Review Article | Published:

Applications of CRISPR technologies in research and beyond

Nature Biotechnology volume 34, pages 933941 (2016) | Download Citation

Abstract

Programmable DNA cleavage using CRISPR–Cas9 enables efficient, site-specific genome engineering in single cells and whole organisms. In the research arena, versatile CRISPR-enabled genome editing has been used in various ways, such as controlling transcription, modifying epigenomes, conducting genome-wide screens and imaging chromosomes. CRISPR systems are already being used to alleviate genetic disorders in animals and are likely to be employed soon in the clinic to treat human diseases of the eye and blood. Two clinical trials using CRISPR-Cas9 for targeted cancer therapies have been approved in China and the United States. Beyond biomedical applications, these tools are now being used to expedite crop and livestock breeding, engineer new antimicrobials and control disease-carrying insects with gene drives.

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References

  1. 1.

    The CRISPR craze. Science 341, 833–836 (2013).

  2. 2.

    CRISPR, the disruptor. Nature 522, 20–24 (2015).

  3. 3.

    et al. Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. J. Bacteriol. 190, 1390–1400 (2008).

  4. 4.

    et al. Diversity, activity, and evolution of CRISPR loci in Streptococcus thermophilus. J. Bacteriol. 190, 1401–1412 (2008).

  5. 5.

    , , & Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology 155, 733–740 (2009).

  6. 6.

    , , & Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature 513, 569–573 (2014).

  7. 7.

    , & Mechanism of substrate selection by a highly specific CRISPR endoribonuclease. RNA 18, 661–672 (2012).

  8. 8.

    et al. Systematic analysis of CRISPR–Cas9 mismatch tolerance reveals low levels of off-target activity. J. Biotechnol. 211, 56–65 (2015).

  9. 9.

    et al. Direct observation of R-loop formation by single RNA-guided Cas9 and Cascade effector complexes. Proc. Natl. Acad. Sci. USA 111, 9798–9803 (2014).

  10. 10.

    et al. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 468, 67–71 (2010).

  11. 11.

    et al. Dynamics of CRISPR–Cas9 genome interrogation in living cells. Science 350, 823–826 (2015).

  12. 12.

    , , & Conformational control of DNA target cleavage by CRISPR–Cas9. Nature 527, 110–113 (2015).

  13. 13.

    , & ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol. 31, 397–405 (2013).

  14. 14.

    & Group II introns: mobile ribozymes that invade DNA. Cold Spring Harb. Perspect. Biol. 3, a003616 (2011).

  15. 15.

    & Genome editing. The new frontier of genome engineering with CRISPR–Cas9. Science 346, 1258096 (2014).

  16. 16.

    RNA events. Cas9 targeting and the CRISPR revolution. Science 344, 707–708 (2014).

  17. 17.

    , , & DNA repair and genome maintenance in Bacillus subtilis. Microbiol. Mol. Biol. Rev. 76, 530–564 (2012).

  18. 18.

    Alternative end-joining mechanisms: a historical perspective. Front. Genet. 4, 48 (2013).

  19. 19.

    , , & Alternative-NHEJ is a mechanistically distinct pathway of mammalian chromosome break repair. PLoS Genet. 4, e1000110 (2008).

  20. 20.

    , , , & MMEJ-assisted gene knock-in using TALENs and CRISPR–Cas9 with the PITCh systems. Nat. Protoc. 11, 118–133 (2016).

  21. 21.

    , & Development and applications of CRISPR–Cas9 for genome engineering. Cell 157, 1262–1278 (2014).

  22. 22.

    & CRISPR–Cas systems for editing, regulating and targeting genomes. Nat. Biotechnol. 32, 347–355 (2014).

  23. 23.

    , , , & Dimeric CRISPR RNA-guided FokI-dCas9 nucleases directed by truncated gRNAs for highly specific genome editing. Hum. Gene Ther. 26, 425–431 (2015).

  24. 24.

    et al. Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nat. Biotechnol. 32, 569–576 (2014).

  25. 25.

    , , , & Improving CRISPR–Cas nuclease specificity using truncated guide RNAs. Nat. Biotechnol. 32, 279–284 (2014).

  26. 26.

    et al. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat. Biotechnol. 31, 833–838 (2013).

  27. 27.

    & Genome editing using Cas9 nickases. Methods Enzymol. 546, 161–174 (2014).

  28. 28.

    et al. Genome engineering using the CRISPR–Cas9 system. Nat. Protoc. 8, 2281–2308 (2013).

  29. 29.

    et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR–Cas system. Cell 163, 759–771 (2015).

  30. 30.

    et al. Discovery and functional characterization of diverse class 2 CRISPR–Cas systems. Mol. Cell 60, 385–397 (2015).

  31. 31.

    et al. Genome-scale CRISPR-mediated control of gene repression and activation. Cell 159, 647–661 (2014).

  32. 32.

    et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, 442–451 (2013).

  33. 33.

    et al. CRISPR interference (CRISPRi) for sequence-specific control of gene expression. Nat. Protoc. 8, 2180–2196 (2013).

  34. 34.

    et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 1173–1183 (2013).

  35. 35.

    et al. Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell 155, 1479–1491 (2013).

  36. 36.

    et al. Multiplexed labeling of genomic loci with dCas9 and engineered sgRNAs using CRISPRainbow. Nat. Biotechnol. 34, 528–530 (2016).

  37. 37.

    et al. Epigenome editing by a CRISPR–Cas9-based acetyltransferase activates genes from promoters and enhancers. Nat. Biotechnol. 33, 510–517 (2015).

  38. 38.

    et al. Functional annotation of native enhancers with a Cas9-histone demethylase fusion. Nat. Methods 12, 401–403 (2015).

  39. 39.

    & Enabling functional genomics with genome engineering. Genome Res. 25, 1442–1455 (2015).

  40. 40.

    et al. Guide RNA functional modules direct Cas9 activity and orthogonality. Mol. Cell 56, 333–339 (2014).

  41. 41.

    et al. Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nat. Methods 10, 1116–1121 (2013).

  42. 42.

    et al. Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR–Cas systems. Nucleic Acids Res. 42, 2577–2590 (2014).

  43. 43.

    et al. Orthogonal gene knockout and activation with a catalytically active Cas9 nuclease. Nat. Biotechnol. 33, 1159–1161 (2015).

  44. 44.

    et al. Chemically modified guide RNAs enhance CRISPR–Cas genome editing in human primary cells. Nat. Biotechnol. 33, 985–989 (2015).

  45. 45.

    et al. Synthetic CRISPR RNA-Cas9-guided genome editing in human cells. Proc. Natl. Acad. Sci. USA 112, E7110–E7117 (2015).

  46. 46.

    et al. Streptococcus thermophilus CRISPR–Cas9 systems enable specific editing of the human genome. Mol. Ther. 24, 636–644 (2016).

  47. 47.

    et al. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 156, 935–949 (2014).

  48. 48.

    , & In vitro reconstitution and crystallization of Cas9 endonuclease bound to a guide RNA and a DNA target. Methods Enzymol. 558, 515–537 (2015).

  49. 49.

    & In vitro enzymology of Cas9. Methods Enzymol. 546, 1–20 (2014).

  50. 50.

    et al. Structures of Cas9 endonucleases reveal RNA-mediated conformational activation. Science 343, 1247997 (2014).

  51. 51.

    et al. Engineered CRISPR–Cas9 nucleases with altered PAM specificities. Nature 523, 481–485 (2015).

  52. 52.

    & The structural biology of CRISPR–Cas systems. Curr. Opin. Struct. Biol. 30, 100–111 (2015).

  53. 53.

    , , , & STRUCTURAL BIOLOGY. A Cas9-guide RNA complex preorganized for target DNA recognition. Science 348, 1477–1481 (2015).

  54. 54.

    et al. Crystal structure of Cpf1 in complex with guide RNA and target DNA. Cell 165, 949–962 (2016).

  55. 55.

    et al. The crystal structure of Cpf1 in complex with CRISPR RNA. Nature 532, 522–526 (2016).

  56. 56.

    et al. Rationally engineered Cas9 nucleases with improved specificity. Science 351, 84–88 (2016).

  57. 57.

    et al. High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529, 490–495 (2016).

  58. 58.

    et al. Identifying and visualizing functional PAM diversity across CRISPR–Cas systems. Mol. Cell 62, 137–147 (2016).

  59. 59.

    et al. Rational design of a split-Cas9 enzyme complex. Proc. Natl. Acad. Sci. USA 112, 2984–2989 (2015).

  60. 60.

    , & A split-Cas9 architecture for inducible genome editing and transcription modulation. Nat. Biotechnol. 33, 139–142 (2015).

  61. 61.

    , , , & Enhancing homology-directed genome editing by catalytically active and inactive CRISPR–Cas9 using asymmetric donor DNA. Nat. Biotechnol. 34, 339–344 (2016).

  62. 62.

    et al. DNA-binding-domain fusions enhance the targeting range and precision of Cas9. Nat. Methods 12, 1150–1156 (2015).

  63. 63.

    , , & Photoactivatable CRISPR–Cas9 for optogenetic genome editing. Nat. Biotechnol. 33, 755–760 (2015).

  64. 64.

    , , , & CRISPR–Cas9-based photoactivatable transcription system. Chem. Biol. 22, 169–174 (2015).

  65. 65.

    et al. Inducible in vivo genome editing with CRISPR–Cas9. Nat. Biotechnol. 33, 390–394 (2015).

  66. 66.

    et al. Increasing the efficiency of precise genome editing with CRISPR–Cas9 by inhibition of nonhomologous end joining. Nat. Biotechnol. 33, 538–542 (2015).

  67. 67.

    et al. Increasing the efficiency of homology-directed repair for CRISPR–Cas9-induced precise gene editing in mammalian cells. Nat. Biotechnol. 33, 543–548 (2015).

  68. 68.

    , & High-throughput functional genomics using CRISPR–Cas9. Nat. Rev. Genet. 16, 299–311 (2015).

  69. 69.

    et al. Small molecules enhance CRISPR genome editing in pluripotent stem cells. Cell Stem Cell 16, 142–147 (2015).

  70. 70.

    et al. Advances in CRISPR–Cas9 genome engineering: lessons learned from RNA interference. Nucleic Acids Res. 43, 3407–3419 (2015).

  71. 71.

    et al. Genome-wide CRISPR screen in a mouse model of tumor growth and metastasis. Cell 160, 1246–1260 (2015).

  72. 72.

    et al. Next-generation libraries for robust RNA interference-based genome-wide screens. Proc. Natl. Acad. Sci. USA 112, E3384–E3391 (2015).

  73. 73.

    & Functional genetics for all: engineered nucleases, CRISPR and the gene editing revolution. Evodevo 5, 43 (2014).

  74. 74.

    , & Functional genomics platform for pooled screening and generation of mammalian genetic interaction maps. Nat. Protoc. 9, 1825–1847 (2014).

  75. 75.

    et al. MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens. Genome Biol. 15, 554 (2014).

  76. 76.

    et al. Adapting CRISPR/Cas9 for functional genomics screens. Methods Enzymol. 546, 193–213 (2014).

  77. 77.

    , , & Genetic screens in human cells using the CRISPR–Cas9 system. Science 343, 80–84 (2014).

  78. 78.

    , , , & Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library. Nat. Biotechnol. 32, 267–273 (2014).

  79. 79.

    et al. Genome-scale transcriptional activation by an engineered CRISPR–Cas9 complex. Nature 517, 583–588 (2015).

  80. 80.

    et al. Identification and characterization of essential genes in the human genome. Science 350, 1096–1101 (2015).

  81. 81.

    et al. CRISPR knockout screening outperforms shRNA and CRISPRi in identifying essential genes. Nat. Biotechnol. 34, 631–633 (2016).

  82. 82.

    et al. Identification of potential drug targets for tuberous sclerosis complex by synthetic screens combining CRISPR-based knockouts with RNAi. Sci. Signal. 8, rs9 (2015).

  83. 83.

    et al. A genome-wide CRISPR screen in primary immune cells to dissect regulatory networks. Cell 162, 675–686 (2015).

  84. 84.

    et al. High-resolution CRISPR screens reveal fitness genes and genotype-specific cancer liabilities. Cell 163, 1515–1526 (2015).

  85. 85.

    et al. ASXL1 mutation correction by CRISPR/Cas9 restores gene function in leukemia cells and increases survival in mouse xenografts. Oncotarget 6, 44061–44071 (2015).

  86. 86.

    et al. CRISPR/Cas9 somatic multiplex-mutagenesis for high-throughput functional cancer genomics in mice. Proc. Natl. Acad. Sci. USA 112, 13982–13987 (2015).

  87. 87.

    et al. Functional genetic screens for enhancer elements in the human genome using CRISPR–Cas9. Nat. Biotechnol. 34, 192–198 (2016).

  88. 88.

    et al. High-throughput mapping of regulatory DNA. Nat. Biotechnol. 34, 167–174 (2016).

  89. 89.

    et al. Tailored pig models for preclinical efficacy and safety testing of targeted therapies. Toxicol. Pathol. 44, 346–357 (2016).

  90. 90.

    et al. Genome-wide inactivation of porcine endogenous retroviruses (PERVs). Science 350, 1101–1104 (2015).

  91. 91.

    et al. Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos. Cell 156, 836–843 (2014).

  92. 92.

    & Unraveling the potential of CRISPR–Cas9 for gene therapy. Expert Opin. Biol. Ther. 15, 311–314 (2015).

  93. 93.

    et al. Functional gene correction for cystic fibrosis in lung epithelial cells generated from patient iPSCs. Cell Rep. 12, 1385–1390 (2015).

  94. 94.

    et al. Correction of a genetic disease in mouse via use of CRISPR–Cas9. Cell Stem Cell 13, 659–662 (2013).

  95. 95.

    et al. Prevention of muscular dystrophy in mice by CRISPR/Cas9-mediated editing of germline DNA. Science 345, 1184–1188 (2014).

  96. 96.

    et al. Fanconi anemia gene editing by the CRISPR/Cas9 system. Hum. Gene Ther. 26, 114–126 (2015).

  97. 97.

    et al. In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy. Science 351, 403–407 (2016).

  98. 98.

    et al. Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy. Science 351, 400–403 (2016).

  99. 99.

    et al. In vivo gene editing in dystrophic mouse muscle and muscle stem cells. Science 351, 407–411 (2016).

  100. 100.

    et al. Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nat. Biotechnol. 32, 551–553 (2014).

  101. 101.

    et al. Permanent alteration of PCSK9 with in vivo CRISPR–Cas9 genome editing. Circ. Res. 115, 488–492 (2014).

  102. 102.

    et al. In vivo CRISPR/Cas9 gene editing corrects retinal dystrophy in the S334ter-3 rat model of autosomal dominant retinitis Pigmentosa. Mol. Ther. 24, 556–563 (2016).

  103. 103.

    et al. Modelling kidney disease with CRISPR-mutant kidney organoids derived from human pluripotent epiblast spheroids. Nat. Commun. 6, 8715 (2015).

  104. 104.

    et al. CRISPR/Cas9-mediated heterozygous knockout of the autism gene CHD8 and characterization of its transcriptional networks in neurodevelopment. Mol. Autism 6, 55 (2015).

  105. 105.

    & Applications of CRISPR–Cas systems in neuroscience. Nat. Rev. Neurosci. 17, 36–44 (2016).

  106. 106.

    et al. In vivo interrogation of gene function in the mammalian brain using CRISPR–Cas9. Nat. Biotechnol. 33, 102–106 (2015).

  107. 107.

    , & CRISPR/Cas9-mediated genome editing of epigenetic factors for cancer therapy. Hum. Gene Ther. 26, 463–471 (2015).

  108. 108.

    et al. CRISPR-mediated direct mutation of cancer genes in the mouse liver. Nature 514, 380–384 (2014).

  109. 109.

    et al. Adenovirus-mediated somatic genome editing of Pten by CRISPR/Cas9 in mouse liver in spite of Cas9-specific immune responses. Hum. Gene Ther. 26, 432–442 (2015).

  110. 110.

    & Genome engineering using adeno-associated virus (AAV). Methods Mol. Biol. 1239, 75–103 (2015).

  111. 111.

    , , & Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. eLife 3, e04766 (2014).

  112. 112.

    et al. Generation of knock-in primary human T cells using Cas9 ribonucleoproteins. Proc. Natl. Acad. Sci. USA 112, 10437–10442 (2015).

  113. 113.

    et al. Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat. Biotechnol. 33, 73–80 (2015).

  114. 114.

    , , & TALEN- and CRISPR/Cas9-mediated gene editing in human pluripotent stem cells using lipid-based transfection. Curr Protoc Stem Cell Biol 34, 5B 3.1–5B 3.25 (2015).

  115. 115.

    , , & High efficiency, homology-directed genome editing in Caenorhabditis elegans using CRISPR–Cas9 ribonucleoprotein complexes. Genetics 201, 47–54 (2015).

  116. 116.

    et al. DNA-free genome editing in plants with preassembled CRISPR–Cas9 ribonucleoproteins. Nat. Biotechnol. 33, 1162–1164 (2015).

  117. 117.

    , , , & Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res. 24, 1012–1019 (2014).

  118. 118.

    et al. Self-assembled DNA nanoclews for the efficient delivery of CRISPR–Cas9 for genome editing. Angew. Chem. Int. Edn Engl. 54, 12029–12033 (2015).

  119. 119.

    , & A CRISPR design for next-generation antimicrobials. Genome Biol. 15, 516 (2014).

  120. 120.

    et al. Programmable removal of bacterial strains by use of genome-targeting CRISPR–Cas systems. MBio 5, e00928–e13 (2014).

  121. 121.

    et al. Exploiting CRISPR–Cas nucleases to produce sequence-specific antimicrobials. Nat. Biotechnol. 32, 1146–1150 (2014).

  122. 122.

    , & Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases. Nat. Biotechnol. 32, 1141–1145 (2014).

  123. 123.

    et al. Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system. EMBO J. 30, 1335–1342 (2011).

  124. 124.

    et al. In vitro reconstitution of Cascade-mediated CRISPR immunity in Streptococcus thermophilus. EMBO J. 32, 385–394 (2013).

  125. 125.

    et al. RNA-directed gene editing specifically eradicates latent and prevents new HIV-1 infection. Proc. Natl. Acad. Sci. USA 111, 11461–11466 (2014).

  126. 126.

    et al. Inhibition of HIV-1 infection of primary CD4+ T-cells by gene editing of CCR5 using adenovirus-delivered CRISPR/Cas9. J. Gen. Virol. 96, 2381–2393 (2015).

  127. 127.

    et al. CCR5 gene disruption via lentiviral vectors expressing Cas9 and single guided RNA renders cells resistant to HIV-1 infection. PLoS One 9, e115987 (2014).

  128. 128.

    et al. Seamless modification of wild-type induced pluripotent stem cells to the natural CCR5Δ32 mutation confers resistance to HIV infection. Proc. Natl. Acad. Sci. USA 111, 9591–9596 (2014).

  129. 129.

    & Efficient human immunodeficiency virus (HIV-1) infection of cells lacking PDZD8. Virology 481, 73–78 (2015).

  130. 130.

    et al. Use of the CRISPR/Cas9 system as an intracellular defense against HIV-1 infection in human cells. Nat. Commun. 6, 6413 (2015).

  131. 131.

    et al. Genome editing of CXCR4 by CRISPR/cas9 confers cells resistant to HIV-1 infection. Sci. Rep. 5, 15577 (2015).

  132. 132.

    & RNA-guided endonuclease provides a therapeutic strategy to cure latent herpesviridae infection. Proc. Natl. Acad. Sci. USA 111, 13157–13162 (2014).

  133. 133.

    et al. Disruption of HPV16–E7 by CRISPR/Cas system induces apoptosis and growth inhibition in HPV16 positive human cervical cancer cells. BioMed Res. Int. 2014, 612823 (2014).

  134. 134.

    et al. Inactivation of the human papillomavirus E6 or E7 gene in cervical carcinoma cells by using a bacterial CRISPR/Cas RNA-guided endonuclease. J. Virol. 88, 11965–11972 (2014).

  135. 135.

    et al. Disruption of human papillomavirus 16 E6 gene by clustered regularly interspaced short palindromic repeat/Cas system in human cervical cancer cells. Onco Targets Ther. 8, 37–44 (2014).

  136. 136.

    et al. Targeting hepatitis B virus cccDNA by CRISPR/Cas9 nuclease efficiently inhibits viral replication. Antiviral Res. 118, 110–117 (2015).

  137. 137.

    et al. Suppression of hepatitis B virus DNA accumulation in chronically infected cells using a bacterial CRISPR/Cas RNA-guided DNA endonuclease. Virology 476, 196–205 (2015).

  138. 138.

    & Bacterial CRISPR/Cas DNA endonucleases: a revolutionary technology that could dramatically impact viral research and treatment. Virology 479–480, 213–220 (2015).

  139. 139.

    , , , & Inhibition of hepatitis B virus by the CRISPR/Cas9 system via targeting the conserved regions of the viral genome. J. Gen. Virol. 96, 2252–2261 (2015).

  140. 140.

    et al. CRISPR/Cas9 cleavage of viral DNA efficiently suppresses hepatitis B virus. Sci. Rep. 5, 10833 (2015).

  141. 141.

    et al. Dual gRNAs guided CRISPR/Cas9 system inhibits hepatitis B virus replication. World J. Gastroenterol. 21, 9554–9565 (2015).

  142. 142.

    et al. The CRISPR/Cas9 system facilitates clearance of the intrahepatic HBV templates in vivo. Mol. Ther. Nucleic Acids 3, e186 (2014).

  143. 143.

    et al. Harnessing the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated Cas9 system to disrupt the hepatitis B virus. Gene Ther. 22, 404–412 (2015).

  144. 144.

    et al. Gene-edited pigs are protected from porcine reproductive and respiratory syndrome virus. Nat. Biotechnol. 34, 20–22 (2016).

  145. 145.

    et al. Production of hornless dairy cattle from genome-edited cell lines. Nat. Biotechnol. 34, 479–481 (2016).

  146. 146.

    et al. Production of human albumin in pigs through CRISPR/Cas9-mediated knockin of human cDNA into swine albumin locus in the zygotes. Sci. Rep. 5, 16705 (2015).

  147. 147.

    , , , & Editing plant genomes with CRISPR/Cas9. Curr. Opin. Biotechnol. 32, 76–84 (2015).

  148. 148.

    & Next biotech plants: new traits, crops, developers and technologies for addressing global challenges. Crit. Rev. Biotechnol. 36, 675–690 (2016).

  149. 149.

    et al. Targeted mutagenesis, precise gene editing, and site-specific gene insertion in maize using Cas9 and guide RNA. Plant Physiol. 169, 931–945 (2015).

  150. 150.

    et al. Cas9-guide RNA directed genome editing in soybean. Plant Physiol. 169, 960–970 (2015).

  151. 151.

    et al. Efficient virus-mediated genome editing in plants using the CRISPR/Cas9 system. Mol. Plant 8, 1288–1291 (2015).

  152. 152.

    Gene-edited CRISPR mushroom escapes US regulation. Nature 532, 293 (2016).

  153. 153.

    & CRISPR-based technologies and the future of food science. J. Food Sci. 80, R2367–R2372 (2015).

  154. 154.

    & Harnessing CRISPR–Cas systems for bacterial genome editing. Trends Microbiol. 23, 225–232 (2015).

  155. 155.

    et al. Genomic impact of CRISPR immunization against bacteriophages. Biochem. Soc. Trans. 41, 1383–1391 (2013).

  156. 156.

    & CRISPR: new horizons in phage resistance and strain identification. Annu. Rev. Food Sci. Technol. 3, 143–162 (2012).

  157. 157.

    & CRISPR–Cas9-assisted recombineering in Lactobacillus reuteri. Nucleic Acids Res. 42, e131 (2014).

  158. 158.

    & Precision genome engineering in lactic acid bacteria. Microb. Cell Fact. 13 (Suppl. 1), S10 (2014).

  159. 159.

    & Exploiting CRISPR–Cas immune systems for genome editing in bacteria. Curr. Opin. Biotechnol. 37, 61–68 (2016).

  160. 160.

    et al. Selection of chromosomal DNA libraries using a multiplex CRISPR system. eLife 3, e03703 (2014).

  161. 161.

    , & CRISPR-based screening of genomic island excision events in bacteria. Proc. Natl. Acad. Sci. USA 112, 8076–8081 (2015).

  162. 162.

    , , & Molecular recordings by directed CRISPR spacer acquisition. Science 353, aaf1175 (2016).

  163. 163.

    , , & Concerning RNA-guided gene drives for the alteration of wild populations. eLife 3, e03401 (2014).

  164. 164.

    et al. Highly efficient Cas9-mediated gene drive for population modification of the malaria vector mosquito Anopheles stephensi. Proc. Natl. Acad. Sci. USA 112, E6736–E6743 (2015).

  165. 165.

    , , , & Safeguarding CRISPR–Cas9 gene drives in yeast. Nat. Biotechnol. 33, 1250–1255 (2015).

  166. 166.

    et al. A CRISPR–Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae. Nat. Biotechnol. 34, 78–83 (2016).

  167. 167.

    et al. Discovery of cancer drug targets by CRISPR–Cas9 screening of protein domains. Nat. Biotechnol. 33, 661–667 (2015).

  168. 168.

    et al. Efficient generation of genetically distinct pigs in a single pregnancy using multiplexed single-guide RNA and carbohydrate selection. Xenotransplantation 22, 20–31 (2015).

  169. 169.

    et al. Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients. Cell Stem Cell 13, 653–658 (2013).

  170. 170.

    et al. Multiplex CRISPR/Cas9-based genome editing for correction of dystrophin mutations that cause Duchenne muscular dystrophy. Nat. Commun. 6, 6244 (2015).

  171. 171.

    et al. Functional disruption of the dystrophin gene in rhesus monkey using CRISPR/Cas9. Hum. Mol. Genet. 24, 3764–3774 (2015).

  172. 172.

    et al. Improved hematopoietic differentiation efficiency of gene-corrected beta-thalassemia induced pluripotent stem cells by CRISPR/Cas9 system. Stem Cells Dev. 24, 1053–1065 (2015).

  173. 173.

    et al. Seamless gene correction of β-thalassemia mutations in patient-specific iPSCs using CRISPR/Cas9 and piggyBac. Genome Res. 24, 1526–1533 (2014).

  174. 174.

    et al. Both TALENs and CRISPR/Cas9 directly target the HBB IVS2–654 (C>T) mutation in β-thalassemia-derived iPSCs. Sci. Rep. 5, 12065 (2015).

  175. 175.

    et al. Naïve induced pluripotent stem cells generated from β-thalassemia fibroblasts allow efficient gene correction with CRISPR/Cas9. Stem Cells Transl. Med. 5, 8–19 (2016).

  176. 176.

    et al. CRISPR germline engineering–the community speaks. Nat. Biotechnol. 33, 478–486 (2015).

  177. 177.

    et al. Biotechnology. A prudent path forward for genomic engineering and germline gene modification. Science 348, 36–38 (2015).

  178. 178.

    & Annotation and classification of CRISPR–Cas systems. Methods Mol. Biol. 1311, 47–75 (2015).

  179. 179.

    et al. Outbred genome sequencing and CRISPR/Cas9 gene editing in butterflies. Nat. Commun. 6, 8212 (2015).

  180. 180.

    et al. Crystal structure of Staphylococcus aureus Cas9. Cell 162, 1113–1126 (2015).

  181. 181.

    et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186–191 (2015).

  182. 182.

    et al. CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes. Protein Cell 6, 363–372 (2015).

  183. 183.

    et al. Characterization of human induced pluripotent stem cell-derived retinal pigment epithelium cell sheets aiming for clinical application. Stem Cell Reports 2, 205–218 (2014).

  184. 184.

    & From genomics to gene therapy: induced pluripotent stem cells meet genome editing. Annu. Rev. Genet. 49, 47–70 (2015).

  185. 185.

    Genomic engineering and the future of medicine. J. Am. Med. Assoc. 313, 791–792 (2015).

  186. 186.

    , , , & CRISPR-based adaptive and heritable immunity in prokaryotes. Trends Biochem. Sci. 34, 401–407 (2009).

  187. 187.

    CRISPR–Cas immunity in prokaryotes. Nature 526, 55–61 (2015).

  188. 188.

    & The bacterial origins of the CRISPR genome-editing revolution. Hum. Gene Ther. 26, 413–424 (2015).

  189. 189.

    & CRISPR–Cas systems: prokaryotes upgrade to adaptive immunity. Mol. Cell 54, 234–244 (2014).

  190. 190.

    et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science 315, 1709–1712 (2007).

  191. 191.

    et al. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321, 960–964 (2008).

  192. 192.

    & CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science 322, 1843–1845 (2008).

  193. 193.

    et al. RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex. Cell 139, 945–956 (2009).

  194. 194.

    , , & Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc. Natl. Acad. Sci. USA 109, E2579–E2586 (2012).

  195. 195.

    et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).

  196. 196.

    et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 471, 602–607 (2011).

  197. 197.

    RNA-mediated programmable DNA cleavage. Nat. Biotechnol. 30, 836–838 (2012).

  198. 198.

    et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).

  199. 199.

    et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).

  200. 200.

    , , , & RNA-guided editing of bacterial genomes using CRISPR–Cas systems. Nat. Biotechnol. 31, 233–239 (2013).

  201. 201.

    , , & Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat. Biotechnol. 31, 230–232 (2013).

  202. 202.

    et al. Efficient genome editing in zebrafish using a CRISPR–Cas system. Nat. Biotechnol. 31, 227–229 (2013).

  203. 203.

    et al. RNA-programmed genome editing in human cells. eLife 2, e00471 (2013).

  204. 204.

    & Expanding the biologist's toolkit with CRISPR–Cas9. Mol. Cell 58, 568–574 (2015).

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Acknowledgements

The authors would like to acknowledge their laboratory members, collaborators and colleagues throughout the CRISPR community for fruitful discussions and insightful opinions. We also thank A. Briner for assistance with figures and graphic design, and M. Perry and C. Desplan for providing the picture of butterflies. Data regarding CRISPR deposits and distributions through Addgene were provided courtesy of N. Waxmonsky and J. Welch at Addgene, Inc. (http://www.addgene.org/).

Author information

Affiliations

  1. Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, North Carolina, USA.

    • Rodolphe Barrangou
  2. Howard Hughes Medical Institute, Innovative Genomics Initiative, Department of Molecular and Cell Biology, University of California, Berkeley, California, USA.

    • Jennifer A Doudna
  3. Department of Chemistry, University of California, Berkeley, California, USA.

    • Jennifer A Doudna
  4. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.

    • Jennifer A Doudna

Authors

  1. Search for Rodolphe Barrangou in:

  2. Search for Jennifer A Doudna in:

Competing interests

R.B. and J.A.D. are inventors on several patents related to various uses of CRISPR–Cas systems. R.B. is a board member of Caribou Biosciences, a founder and advisor of Intellia Therapeutics, a founder and advisor of Locus Biosciences; J.A.D. is a co-founder and advisor of Caribou Biosciences and Intellia Therapeutics, and a co-founder of Editas Medicine; these companies are involved in commercialization of CRISPR applications

Corresponding authors

Correspondence to Rodolphe Barrangou or Jennifer A Doudna.

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DOI

https://doi.org/10.1038/nbt.3659