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The next generation of CRISPR–Cas technologies and applications

Abstract

The prokaryote-derived CRISPR–Cas genome editing systems have transformed our ability to manipulate, detect, image and annotate specific DNA and RNA sequences in living cells of diverse species. The ease of use and robustness of this technology have revolutionized genome editing for research ranging from fundamental science to translational medicine. Initial successes have inspired efforts to discover new systems for targeting and manipulating nucleic acids, including those from Cas9, Cas12, Cascade and Cas13 orthologues. Genome editing by CRISPR–Cas can utilize non-homologous end joining and homology-directed repair for DNA repair, as well as single-base editing enzymes. In addition to targeting DNA, CRISPR–Cas-based RNA-targeting tools are being developed for research, medicine and diagnostics. Nuclease-inactive and RNA-targeting Cas proteins have been fused to a plethora of effector proteins to regulate gene expression, epigenetic modifications and chromatin interactions. Collectively, the new advances are considerably improving our understanding of biological processes and are propelling CRISPR–Cas-based tools towards clinical use in gene and cell therapies.

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Fig. 1: Overview of the main CRISPR–Cas gene editing tools.
Fig. 2: Genome editing strategies.
Fig. 3: RNA-targeting tools and their applications.
Fig. 4: Targeted gene regulation and other applications.

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References

  1. Danna, K. & Nathans, D. Specific cleavage of simian virus 40 DNA by restriction endonuclease of Hemophilus influenzae. Proc. Natl Acad. Sci. USA 68, 2913–2917 (1971).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Roberts, R. J. How restriction enzymes became the workhorses of molecular biology. Proc. Natl Acad. Sci. USA 102, 5905–5908 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Olorunniji, F. J., Rosser, S. J. & Stark, W. M. Site-specific recombinases: molecular machines for the genetic revolution. Biochem. J. 473, 673–684 (2016).

    CAS  PubMed  Google Scholar 

  4. Chandrasegaran, S. & Carroll, D. Origins of programmable nucleases for genome engineering. J. Mol. Biol. 428, 963–989 (2016).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  6. Makarova, K. S. et al. An updated evolutionary classification of CRISPR-Cas systems. Nat. Rev. Microbiol. 13, 722–736 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Shmakov, S. et al. Diversity and evolution of class 2 CRISPR-Cas systems. Nat. Rev. Microbiol. 15, 169–182 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Koonin, E. V., Makarova, K. S. & Zhang, F. Diversity, classification and evolution of CRISPR-Cas systems. Curr. Opin. Microbiol. 37, 67–78 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Hsu, P. D., Lander, E. S. & Zhang, F. Development and applications of CRISPR-Cas9 for genome engineering. Cell 157, 1262–1278 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  14. Mojica, F. J., Diez-Villasenor, C., Garcia-Martinez, J. & Almendros, C. Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology 155, 733–740 (2009).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Gasiunas, G., Barrangou, R., Horvath, P. & Siksnys, V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc. Natl Acad. Sci. USA 109, E2579–E2586 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    PubMed  PubMed Central  Google Scholar 

  18. Hu, J. H. et al. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature 556, 57–63 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Chen, J. S. et al. Enhanced proofreading governs CRISPR-Cas9 targeting accuracy. Nature 550, 407–410 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Casini, A. et al. A highly specific SpCas9 variant is identified by in vivo screening in yeast. Nat. Biotechnol. 36, 265–271 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Vakulskas, C. A. et al. A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells. Nat. Med. 24, 1216–1224 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Kocak, D. D. et al. Increasing the specificity of CRISPR systems with engineered RNA secondary structures. Nat. Biotechnol. https://doi.org/10.1038/s41587-019-0095-1 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Zhang, Y. et al. Processing-independent CRISPR RNAs limit natural transformation in Neisseria meningitidis. Mol. Cell 50, 488–503 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Hou, Z. et al. Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis. Proc. Natl Acad. Sci. USA 110, 15644–15649 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Yamada, M. et al. Crystal structure of the minimal Cas9 from Campylobacter jejuni reveals the molecular diversity in the CRISPR-Cas9 systems. Mol. Cell 65, 1109–1121 (2017).

    CAS  PubMed  Google Scholar 

  31. Kim, E. et al. In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni. Nat. Commun. 8, 14500 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Burstein, D. et al. New CRISPR-Cas systems from uncultivated microbes. Nature 542, 237–241 (2017).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Zetsche, B. et al. Multiplex gene editing by CRISPR-Cpf1 using a single crRNA array. Nat. Biotechnol. 35, 31–34 (2017).

    CAS  PubMed  Google Scholar 

  35. Kleinstiver, B. P. et al. Engineered CRISPR–Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing. Nat. Biotechnol. 37, 276–282 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Gao, L. et al. Engineered Cpf1 variants with altered PAM specificities. Nat. Biotechnol. 35, 789–792 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Jore, M. M. et al. Structural basis for CRISPR RNA-guided DNA recognition by Cascade. Nat. Struct. Mol. Biol. 18, 529–536 (2011).

    CAS  PubMed  Google Scholar 

  38. Westra, E. R. et al. CRISPR immunity relies on the consecutive binding and degradation of negatively supercoiled invader DNA by Cascade and Cas3. Mol. Cell 46, 595–605 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Hochstrasser, M. L. et al. CasA mediates Cas3-catalyzed target degradation during CRISPR RNA-guided interference. Proc. Natl Acad. Sci. USA 111, 6618–6623 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Brendel, J. et al. A complex of Cas proteins 5, 6, and 7 is required for the biogenesis and stability of clustered regularly interspaced short palindromic repeats (CRISPR)-derived RNAs (crRNAs) in Haloferax volcanii. J. Biol. Chem. 289, 7164–7177 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Richter, C., Gristwood, T., Clulow, J. S. & Fineran, P. C. In vivo protein interactions and complex formation in the Pectobacterium atrosepticum subtype I-F CRISPR/Cas system. PLOS ONE 7, e49549 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Wiedenheft, B. et al. RNA-guided complex from a bacterial immune system enhances target recognition through seed sequence interactions. Proc. Natl Acad. Sci. USA 108, 10092–10097 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Hayes, R. P. et al. Structural basis for promiscuous PAM recognition in type I-E Cascade from E. coli. Nature 530, 499–503 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Sinkunas, T. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Jackson, R. N., Lavin, M., Carter, J. & Wiedenheft, B. Fitting CRISPR-associated Cas3 into the helicase family tree. Curr. Opin. Struct. Biol. 24, 106–114 (2014).

    CAS  PubMed  Google Scholar 

  46. Gomaa, A. A. et al. Programmable removal of bacterial strains by use of genome-targeting CRISPR-Cas systems. mBio 5, e00928-00913 (2014).

    PubMed  PubMed Central  Google Scholar 

  47. Haurwitz, R. E., Jinek, M., Wiedenheft, B., Zhou, K. & Doudna, J. A. Sequence- and structure-specific RNA processing by a CRISPR endonuclease. Science 329, 1355–1358 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Nissim, L., Perli, S. D., Fridkin, A., Perez-Pinera, P. & Lu, T. K. Multiplexed and programmable regulation of gene networks with an integrated RNA and CRISPR/Cas toolkit in human cells. Mol. Cell 54, 698–710 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Wyman, C. & Kanaar, R. DNA double-strand break repair: all’s well that ends well. Annu. Rev. Genet. 40, 363–383 (2006).

    CAS  PubMed  Google Scholar 

  51. Lin, S., Staahl, B. T., Alla, R. K. & Doudna, J. A. Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. eLife 3, e04766 (2014).

    PubMed  PubMed Central  Google Scholar 

  52. Pinder, J., Salsman, J. & Dellaire, G. Nuclear domain ‘knock-in’ screen for the evaluation and identification of small molecule enhancers of CRISPR-based genome editing. Nucleic Acids Res. 43, 9379–9392 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Song, J. et al. RS-1 enhances CRISPR/Cas9- and TALEN-mediated knock-in efficiency. Nat. Commun. 7, 10548 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Gutschner, T., Haemmerle, M., Genovese, G., Draetta, G. F. & Chin, L. Post-translational regulation of Cas9 during G1 enhances homology-directed repair. Cell Rep. 14, 1555–1566 (2016).

    CAS  PubMed  Google Scholar 

  55. Aird, E. J., Lovendahl, K. N., St Martin, A., Harris, R. S. & Gordon, W. R. Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template. Commun. Biol. 1, 54 (2018).

    PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  57. Ran, F. A. et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154, 1380–1389 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. Zhu, S. et al. Genome-scale deletion screening of human long non-coding RNAs using a paired-guide RNA CRISPR-Cas9 library. Nat. Biotechnol. 34, 1279–1286 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. Essletzbichler, P. et al. Megabase-scale deletion using CRISPR/Cas9 to generate a fully haploid human cell line. Genome Res. 24, 2059–2065 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  60. Canver, M. C. et al. Characterization of genomic deletion efficiency mediated by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 nuclease system in mammalian cells. J. Biol. Chem. 289, 21312–21324 (2014).

    PubMed  PubMed Central  Google Scholar 

  61. Xiao, A. et al. Chromosomal deletions and inversions mediated by TALENs and CRISPR/Cas in zebrafish. Nucleic Acids Res. 41, e141 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  62. Zhou, J. et al. Dual sgRNAs facilitate CRISPR/Cas9-mediated mouse genome targeting. FEBS J. 281, 1717–1725 (2014).

    CAS  PubMed  Google Scholar 

  63. Yang, H. et al. One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell 154, 1370–1379 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  64. Kraft, K. et al. Deletions, inversions, duplications: engineering of structural variants using CRISPR/Cas in mice. Cell Rep. 10, 833–839 (2015).

    CAS  PubMed  Google Scholar 

  65. Suzuki, K. et al. In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration. Nature 540, 144–149 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  66. Lackner, D. H. et al. A generic strategy for CRISPR-Cas9-mediated gene tagging. Nat. Commun. 6, 10237 (2015).

    CAS  PubMed  Google Scholar 

  67. Schmid-Burgk, J. L., Honing, K., Ebert, T. S. & Hornung, V. CRISPaint allows modular base-specific gene tagging using a ligase-4-dependent mechanism. Nat. Commun. 7, 12338 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  68. Leonetti, M. D., Sekine, S., Kamiyama, D., Weissman, J. S. & Huang, B. A scalable strategy for high-throughput GFP tagging of endogenous human proteins. Proc. Natl Acad. Sci. USA 113, E3501–E3508 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  69. Wang, H. et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153, 910–918 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  70. Platt, R. J. et al. CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell 159, 440–455 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  71. Quadros, R. M. et al. Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins. Genome Biol. 18, 92 (2017).

    PubMed  PubMed Central  Google Scholar 

  72. Paquet, D. et al. Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9. Nature 533, 125–129 (2016).

    CAS  PubMed  Google Scholar 

  73. Kwart, D., Paquet, D., Teo, S. & Tessier-Lavigne, M. Precise and efficient scarless genome editing in stem cells using CORRECT. Nat. Protoc. 12, 329–354 (2017).

    CAS  PubMed  Google Scholar 

  74. Choi, P. S. & Meyerson, M. Targeted genomic rearrangements using CRISPR/Cas technology. Nat. Commun. 5, 3728 (2014).

    CAS  PubMed  Google Scholar 

  75. Torres, R. et al. Engineering human tumour-associated chromosomal translocations with the RNA-guided CRISPR-Cas9 system. Nat. Commun. 5, 3964 (2014).

    CAS  PubMed  Google Scholar 

  76. Maddalo, D. et al. In vivo engineering of oncogenic chromosomal rearrangements with the CRISPR/Cas9 system. Nature 516, 423–427 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  77. Haapaniemi, E., Botla, S., Persson, J., Schmierer, B. & Taipale, J. CRISPR-Cas9 genome editing induces a p53-mediated DNA damage response. Nat. Med. 24, 927–930 (2018).

    CAS  PubMed  Google Scholar 

  78. Ihry, R. J. et al. p53 inhibits CRISPR-Cas9 engineering in human pluripotent stem cells. Nat. Med. 24, 939–946 (2018).

    CAS  PubMed  Google Scholar 

  79. Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A. & Liu, D. R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420–424 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  80. Nishida, K. et al. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science 353, aaf8729 (2016).

    PubMed  Google Scholar 

  81. Ma, Y. et al. Targeted AID-mediated mutagenesis (TAM) enables efficient genomic diversification in mammalian cells. Nat. Methods 13, 1029–1035 (2016).

    CAS  PubMed  Google Scholar 

  82. Kim, K. et al. Highly efficient RNA-guided base editing in mouse embryos. Nat. Biotechnol. 35, 435–437 (2017).

    CAS  PubMed  Google Scholar 

  83. Rees, H. A. et al. Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery. Nat. Commun. 8, 15790 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  84. Rossidis, A. C. et al. In utero CRISPR-mediated therapeutic editing of metabolic genes. Nat. Med. 24, 1513–1518 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  85. Liang, P. et al. Correction of beta-thalassemia mutant by base editor in human embryos. Protein Cell 8, 811–822 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  86. Li, G. et al. Highly efficient and precise base editing in discarded human tripronuclear embryos. Protein Cell 8, 776–779 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  87. Chadwick, A. C., Wang, X. & Musunuru, K. In vivo base editing of PCSK9 (proprotein convertase subtilisin/kexin type 9) as a therapeutic alternative to genome editing. Arterioscler. Thromb. Vasc. Biol. 37, 1741–1747 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  88. Komor, A. C. et al. Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity. Sci. Adv. 3, eaao4774 (2017).

    PubMed  PubMed Central  Google Scholar 

  89. Li, X. et al. Base editing with a Cpf1-cytidine deaminase fusion. Nat. Biotechnol. 36, 324–327 (2018).

    CAS  PubMed  Google Scholar 

  90. Gehrke, J. M. et al. An APOBEC3A-Cas9 base editor with minimized bystander and off-target activities. Nat. Biotechnol. 36, 977–982 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  91. Wang, X. et al. Efficient base editing in methylated regions with a human APOBEC3A-Cas9 fusion. Nat. Biotechnol. 36, 946–949 (2018).

    CAS  PubMed  Google Scholar 

  92. Zong, Y. et al. Efficient C-to-T base editing in plants using a fusion of nCas9 and human APOBEC3A. Nat. Biotechnol. 36, 950–953 (2018).

    CAS  Google Scholar 

  93. Gaudelli, N. M. et al. Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage. Nature 551, 464–471 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  94. Koblan, L. W. et al. Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction. Nat. Biotechnol. 36, 843–846 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  95. Kim, D. et al. Genome-wide target specificities of CRISPR RNA-guided programmable deaminases. Nat. Biotechnol. 35, 475–480 (2017).

    CAS  PubMed  Google Scholar 

  96. Jin, S. et al. Cytosine, but not adenine, base editors induce genome-wide off-target mutations in rice. Science 364, 292–295 (2019).

  97. Zuo, E. et al. Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos. Science 364, 289–292 (2019).

  98. Song, C. Q. et al. Adenine base editing in an adult mouse model of tyrosinaemia. Nat. Biomed. Eng. https://doi.org/10.1038/s41551-019-0357-8 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  99. Shalem, O. et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 343, 84–87 (2014).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  103. Koike-Yusa, H., Li, Y., Tan, E. P., Velasco-Herrera Mdel, C. & Yusa, K. Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library. Nat. Biotechnol. 32, 267–273 (2014).

    CAS  PubMed  Google Scholar 

  104. Wang, T., Wei, J. J., Sabatini, D. M. & Lander, E. S. Genetic screens in human cells using the CRISPR-Cas9 system. Science 343, 80–84 (2014).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  106. Dixit, A. et al. Perturb-seq: dissecting molecular circuits with scalable single-cell RNA profiling of pooled genetic screens. Cell 167, 1853–1866 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  107. Jaitin, D. A. et al. Dissecting immune circuits by linking CRISPR-pooled screens with single-cell RNA-seq. Cell 167, 1883–1896 (2016).

    CAS  PubMed  Google Scholar 

  108. Datlinger, P. et al. Pooled CRISPR screening with single-cell transcriptome readout. Nat. Methods 14, 297–301 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  109. Sanson, K. R. et al. Optimized libraries for CRISPR-Cas9 genetic screens with multiple modalities. Nat. Commun. 9, 5416 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  110. Han, K. et al. Synergistic drug combinations for cancer identified in a CRISPR screen for pairwise genetic interactions. Nat. Biotechnol. 35, 463–474 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  111. Shen, J. P. et al. Combinatorial CRISPR-Cas9 screens for de novo mapping of genetic interactions. Nat. Methods 14, 573–576 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  112. Billon, P. et al. CRISPR-mediated base editing enables efficient disruption of eukaryotic genes through induction of STOP codons. Mol. Cell 67, 1068–1079 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  113. Canver, M. C. et al. BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis. Nature 527, 192–197 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  114. Wu, Y. et al. Highly efficient therapeutic gene editing of human hematopoietic stem cells. Nat. Med. 25, 776–783 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  115. Findlay, G. M., Boyle, E. A., Hause, R. J., Klein, J. C. & Shendure, J. Saturation editing of genomic regions by multiplex homology-directed repair. Nature 513, 120–123 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  116. Vierstra, J. et al. Functional footprinting of regulatory DNA. Nat. Methods 12, 927–930 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  118. Diao, Y. et al. A tiling-deletion-based genetic screen for cis-regulatory element identification in mammalian cells. Nat. Methods 14, 629–635 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  119. Gasperini, M. et al. CRISPR/Cas9-mediated scanning for regulatory elements required for HPRT1 expression via thousands of large, programmed genomic deletions. Am. J. Hum. Genet. 101, 192–205 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  121. Sen, D. R. et al. The epigenetic landscape of T cell exhaustion. Science 354, 1165–1169 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  122. Canver, M. C. et al. Variant-aware saturating mutagenesis using multiple Cas9 nucleases identifies regulatory elements at trait-associated loci. Nat. Genet. 49, 625–634 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  123. Klann, T. S., Black, J. B. & Gersbach, C. A. CRISPR-based methods for high-throughput annotation of regulatory DNA. Curr. Opin. Biotechnol. 52, 32–41 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  124. Liu, Y. et al. Genome-wide screening for functional long noncoding RNAs in human cells by Cas9 targeting of splice sites. Nat. Biotechnol. 36, 1203–1210 (2018).

    CAS  Google Scholar 

  125. Perli, S. D., Cui, C. H. & Lu, T. K. Continuous genetic recording with self-targeting CRISPR-Cas in human cells. Science 353, aag0511 (2016).

    PubMed  Google Scholar 

  126. Kalhor, R., Mali, P. & Church, G. M. Rapidly evolving homing CRISPR barcodes. Nat. Methods 14, 195–200 (2017).

    CAS  PubMed  Google Scholar 

  127. Tang, W. & Liu, D. R. Rewritable multi-event analog recording in bacterial and mammalian cells. Science 360, eaap8992 (2018).

    PubMed  PubMed Central  Google Scholar 

  128. McKenna, A. et al. Whole-organism lineage tracing by combinatorial and cumulative genome editing. Science 353, aaf7907 (2016).

    PubMed  PubMed Central  Google Scholar 

  129. Frieda, K. L. et al. Synthetic recording and in situ readout of lineage information in single cells. Nature 541, 107–111 (2017).

    CAS  PubMed  Google Scholar 

  130. Yosef, I., Goren, M. G. & Qimron, U. Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli. Nucleic Acids Res. 40, 5569–5576 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  131. Shipman, S. L., Nivala, J., Macklis, J. D. & Church, G. M. Molecular recordings by directed CRISPR spacer acquisition. Science 353, aaf1175 (2016).

    PubMed  PubMed Central  Google Scholar 

  132. Shipman, S. L., Nivala, J., Macklis, J. D. & Church, G. M. CRISPR-Cas encoding of a digital movie into the genomes of a population of living bacteria. Nature 547, 345–349 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  133. Sternberg, S. H., Redding, S., Jinek, M., Greene, E. C. & Doudna, J. A. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature 507, 62–67 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  134. O’Connell, M. R. et al. Programmable RNA recognition and cleavage by CRISPR/Cas9. Nature 516, 263–266 (2014).

    PubMed  PubMed Central  Google Scholar 

  135. Nelles, D. A. et al. Programmable RNA tracking in live cells with CRISPR/Cas9. Cell 165, 488–496 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  136. Batra, R. et al. Elimination of toxic microsatellite repeat expansion RNA by RNA-targeting Cas9. Cell 170, 899–912 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  137. Naldini, L. Gene therapy returns to centre stage. Nature 526, 351–360 (2015).

    CAS  PubMed  Google Scholar 

  138. Strutt, S. C., Torrez, R. M., Kaya, E., Negrete, O. A. & Doudna, J. A. RNA-dependent RNA targeting by CRISPR-Cas9. eLife 7, e32724 (2018).

    PubMed  PubMed Central  Google Scholar 

  139. Dugar, G. et al. CRISPR RNA-dependent binding and cleavage of endogenous RNAs by the Campylobacter jejuni Cas9. Mol. Cell 69, 893–905 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  140. Sampson, T. R., Saroj, S. D., Llewellyn, A. C., Tzeng, Y. L. & Weiss, D. S. A. CRISPR/Cas system mediates bacterial innate immune evasion and virulence. Nature 497, 254–257 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  141. Price, A. A., Sampson, T. R., Ratner, H. K., Grakoui, A. & Weiss, D. S. Cas9-mediated targeting of viral RNA in eukaryotic cells. Proc. Natl Acad. Sci. USA 112, 6164–6169 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  142. Abudayyeh, O. O. et al. C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science 353, aaf5573 (2016).

    PubMed  PubMed Central  Google Scholar 

  143. Abudayyeh, O. O. et al. RNA targeting with CRISPR-Cas13. Nature 550, 280–284 (2017).

    PubMed  PubMed Central  Google Scholar 

  144. Cox, D. B. T. et al. RNA editing with CRISPR-Cas13. Science 358, 1019–1027 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  145. East-Seletsky, A. et al. Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection. Nature 538, 270–273 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  146. Gootenberg, J. S. et al. Nucleic acid detection with CRISPR-Cas13a/C2c2. Science 356, 438–442 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  147. Gootenberg, J. S. et al. Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6. Science 360, 439–444 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  148. Chen, J. S. et al. CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science 360, 436–439 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  149. Nishikura, K. Functions and regulation of RNA editing by ADAR deaminases. Annu. Rev. Biochem. 79, 321–349 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  150. Tan, M. H. et al. Dynamic landscape and regulation of RNA editing in mammals. Nature 550, 249–254 (2017).

    PubMed  PubMed Central  Google Scholar 

  151. Yan, W. X. et al. Cas13d is a compact RNA-targeting type VI CRISPR effector positively modulated by a WYL-domain-containing accessory protein. Mol. Cell 70, 327–339 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  152. Konermann, S. et al. Transcriptome engineering with RNA-targeting type VI-D CRISPR effectors. Cell 173, 665–676 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  153. Thakore, P. I., Black, J. B., Hilton, I. B. & Gersbach, C. A. Editing the epigenome: technologies for programmable transcription and epigenetic modulation. Nat. Methods 13, 127–137 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  157. Margolin, J. F. et al. Krüppel-associated boxes are potent transcriptional repression domains. Proc. Natl Acad. Sci. USA 91, 4509–4513 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  158. Kearns, N. A. et al. Cas9 effector-mediated regulation of transcription and differentiation in human pluripotent stem cells. Development 141, 219–223 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  159. Gao, X. et al. Comparison of TALE designer transcription factors and the CRISPR/dCas9 in regulation of gene expression by targeting enhancers. Nucleic Acids Res. 42, e155 (2014).

    PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  161. Thakore, P. I. et al. Highly specific epigenome editing by CRISPR-Cas9 repressors for silencing of distal regulatory elements. Nat. Methods 12, 1143–1149 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  162. Yeo, N. C. et al. An enhanced CRISPR repressor for targeted mammalian gene regulation. Nat. Methods 15, 611–616 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  163. Perez-Pinera, P. et al. RNA-guided gene activation by CRISPR-Cas9-based transcription factors. Nat. Methods 10, 973–976 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  164. Maeder, M. L. et al. CRISPR RNA-guided activation of endogenous human genes. Nat. Methods 10, 977–979 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  165. Farzadfard, F., Perli, S. D. & Lu, T. K. Tunable and multifunctional eukaryotic transcription factors based on CRISPR/Cas. ACS Synth. Biol. 2, 604–613 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  166. Cheng, A. W. et al. Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system. Cell Res. 23, 1163–1171 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  167. Chakraborty, S. et al. A CRISPR/Cas9-based system for reprogramming cell lineage specification. Stem Cell Rep. 3, 940–947 (2014).

    CAS  Google Scholar 

  168. Tanenbaum, M. E., Gilbert, L. A., Qi, L. S., Weissman, J. S. & Vale, R. D. A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell 159, 635–646 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  169. Chavez, A. et al. Highly efficient Cas9-mediated transcriptional programming. Nat. Methods 12, 326–328 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  171. Black, J. B. & Gersbach, C. A. Synthetic transcription factors for cell fate reprogramming. Curr. Opin. Genet. Dev. 52, 13–21 (2018).

    CAS  PubMed  Google Scholar 

  172. Black, J. B. et al. Targeted epigenetic remodeling of endogenous loci by CRISPR/Cas9-based transcriptional activators directly converts fibroblasts to neuronal cells. Cell Stem Cell 19, 406–414 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  173. Liu, P., Chen, M., Liu, Y., Qi, L. S. & Ding, S. CRISPR-based chromatin remodeling of the endogenous Oct4 or Sox2 locus enables reprogramming to pluripotency. Cell Stem Cell 22, 252–261 (2018).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  175. Choudhury, S. R., Cui, Y., Lubecka, K., Stefanska, B. & Irudayaraj, J. CRISPR-dCas9 mediated TET1 targeting for selective DNA demethylation at BRCA1 promoter. Oncotarget 7, 46545–46556 (2016).

    PubMed  PubMed Central  Google Scholar 

  176. Liu, X. S. et al. Rescue of fragile X syndrome neurons by DNA methylation editing of the FMR1 gene. Cell 172, 979–992 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  177. Amabile, A. et al. Inheritable silencing of endogenous genes by hit-and-run targeted epigenetic editing. Cell 167, 219–232 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  178. Gonzalez, F. et al. An iCRISPR platform for rapid, multiplexable, and inducible genome editing in human pluripotent stem cells. Cell Stem Cell 15, 215–226 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  179. Chen, Y. et al. Engineering human stem cell lines with inducible gene knockout using CRISPR/Cas9. Cell Stem Cell 17, 233–244 (2015).

    PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  181. Mandegar, M. A. et al. CRISPR interference efficiently induces specific and reversible gene silencing in human iPSCs. Cell Stem Cell 18, 541–553 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  182. Balboa, D. et al. Conditionally stabilized dCas9 activator for controlling gene expression in human cell reprogramming and differentiation. Stem Cell Rep. 5, 448–459 (2015).

    CAS  Google Scholar 

  183. Zetsche, B., Volz, S. E. & Zhang, F. A split-Cas9 architecture for inducible genome editing and transcription modulation. Nat. Biotechnol. 33, 139–142 (2015).

    CAS  PubMed  Google Scholar 

  184. Bao, Z., Jain, S., Jaroenpuntaruk, V. & Zhao, H. Orthogonal genetic regulation in human cells using chemically induced CRISPR/Cas9 activators. ACS Synth. Biol. 6, 686–693 (2017).

    CAS  PubMed  Google Scholar 

  185. Nihongaki, Y., Yamamoto, S., Kawano, F., Suzuki, H. & Sato, M. CRISPR-Cas9-based photoactivatable transcription system. Chem. Biol. 22, 169–174 (2015).

    CAS  PubMed  Google Scholar 

  186. Polstein, L. R. & Gersbach, C. A. A light-inducible CRISPR-Cas9 system for control of endogenous gene activation. Nat. Chem. Biol. 11, 198–200 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  187. Bubeck, F. et al. Engineered anti-CRISPR proteins for optogenetic control of CRISPR-Cas9. Nat. Methods 15, 924–927 (2018).

    CAS  PubMed  Google Scholar 

  188. Nihongaki, Y. et al. CRISPR-Cas9-based photoactivatable transcription systems to induce neuronal differentiation. Nat. Methods 14, 963–966 (2017).

    CAS  PubMed  Google Scholar 

  189. Gao, Y. et al. Complex transcriptional modulation with orthogonal and inducible dCas9 regulators. Nat. Methods 13, 1043–1049 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  190. Fulco, C. P. et al. Systematic mapping of functional enhancer-promoter connections with CRISPR interference. Science 354, 769–773 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  191. Xie, S., Duan, J., Li, B., Zhou, P. & Hon, G. C. Multiplexed engineering and analysis of combinatorial enhancer activity in single cells. Mol. Cell 66, 285–299 (2017).

    CAS  PubMed  Google Scholar 

  192. Simeonov, D. R. et al. Discovery of stimulation-responsive immune enhancers with CRISPR activation. Nature 549, 111–115 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  193. Klann, T. S. et al. CRISPR-Cas9 epigenome editing enables high-throughput screening for functional regulatory elements in the human genome. Nat. Biotechnol. 35, 561–568 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  194. Joung, J. et al. Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening. Nat. Protoc. 12, 828–863 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  195. Liu, S. J. et al. CRISPRi-based genome-scale identification of functional long noncoding RNA loci in human cells. Science 355, aah7111 (2017).

    PubMed  Google Scholar 

  196. Joung, J. et al. Genome-scale activation screen identifies a lncRNA locus regulating a gene neighbourhood. Nature 548, 343–346 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  197. Fujita, T. & Fujii, H. Efficient isolation of specific genomic regions and identification of associated proteins by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR. Biochem. Biophys. Res. Commun. 439, 132–136 (2013).

    CAS  PubMed  Google Scholar 

  198. Fujita, T. & Fujii, H. Identification of proteins associated with an IFNγ-responsive promoter by a retroviral expression system for enChIP using CRISPR. PLOS ONE 9, e103084 (2014).

    PubMed  PubMed Central  Google Scholar 

  199. Lam, S. S. et al. Directed evolution of APEX2 for electron microscopy and proximity labeling. Nat. Methods 12, 51–54 (2015).

    CAS  PubMed  Google Scholar 

  200. Myers, S. A. et al. Discovery of proteins associated with a predefined genomic locus via dCas9-APEX-mediated proximity labeling. Nat. Methods 15, 437–439 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  201. Liu, X. et al. In situ capture of chromatin interactions by biotinylated dCas9. Cell 170, 1028–1043 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  202. Morgan, S. L. et al. Manipulation of nuclear architecture through CRISPR-mediated chromosomal looping. Nat. Commun. 8, 15993 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  203. Rege, M. et al. LADL: light-activated dynamic looping for endogenous gene expression control. Preprint at bioRxiv https://doi.org/10.1101/349340 (2018).

  204. Wang, H. et al. CRISPR-mediated programmable 3D genome positioning and nuclear organization. Cell 175, 1405–1417 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  205. D’Ippolito, A. M. et al. Pre-established chromatin interactions mediate the genomic response to glucocorticoids. Cell Syst. 7, 146–160 (2018).

    PubMed  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  207. Zhou, Y. et al. Painting a specific chromosome with CRISPR/Cas9 for live-cell imaging. Cell Res. 27, 298–301 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  208. Ma, H. et al. Multicolor CRISPR labeling of chromosomal loci in human cells. Proc. Natl Acad. Sci. USA 112, 3002–3007 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  209. Chen, B. et al. Expanding the CRISPR imaging toolset with Staphylococcus aureus Cas9 for simultaneous imaging of multiple genomic loci. Nucleic Acids Res. 44, e75 (2016).

    PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  211. Shao, S. et al. Long-term dual-color tracking of genomic loci by modified sgRNAs of the CRISPR/Cas9 system. Nucleic Acids Res. 44, e86 (2016).

    PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  213. Wang, X. et al. CRISPR-Cas9 targeting of PCSK9 in human hepatocytes in vivo-brief report. Arterioscler. Thromb. Vasc. Biol. 36, 783–786 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  214. Thakore, P. I. et al. RNA-guided transcriptional silencing in vivo with S. aureus CRISPR-Cas9 repressors. Nat. Commun. 9, 1674 (2018).

    PubMed  PubMed Central  Google Scholar 

  215. Yu, W. et al. Nrl knockdown by AAV-delivered CRISPR/Cas9 prevents retinal degeneration in mice. Nat. Commun. 8, 14716 (2017).

    PubMed  PubMed Central  Google Scholar 

  216. Young, C. S. et al. A single CRISPR-Cas9 deletion strategy that targets the majority of DMD patients restores dystrophin function in hiPSC-derived muscle cells. Cell Stem Cell 18, 533–540 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  220. Hakim, C. H. et al. AAV CRISPR editing rescues cardiac and muscle function for 18 months in dystrophic mice. JCI Insight 3, 124297 (2018).

    PubMed  Google Scholar 

  221. Nelson, C. E. et al. Long-term evaluation of AAV-CRISPR genome editing for Duchenne muscular dystrophy. Nat. Med. 25, 427–432 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  222. Amoasii, L. et al. Gene editing restores dystrophin expression in a canine model of Duchenne muscular dystrophy. Science 362, 86–91 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  223. Gapinske, M. et al. CRISPR-SKIP: programmable gene splicing with single base editors. Genome Biol. 19, 107 (2018).

    PubMed  PubMed Central  Google Scholar 

  224. Yuan, J. et al. Genetic modulation of RNA splicing with a CRISPR-guided cytidine deaminase. Mol. Cell 72, 380–394 (2018).

    CAS  PubMed  Google Scholar 

  225. Finn, J. D. et al. A single administration of CRISPR/Cas9 Lipid nanoparticles achieves robust and persistent in vivo genome editing. Cell Rep. 22, 2227–2235 (2018).

    CAS  PubMed  Google Scholar 

  226. Nelson, C. E. & Gersbach, C. A. Engineering delivery vehicles for genome editing. Annu. Rev. Chem. Biomol. Eng. 7, 637–662 (2016).

    CAS  PubMed  Google Scholar 

  227. Maeder, M. L. et al. Development of a gene-editing approach to restore vision loss in Leber congenital amaurosis type 10. Nat. Med. 25, 229–233 (2019).

    CAS  PubMed  Google Scholar 

  228. Miller, J. F. & Sadelain, M. The journey from discoveries in fundamental immunology to cancer immunotherapy. Cancer Cell 27, 439–449 (2015).

    CAS  PubMed  Google Scholar 

  229. Eyquem, J. et al. Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection. Nature 543, 113–117 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  230. Ren, J. et al. A versatile system for rapid multiplex genome-edited CAR T cell generation. Oncotarget 8, 17002–17011 (2017).

    PubMed  PubMed Central  Google Scholar 

  231. Qasim, W. et al. Molecular remission of infant B-ALL after infusion of universal TALEN gene-edited CAR T cells. Sci. Transl Med. 9, eaaj2013 (2017).

    PubMed  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  233. Ren, J. et al. Multiplex genome editing to generate universal CAR T cells resistant to PD1 inhibition. Clin. Cancer Res. 23, 2255–2266 (2017).

    CAS  PubMed  Google Scholar 

  234. Torikai, H. et al. Toward eliminating HLA class I expression to generate universal cells from allogeneic donors. Blood 122, 1341–1349 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  235. Maeder, M. L. & Gersbach, C. A. Genome-editing technologies for gene and cell therapy. Mol. Ther. 24, 430–446 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  236. Zhou, Y. et al. High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells. Nature 509, 487–491 (2014).

    CAS  PubMed  Google Scholar 

  237. Wang, T. et al. Gene essentiality profiling reveals gene networks and synthetic lethal interactions with oncogenic Ras. Cell 168, 890–903 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  238. Aguirre, A. J. et al. Genomic copy number dictates a gene-independent cell response to CRISPR/Cas9 targeting. Cancer Discov. 6, 914–929 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  239. Han, J. et al. Genome-wide CRISPR/Cas9 screen identifies host factors essential for influenza virus replication. Cell Rep. 23, 596–607 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  240. Liu, Y. et al. CRISPR activation screens systematically identify factors that drive neuronal fate and reprogramming. Cell Stem Cell 23, 758–771 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  241. Akcakaya, P. et al. In vivo CRISPR editing with no detectable genome-wide off-target mutations. Nature 561, 416–419 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  242. Tsai, S. Q. et al. CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR-Cas9 nuclease off-targets. Nat. Methods 14, 607–614 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  243. Chew, W. L. et al. A multifunctional AAV-CRISPR-Cas9 and its host response. Nat. Methods 13, 868–874 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  244. Simhadri, V. L. et al. Prevalence of pre-existing antibodies to CRISPR-associated nuclease Cas9 in the USA population. Mol. Ther. Methods Clin. Dev. 10, 105–112 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  245. Wagner, D. L. et al. High prevalence of Streptococcus pyogenes Cas9-reactive T cells within the adult human population. Nat. Med. 25, 242–248 (2019).

    CAS  PubMed  Google Scholar 

  246. Charlesworth, C. T. et al. Identification of preexisting adaptive immunity to Cas9 proteins in humans. Nat. Med. 25, 249–254 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  247. Wang, J. et al. Homology-driven genome editing in hematopoietic stem and progenitor cells using ZFN mRNA and AAV6 donors. Nat. Biotechnol. 33, 1256–1263 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  248. Hsu, P. D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827–832 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  249. Chatterjee, P., Jakimo, N. & Jacobson, J. M. Minimal PAM specificity of a highly similar SpCas9 ortholog. Sci. Adv. 4, eaau0766 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This work was supported by an Allen Distinguished Investigator Award from the Paul G. Allen Frontiers Group, Open Philanthropy, US National Institutes of Health grants R01DA036865, R01AR069085, R21NS103007, R33DA041878, P30AR066527, R41GM119914, R41AI136755, U01HG007900, UM1HG009428 and UG3TR002142 and National Science Foundation grants EFRI-1830957 and DMR-1709527. A.P.-O. is supported by a Pfizer–NCBiotech Distinguished Postdoctoral Fellowship.

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Nature Reviews Molecular Cell Biology thanks J. Chen, R. Platt and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Both authors equally contributed to researching data for the article, the discussion of content, writing of the manuscript and its editing before submission.

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Correspondence to Charles A. Gersbach.

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Competing interests

C.A.G. and A.P.-O. are inventors on patent applications related to CRISPR technologies. C.A.G. is a scientific adviser to Element Genomics, Locus Biosciences and Sarepta Therapeutics.

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Glossary

Spacer

The interchangeable portion of the guide RNA that is complementary to the targeted sequence.

Directed evolution

Method of generating and selecting for nucleic acid or protein variants with desirable properties.

crRNA arrays

In bacterial genomes, series of spacers flanked by repeats, which are transcribed as a single pre-CRISPR RNA array and subsequently processed into individual CRISPR RNAs. 

Indels

Small insertions or deletions of nucleotides at repair sites of DNA double-strand breaks.

Stress granules

Cytosolic membraneless bodies with high concentrations of RNA and/or proteins that form in different cell stress conditions.

Microsatellite-repeat expansion

Repetitive DNA sequences that can expand between generations and encode RNAs that are toxic to cells and cause neurological disorders.

Anti-CRISPR protein

A protein that interacts with and inhibits CRISPR–Cas activity

DNase I hypersensitive sites

Chromatin regions accessible to the enzyme DNase I; generally denote gene-activity-permissive chromatin.

gRNA scaffolds

The backbone (invariable) portions of guide RNAs, which are recognized by Cas proteins.

gRNA aptamer

RNA structures added to the guide RNA scaffold, which can bind specific effector molecules.

Photobleaching

Reduction in the intensity of fluorescence owing to the imaging of a sample over time.

Chimeric antigen receptors

T cell receptors engineered to recognize a specific antigen.

Universal cell donors

Cells engineered to avoid recognition by a recipient immune system.

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Pickar-Oliver, A., Gersbach, C.A. The next generation of CRISPR–Cas technologies and applications. Nat Rev Mol Cell Biol 20, 490–507 (2019). https://doi.org/10.1038/s41580-019-0131-5

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