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PMID: 24906146 Published · ppublish English Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S. Review

Development and applications of CRISPR-Cas9 for genome engineering.

Cell ·Vol. 157 ·No. 6 ·2014-06-05 ·Pages 1262-1278

Hsu PD, Lander ES, Zhang F

Abstract

Recent advances in genome engineering technologies based on the CRISPR-associated RNA-guided endonuclease Cas9 are enabling the systematic interrogation of mammalian genome function. Analogous to the search function in modern word processors, Cas9 can be guided to specific locations within complex genomes by a short RNA search string. Using this system, DNA sequences within the endogenous genome and their functional outputs are now easily edited or modulated in virtually any organism of choice. Cas9-mediated genetic perturbation is simple and scalable, empowering researchers to elucidate the functional organization of the genome at the systems level and establish causal linkages between genetic variations and biological phenotypes. In this Review, we describe the development and applications of Cas9 for a variety of research or translational applications while highlighting challenges as well as future directions. Derived from a remarkable microbial defense system, Cas9 is driving innovative applications from basic biology to biotechnology and medicine.

MeSH Terms
Animals Bacteria/classification,genetics,immunology,virology CRISPR-Cas Systems Eukaryotic Cells/metabolism Gene Targeting Genetic Engineering Genome Humans Streptococcus pyogenes/enzymology,genetics
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Hsu Patrick D
Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, MA 02141, USA; McGovern Institute for Brain Research, Department of Brain and Cognitive Sciences, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA.
Lander Eric S
Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, MA 02141, USA.
Zhang Feng
Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, MA 02141, USA; McGovern Institute for Brain Research, Department of Brain and Cognitive Sciences, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Electronic address: zhang@broadinstitute.org.
References (98)
98 references, click to expand
  1. DNA targeting specificity of RNA-guided Cas9 nucleases.
    Nat Biotechnol. 2013 Sep;31(9):827-32 PMID: 23873081
  2. Comprehensive analysis of the specificity of transcription activator-like effector nucleases.
    Nucleic Acids Res. 2014 Apr;42(8):5390-402 PMID: 24569350
  3. Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery.
    Nat Biotechnol. 2007 Nov;25(11):1298-306 PMID: 17965707
  4. Site-specific recombination determined by I-SceI, a mitochondrial group I intron-encoded endonuclease expressed in the yeast nucleus.
    Genetics. 1992 Mar;130(3):451-60 PMID: 1551570
  5. Targeted DNA demethylation and activation of endogenous genes using programmable TALE-TET1 fusion proteins.
    Nat Biotechnol. 2013 Dec;31(12):1137-42 PMID: 24108092
  6. Highly efficient endogenous human gene correction using designed zinc-finger nucleases.
    Nature. 2005 Jun 2;435(7042):646-51 PMID: 15806097
  7. Breaking the code of DNA binding specificity of TAL-type III effectors.
    Science. 2009 Dec 11;326(5959):1509-12 PMID: 19933107
  8. Comparative analysis of CRISPR loci in lactic acid bacteria genomes.
    Int J Food Microbiol. 2009 Apr 30;131(1):62-70 PMID: 18635282
  9. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity.
    Nat Biotechnol. 2013 Sep;31(9):839-43 PMID: 23934178
  10. Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV.
    N Engl J Med. 2014 Mar 6;370(10):901-10 PMID: 24597865
  11. CRISPR RNA-guided activation of endogenous human genes.
    Nat Methods. 2013 Oct;10(10):977-9 PMID: 23892898
  12. Increased brain penetration and potency of a therapeutic antibody using a monovalent molecular shuttle.
    Neuron. 2014 Jan 8;81(1):49-60 PMID: 24411731
  13. Nucleotide-resolution DNA double-strand break mapping by next-generation sequencing.
    Nat Methods. 2013 Apr;10(4):361-5 PMID: 23503052
  14. RNA-guided gene activation by CRISPR-Cas9-based transcription factors.
    Nat Methods. 2013 Oct;10(10):973-6 PMID: 23892895
  15. Identification of genes that are associated with DNA repeats in prokaryotes.
    Mol Microbiol. 2002 Mar;43(6):1565-75 PMID: 11952905
  16. Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis.
    Proc Natl Acad Sci U S A. 2013 Sep 24;110(39):15644-9 PMID: 23940360
  17. Simultaneous generation and germline transmission of multiple gene mutations in rat using CRISPR-Cas systems.
    Nat Biotechnol. 2013 Aug;31(8):684-6 PMID: 23929337
  18. Phage response to CRISPR-encoded resistance in Streptococcus thermophilus.
    J Bacteriol. 2008 Feb;190(4):1390-400 PMID: 18065545
  19. Essential features and rational design of CRISPR RNAs that function with the Cas RAMP module complex to cleave RNAs.
    Mol Cell. 2012 Feb 10;45(3):292-302 PMID: 22227116
  20. Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin.
    Microbiology (Reading). 2005 Aug;151(Pt 8):2551-2561 PMID: 16079334
  21. Identification of 86 candidates for small non-messenger RNAs from the archaeon Archaeoglobus fulgidus.
    Proc Natl Acad Sci U S A. 2002 May 28;99(11):7536-41 PMID: 12032318
  22. Low LDL cholesterol in individuals of African descent resulting from frequent nonsense mutations in PCSK9.
    Nat Genet. 2005 Feb;37(2):161-5 PMID: 15654334
  23. Genome-scale CRISPR-Cas9 knockout screening in human cells.
    Science. 2014 Jan 3;343(6166):84-87 PMID: 24336571
  24. Modular system for the construction of zinc-finger libraries and proteins.
    Nat Protoc. 2010 Apr;5(4):791-810 PMID: 20360772
  25. An improved zinc-finger nuclease architecture for highly specific genome editing.
    Nat Biotechnol. 2007 Jul;25(7):778-85 PMID: 17603475
  26. RNA-guided editing of bacterial genomes using CRISPR-Cas systems.
    Nat Biotechnol. 2013 Mar;31(3):233-9 PMID: 23360965
  27. A TALE nuclease architecture for efficient genome editing.
    Nat Biotechnol. 2011 Feb;29(2):143-8 PMID: 21179091
  28. Targeting DNA double-strand breaks with TAL effector nucleases.
    Genetics. 2010 Oct;186(2):757-61 PMID: 20660643
  29. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering.
    Cell. 2013 May 9;153(4):910-8 PMID: 23643243
  30. CRISPR-Cas systems for editing, regulating and targeting genomes.
    Nat Biotechnol. 2014 Apr;32(4):347-55 PMID: 24584096
  31. A combinatorial approach to create artificial homing endonucleases cleaving chosen sequences.
    Nucleic Acids Res. 2006;34(22):e149 PMID: 17130168
  32. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria.
    Proc Natl Acad Sci U S A. 2012 Sep 25;109(39):E2579-86 PMID: 22949671
  33. Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system.
    Cell. 2013 Dec 19;155(7):1479-91 PMID: 24360272
  34. Diversity, activity, and evolution of CRISPR loci in Streptococcus thermophilus.
    J Bacteriol. 2008 Feb;190(4):1401-12 PMID: 18065539
  35. Positive and negative regulation of endogenous genes by designed transcription factors.
    Proc Natl Acad Sci U S A. 2000 Feb 15;97(4):1495-500 PMID: 10660690
  36. Locus-specific editing of histone modifications at endogenous enhancers.
    Nat Biotechnol. 2013 Dec;31(12):1133-6 PMID: 24013198
  37. CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies.
    Microbiology (Reading). 2005 Mar;151(Pt 3):653-663 PMID: 15758212
  38. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9.
    Nature. 2014 Mar 6;507(7490):62-7 PMID: 24476820
  39. CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA.
    Science. 2008 Dec 19;322(5909):1843-5 PMID: 19095942
  40. A simple cipher governs DNA recognition by TAL effectors.
    Science. 2009 Dec 11;326(5959):1501 PMID: 19933106
  41. Cytosine methylation targetted to pre-determined sequences.
    Nat Genet. 1997 Dec;17(4):376-8 PMID: 9398832
  42. CRISPR provides acquired resistance against viruses in prokaryotes.
    Science. 2007 Mar 23;315(5819):1709-12 PMID: 17379808
  43. Protospacer recognition motifs: mixed identities and functional diversity.
    RNA Biol. 2013 May;10(5):891-9 PMID: 23403393
  44. Timely translation during the mouse oocyte-to-embryo transition.
    Development. 2000 Sep;127(17):3795-803 PMID: 10934024
  45. Breakthrough of the year 2013. Cancer immunotherapy.
    Science. 2013 Dec 20;342(6165):1432-3 PMID: 24357284
  46. Enhancing gene targeting with designed zinc finger nucleases.
    Science. 2003 May 2;300(5620):764 PMID: 12730594
  47. Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells.
    Nat Biotechnol. 2014 Jul;32(7):670-6 PMID: 24752079
  48. Biological significance of a family of regularly spaced repeats in the genomes of Archaea, Bacteria and mitochondria.
    Mol Microbiol. 2000 Apr;36(1):244-6 PMID: 10760181
  49. Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases.
    Genetics. 2002 Jul;161(3):1169-75 PMID: 12136019
  50. Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease.
    Nat Biotechnol. 2013 Mar;31(3):230-2 PMID: 23360966
  51. Introduction of double-strand breaks into the genome of mouse cells by expression of a rare-cutting endonuclease.
    Mol Cell Biol. 1994 Dec;14(12):8096-106 PMID: 7969147
  52. Crystal structure of Cas9 in complex with guide RNA and target DNA.
    Cell. 2014 Feb 27;156(5):935-49 PMID: 24529477
  53. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression.
    Cell. 2013 Feb 28;152(5):1173-83 PMID: 23452860
  54. Structures of Cas9 endonucleases reveal RNA-mediated conformational activation.
    Science. 2014 Mar 14;343(6176):1247997 PMID: 24505130
  55. Processing-independent CRISPR RNAs limit natural transformation in Neisseria meningitidis.
    Mol Cell. 2013 May 23;50(4):488-503 PMID: 23706818
  56. The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems.
    RNA Biol. 2013 May;10(5):726-37 PMID: 23563642
  57. Correction of a genetic disease in mouse via use of CRISPR-Cas9.
    Cell Stem Cell. 2013 Dec 5;13(6):659-62 PMID: 24315440
  58. One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering.
    Cell. 2013 Sep 12;154(6):1370-9 PMID: 23992847
  59. Rapid "open-source" engineering of customized zinc-finger nucleases for highly efficient gene modification.
    Mol Cell. 2008 Jul 25;31(2):294-301 PMID: 18657511
  60. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes.
    Cell. 2013 Jul 18;154(2):442-51 PMID: 23849981
  61. 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.
    Biol Direct. 2006 Mar 16;1:7 PMID: 16545108
  62. An unbiased genome-wide analysis of zinc-finger nuclease specificity.
    Nat Biotechnol. 2011 Aug 07;29(9):816-23 PMID: 21822255
  63. Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems.
    Nucleic Acids Res. 2014 Feb;42(4):2577-90 PMID: 24270795
  64. Chemically regulated zinc finger transcription factors.
    J Biol Chem. 2000 Oct 20;275(42):32617-27 PMID: 10924515
  65. Small CRISPR RNAs guide antiviral defense in prokaryotes.
    Science. 2008 Aug 15;321(5891):960-4 PMID: 18703739
  66. Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos.
    Cell. 2014 Feb 13;156(4):836-43 PMID: 24486104
  67. Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype.
    Nat Biotechnol. 2014 Jun;32(6):551-3 PMID: 24681508
  68. Reassembly of shattered chromosomes in Deinococcus radiodurans.
    Nature. 2006 Oct 5;443(7111):569-73 PMID: 17006450
  69. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements.
    J Mol Evol. 2005 Feb;60(2):174-82 PMID: 15791728
  70. Unification of Cas protein families and a simple scenario for the origin and evolution of CRISPR-Cas systems.
    Biol Direct. 2011 Jul 14;6:38 PMID: 21756346
  71. Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA).
    Nat Methods. 2011 Jan;8(1):67-9 PMID: 21151135
  72. A guild of 45 CRISPR-associated (Cas) protein families and multiple CRISPR/Cas subtypes exist in prokaryotic genomes.
    PLoS Comput Biol. 2005 Nov;1(6):e60 PMID: 16292354
  73. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs.
    Nat Biotechnol. 2014 Mar;32(3):279-284 PMID: 24463574
  74. The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli.
    Nucleic Acids Res. 2011 Nov;39(21):9275-82 PMID: 21813460
  75. Induction of homologous recombination in mammalian chromosomes by using the I-SceI system of Saccharomyces cerevisiae.
    Mol Cell Biol. 1995 Apr;15(4):1968-73 PMID: 7891691
  76. Altering the genome by homologous recombination.
    Science. 1989 Jun 16;244(4910):1288-92 PMID: 2660260
  77. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.
    Science. 2012 Aug 17;337(6096):816-21 PMID: 22745249
  78. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA.
    Nature. 2010 Nov 4;468(7320):67-71 PMID: 21048762
  79. Genetic screens in human cells using the CRISPR-Cas9 system.
    Science. 2014 Jan 3;343(6166):80-4 PMID: 24336569
  80. Orthogonal Cas9 proteins for RNA-guided gene regulation and editing.
    Nat Methods. 2013 Nov;10(11):1116-21 PMID: 24076762
  81. Exome sequencing, ANGPTL3 mutations, and familial combined hypolipidemia.
    N Engl J Med. 2010 Dec 2;363(23):2220-7 PMID: 20942659
  82. Classification and evolution of type II CRISPR-Cas systems.
    Nucleic Acids Res. 2014 Jun;42(10):6091-105 PMID: 24728998
  83. Self versus non-self discrimination during CRISPR RNA-directed immunity.
    Nature. 2010 Jan 28;463(7280):568-71 PMID: 20072129
  84. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells.
    Nat Biotechnol. 2013 Sep;31(9):822-6 PMID: 23792628
  85. RNA-guided human genome engineering via Cas9.
    Science. 2013 Feb 15;339(6121):823-6 PMID: 23287722
  86. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.
    Nature. 2011 Mar 31;471(7340):602-7 PMID: 21455174
  87. Genetic and physical analysis of double-strand break repair and recombination in Saccharomyces cerevisiae.
    Genetics. 1989 Jul;122(3):519-34 PMID: 2668114
  88. RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex.
    Cell. 2009 Nov 25;139(5):945-56 PMID: 19945378
  89. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering.
    Nat Biotechnol. 2013 Sep;31(9):833-8 PMID: 23907171
  90. Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product.
    J Bacteriol. 1987 Dec;169(12):5429-33 PMID: 3316184
  91. Evolution and classification of the CRISPR-Cas systems.
    Nat Rev Microbiol. 2011 Jun;9(6):467-77 PMID: 21552286
  92. Multiplex genome engineering using CRISPR/Cas systems.
    Science. 2013 Feb 15;339(6121):819-23 PMID: 23287718
  93. Stimulation of homologous recombination through targeted cleavage by chimeric nucleases.
    Mol Cell Biol. 2001 Jan;21(1):289-97 PMID: 11113203
  94. Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients.
    Cell Stem Cell. 2013 Dec 5;13(6):653-8 PMID: 24315439
  95. Mammalian base excision repair: the forgotten archangel.
    Nucleic Acids Res. 2013 Apr 1;41(6):3483-90 PMID: 23408852
  96. RNA-programmed genome editing in human cells.
    Elife. 2013 Jan 29;2:e00471 PMID: 23386978
  97. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity.
    Cell. 2013 Sep 12;154(6):1380-9 PMID: 23992846
  98. Optical control of mammalian endogenous transcription and epigenetic states.
    Nature. 2013 Aug 22;500(7463):472-476 PMID: 23877069
Article Info
Journal
Cell
Abbr.
Cell
ISSN
1097-4172
DOI
10.1016/j.cell.2014.05.010
Email
zhang@broadinstitute.org
Published
2014-06-05
Pages
1262-1278
Language
English
Region
United States
NLM ID
0413066
PMCID
PMC4343198
Subset
IM
Grants
NCCDPHP CDC HHS · DP1-MH100706 · United States
NINDS NIH HHS · R01-NS 07312401), · United States
NIMH NIH HHS · DP1 MH100706 · United States
NINDS NIH HHS · R01 NS073124 · United States
NIDDK NIH HHS · R01 DK097768 · United States
External Links
PubMed source DOI: 10.1016/j.cell.2014.05.010 PMC Full Text View journal details
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