485
InterProIPR013382
CDDcd09670

Mechanism[edit]

Exogenous DNA is apparently processed by proteins encoded by Cas genes into small elements (~30 base pairs in length), which are then somehow inserted into the CRISPR locus near the leader sequence. RNAs from the CRISPR loci are constitutively expressed and are processed by Cas proteins to small RNAs composed of individual, exogenously-derived sequence elements with a flanking repeat sequence. The RNAs guide other Cas proteins to silence exogenous genetic elements at the RNA or DNA level.[1][30] Evidence suggests functional diversity among CRISPR subtypes. The Cse (Cas subtype Ecoli) proteins (called CasA-E in E. coli) form a functional complex, Cascade, that processes CRISPR RNA transcripts into spacer-repeat units that Cascade retains.[31] In other prokaryotes, Cas6 processes the CRISPR transcripts. Interestingly, CRISPR-based phage inactivation in E. coli requires Cascade and Cas3, but not Cas1 and Cas2. The Cmr (Cas RAMP module) proteins found in Pyrococcus furiosus and other prokaryotes form a functional complex with small CRISPR RNAs that recognizes and cleaves complementary target RNAs. RNA-guided CRISPR enzymes are classified as type V restriction enzymes.

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.[24]

Through the CRISPR/Cas mechanism, bacteria can acquire immunity to certain phages and thus halt further transmission of targeted phages. For this reason, some researchers have proposed that CRISPR/Cas is a Lamarckian inheritance mechanism.[32] Others investigated the coevolution of host and viral genomes by analysis of CRISPR sequences.[33]

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.[34]

Applications[edit]

The proof-of-principle demonstration of selective engineered redirection of the CRISPR/Cas system in 2012[35] provided a first step toward realization of proposals for CRISPR-derived biotechnology:[36]

Therapeutics[edit]

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, sickle-cell anemia and Huntington's disease 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 activate inhibits its expression.[40]

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.[22]

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.[40]

Other pathologies potentially treatable by CRISPR include Huntington’s disease, aging, schizophrenia and autism, not to mention modifying DNA in living embryos.[22]

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.[12]

Mouse models[edit]

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]

Functions[edit]

Editing[edit]

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.[12]

Reversible knockdown[edit]

"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.[12]

Activation[edit]

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.[12]

The genes included some tied to human diseases, including those involved in muscle differentiation, cancer, inflammation and producing fetal hemoglobin.[12]

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.[41] 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.[42]

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.[12]

Patent[edit]

A provisional US patent application on the use of the CRISPR system for editing genes and regulating gene expression was filed by the inventors on May 12, 2012. Subsequent applications were combined and on March 6, 2014 the resulting patent application was published by the USPTO.[43] The patent rights have been assigned by the inventors to the Regents of the University of California and to the University of Vienna.

References[edit]

  1. ^ a b c d Horvath, P.; Barrangou, R. (2010). "CRISPR/Cas, the Immune System of Bacteria and Archaea". Science 327 (5962): 167–170. doi:10.1126/science.1179555. PMID 20056882.  edit
  2. ^ a b c Marraffini, L. A.; Sontheimer, E. J. (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.  edit
  3. ^ 71/79 Archaea, 463/1008 Bacteria CRISPRdb, Date: 19.6.2010
  4. ^ 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.  edit
  5. ^ Barrangou, R.; Fremaux, C.; Deveau, H.; Richards, M.; Boyaval, P.; Moineau, S.; Romero, D. A.; Horvath, P. (2007). "CRISPR Provides Acquired Resistance Against Viruses in Prokaryotes". Science 315 (5819): 1709–1712. doi:10.1126/science.1138140. PMID 17379808.  edit
  6. ^ Marraffini, L. A.; Sontheimer, E. J. (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.  edit
  7. ^ Wiedenheft, B; Sternberg, SH; Doudna, JA (Feb 15, 2012). "RNA-guided genetic silencing systems in bacteria and archaea.". Nature 482 (7385): 331–8. PMID 22337052. 
  8. ^ Overballe-Petersen, S.; Harms, K.; Orlando, L. A. A.; Mayar, J. V. M.; Rasmussen, S.; Dahl, T. W.; Rosing, M. T.; Poole, A. M.; Sicheritz-Ponten, T.; Brunak, S.; Inselmann, S.; De Vries, J.; Wackernagel, W.; Pybus, O. G.; Nielsen, R.; Johnsen, P. J.; Nielsen, K. M.; Willerslev, E. (2013). "Bacterial natural transformation by highly fragmented and damaged DNA". Proceedings of the National Academy of Sciences. doi:10.1073/pnas.1315278110.  edit
    "Bacteria incorporate pieces of old DNA in their own genome, scientists discover". KurzweilAI. doi:10.1073/pnas.1315278110. Retrieved 2013-11-26. 
  9. ^ Ishino, Y.; Shinagawa, H.; Makino, K.; Amemura, M.; Nakata, A. (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.  edit
  10. ^ Mojica, F. J.; Díez-Villaseñor, C.; Soria, E.; Juez, G. (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.  edit
  11. ^ a b Jansen, R.; Embden, J. D.; Gaastra, W.; Schouls, L. M. (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.  edit
  12. ^ a b c d e f g h i j k Pennisi, E. (2013). "The CRISPR Craze". Science 341 (6148): 833–836. doi:10.1126/science.341.6148.833. PMID 23970676.  edit
  13. ^ a b Jinek, M.; Chylinski, K.; Fonfara, I.; Hauer, M.; Doudna, J. A.; Charpentier, E. (2012). "A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity". Science 337 (6096): 816–821. doi:10.1126/science.1225829. PMID 22745249.  edit
  14. ^ "CRISPR gene therapy: Scientists call for more public debate around breakthrough technique - Science - News". The Independent. 2013-11-07. Retrieved 2013-11-25. 
  15. ^ Dicarlo, J. E.; Norville, J. E.; Mali, P; Rios, X; Aach, J; Church, G. M. (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.  edit
  16. ^ Hwang, W. Y.; Fu, Y; Reyon, D; Maeder, M. L.; Tsai, S. Q.; Sander, J. D.; Peterson, R. T.; Yeh, J. R.; Joung, J. K. (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.  edit
  17. ^ Friedland, A. E.; Tzur, Y. B.; Esvelt, K. M.; Colaiácovo, M. P.; Church, G. M.; Calarco, J. A. (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.  edit
  18. ^ Jiang, W; Zhou, H; Bi, H; Fromm, M; Yang, B; Weeks, D. P. (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.  edit
  19. ^ a b Wang, H.; Yang, H.; Shivalila, C. S.; Dawlaty, M. M.; Cheng, A. W.; Zhang, F.; Jaenisch, R. (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.  edit
  20. ^ Larson, M. H.; Gilbert, L. A.; Wang, X; Lim, W. A.; Weissman, J. S.; Qi, L. S. (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.  edit
  21. ^ "Researchers reverse a liver disorder in mice by correcting a mutated gene". PhysOrg. 30 March 2014. Retrieved 31 March 2014. 
  22. ^ 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. 
  23. ^ a b c d Haft, D. H.; Selengut, J.; Mongodin, E. F.; Nelson, K. E. (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.  edit
  24. ^ a b 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.  edit
  25. ^ Mojica, F. J. M.; Díez-Villaseñor, C. S.; García-Martínez, J. S.; Soria, E. (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.  edit
  26. ^ Bolotin, A.; Quinquis, B.; Sorokin, A.; Ehrlich, S. D. (2005). "Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin". Microbiology 151 (8): 2551–2561. doi:10.1099/mic.0.28048-0. PMID 16079334.  edit
  27. ^ Pourcel, C.; Salvignol, G.; Vergnaud, G. (2005). "CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies". Microbiology 151 (3): 653–663. doi:10.1099/mic.0.27437-0. PMID 15758212.  edit
  28. ^ Stern, A.; Keren, L.; Wurtzel, O.; Amitai, G.; Sorek, R. (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.  edit
  29. ^ Tyson, G. W.; Banfield, J. F. (2007). "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.  edit
  30. ^ Makarova, K. S.; Grishin, N. V.; Shabalina, S. A.; Wolf, Y. I.; Koonin, E. V. (2006). "A putative RNA-interference-based immune system in prokaryotes: Computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action". Biology Direct 1: 7. doi:10.1186/1745-6150-1-7. PMC 1462988. PMID 16545108.  edit
  31. ^ Brouns, S. J. J.; Jore, M. M.; Lundgren, M.; Westra, E. R.; Slijkhuis, R. J. H.; Snijders, A. P. L.; Dickman, M. J.; Makarova, K. S.; Koonin, E. V.; Van Der Oost, J. (2008). "Small CRISPR RNAs Guide Antiviral Defense in Prokaryotes". Science 321 (5891): 960–964. doi:10.1126/science.1159689. PMID 18703739.  edit
  32. ^ Koonin, E. V.; Wolf, Y. I. (2009). "Is evolution Darwinian or/and Lamarckian?". Biology Direct 4: 42. doi:10.1186/1745-6150-4-42. PMC 2781790. PMID 19906303.  edit
  33. ^ Heidelberg, J. F.; Nelson, W. C.; Schoenfeld, T.; Bhaya, D. (2009). "Germ Warfare in a Microbial Mat Community: CRISPRs Provide Insights into the Co-Evolution of Host and Viral Genomes". In Ahmed, Niyaz. PLoS ONE 4 (1): e4169. doi:10.1371/journal.pone.0004169. PMC 2612747. PMID 19132092.  edit
  34. ^ Sampson, T. R.; Saroj, S. D.; Llewellyn, A. C.; Tzeng, Y. L.; Weiss, D. S. (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.  edit
  35. ^ Hale, C. R.; Majumdar, S.; Elmore, J.; Pfister, N.; Compton, M.; Olson, S.; Resch, A. M.; Glover Cv, C. V. C.; Graveley, B. R.; Terns, R. M.; Terns, M. P. (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.  edit
  36. ^ Sorek, R.; Kunin, V.; Hugenholtz, P. (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.  edit
  37. ^ Gasiunas, G.; Barrangou, R.; Horvath, P.; Siksnys, V. (2012). "Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria". Proceedings of the National Academy of Sciences 109 (39): E2579–E2586. doi:10.1073/pnas.1208507109. PMC 3465414. PMID 22949671.  edit
  38. ^ Cong, L.; Ran, F. A.; Cox, D.; Lin, S.; Barretto, R.; Habib, N.; Hsu, P. D.; Wu, X.; Jiang, W.; Marraffini, L. A.; Zhang, F. (2013). "Multiplex Genome Engineering Using CRISPR/Cas Systems". Science 339 (6121): 819–823. doi:10.1126/science.1231143. PMC 3795411. PMID 23287718.  edit
  39. ^ Mali, P.; Yang, L.; Esvelt, K. M.; Aach, J.; Guell, M.; Dicarlo, J. E.; Norville, J. E.; Church, G. M. (2013). "RNA-Guided Human Genome Engineering via Cas9". Science 339 (6121): 823–826. doi:10.1126/science.1232033. PMC 3712628. PMID 23287722.  edit
    Hou, Z.; Zhang, Y.; Propson, N. E.; Howden, S. E.; Chu, L. -F.; Sontheimer, E. J.; Thomson, J. A. (2013). "Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis". Proceedings of the National Academy of Sciences 110 (39): 15644. doi:10.1073/pnas.1313587110.  edit
  40. ^ 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. 
  41. ^ Novick, R. P.; Christie, G. E.; Penadés, J. R. (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.  edit
  42. ^ Seed, K. D.; Lazinski, D. W.; Calderwood, S. B.; Camilli, A. (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.  edit
  43. ^ "Methods And Compositions For Rna-directed Target Dna Modification And For Rna-directed Modulation Of Transcription". Freshpatents.com. Retrieved 2014-04-13. 

Further reading[edit]

External links[edit]