Archives of Toxicology

, Volume 89, Issue 7, pp 1023–1034 | Cite as

Application of CRISPR/Cas9 genome editing to the study and treatment of disease

  • Andrea Pellagatti
  • Hamid Dolatshad
  • Simona Valletta
  • Jacqueline BoultwoodEmail author
Review Article


CRISPR/Cas is a microbial adaptive immune system that uses RNA-guided nucleases to cleave foreign genetic elements. The CRISPR/Cas9 method has been engineered from the type II prokaryotic CRISPR system and uses a single-guide RNA to target the Cas9 nuclease to a specific genomic sequence. Cas9 induces double-stranded DNA breaks which are repaired either by imperfect non-homologous end joining to generate insertions or deletions (indels) or, if a repair template is provided, by homology-directed repair. Due to its specificity, simplicity and versatility, the CRISPR/Cas9 system has recently emerged as a powerful tool for genome engineering in various species. This technology can be used to investigate the function of a gene of interest or to correct gene mutations in cells via genome editing, paving the way for future gene therapy approaches. Improvements to the efficiency of CRISPR repair, in particular to increase the rate of gene correction and to reduce undesired off-target effects, and the development of more effective delivery methods will be required for its broad therapeutic application.


CRISPR CRISPR/Cas9 Genome editing Inherited disease Mutation correction Gene therapy 



AP and JB acknowledge the support of Leukaemia and Lymphoma Research (UK).

Conflict of interest

The authors declare that there are no conflicts of interest.


  1. Ain QU, Chung JY, Kim Y-H (2014) Current and future delivery systems for engineered nucleases: ZFN, TALEN and RGEN. J Control Release. doi: 10.1016/j.jconrel.2014.12.036
  2. Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, Romero DA, Horvath P (2007) CRISPR provides acquired resistance against viruses in prokaryotes. Science 315:1709–1712PubMedCrossRefGoogle Scholar
  3. Bolotin A, Quinquis B, Sorokin A, Ehrlich SD (2005) Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology 151:2551–2561PubMedCrossRefGoogle Scholar
  4. Cho SW, Kim S, Kim JM, Kim J-S (2013) Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol 31:230–232PubMedCrossRefGoogle Scholar
  5. Cho SW, Kim S, Kim Y, Kweon J, Kim HS, Bae S, Kim J-S (2014) Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases. Genome Res 24:132–141PubMedCentralPubMedCrossRefGoogle Scholar
  6. Choi PS, Meyerson M (2014) Targeted genomic rearrangements using CRISPR/Cas technology. Nat Commun 5:3728PubMedCentralPubMedGoogle Scholar
  7. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823PubMedCentralPubMedCrossRefGoogle Scholar
  8. Davis L, Maizels N (2014) Homology-directed repair of DNA nicks via pathways distinct from canonical double-strand break repair. Proc Natl Acad Sci 111:E924–E932PubMedCentralPubMedCrossRefGoogle Scholar
  9. Deltcheva E, Chylinski K, Sharma CM, Gonzales K, Chao Y, Pirzada ZA, Eckert MR, Vogel J, Charpentier E (2011) CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 471:602–607PubMedCentralPubMedCrossRefGoogle Scholar
  10. Dianov GL, Hübscher U (2013) Mammalian base excision repair: the forgotten archangel. Nucleic Acids Res 41:3483–3490PubMedCentralPubMedCrossRefGoogle Scholar
  11. Ding Q, Regan SN, Xia Y, Oostrom LA, Cowan CA, Musunuru K (2013) Enhanced efficiency of human pluripotent stem cell genome editing through replacing TALENs with CRISPRs. Cell Stem Cell 12:393–394PubMedCentralPubMedCrossRefGoogle Scholar
  12. Doudna JA, Charpentier E (2014) The new frontier of genome engineering with CRISPR–Cas9. Science. doi: 10.1126/science.1258096 PubMedCentralGoogle Scholar
  13. Folger KR, Wong EA, Wahl G, Capecchi MR (1982) Patterns of integration of DNA microinjected into cultured mammalian cells: evidence for homologous recombination between injected plasmid DNA molecules. Mol Cell Biol 2:1372–1387PubMedCentralPubMedGoogle Scholar
  14. Friedland AE, Tzur YB, Esvelt KM, Colaiacovo MP, Church GM, Calarco JA (2013) Heritable genome editing in C. elegans via a CRISPR–Cas9 system. Nat Methods 10:741–743PubMedCrossRefGoogle Scholar
  15. Fu Y, Foden JA, Khayter C, Maeder ML, Reyon D, Joung JK, Sander JD (2013) High-frequency off-target mutagenesis induced by CRISPR–Cas nucleases in human cells. Nat Biotechnol 31:822–826PubMedCentralPubMedCrossRefGoogle Scholar
  16. Fu Y, Sander JD, Reyon D, Cascio VM, Joung JK (2014) Improving CRISPR–Cas nuclease specificity using truncated guide RNAs. Nat Biotechnol 32:279–284PubMedCentralPubMedCrossRefGoogle Scholar
  17. Gabriel R, Lombardo A, Arens A, Miller JC, Genovese P, Kaeppel C, Nowrouzi A, Bartholomae CC, Wang J, Friedman G, Holmes MC, Gregory PD, Glimm H, Schmidt M, Naldini L, von Kalle C (2011) An unbiased genome-wide analysis of zinc-finger nuclease specificity. Nat Biotechnol 29:816–823PubMedCrossRefGoogle Scholar
  18. Gaj T, Guo J, Kato Y, Sirk SJ, Barbas CF (2012) Targeted gene knockout by direct delivery of zinc-finger nuclease proteins. Nat Methods 9:805–807PubMedCentralPubMedCrossRefGoogle Scholar
  19. Gaj T, Gersbach CA, Barbas Iii CF (2013) ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol 31:397–405PubMedCentralPubMedCrossRefGoogle Scholar
  20. Gasiunas G, Barrangou R, Horvath P, Siksnys V (2012) Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci USA 109:E2579–E2586PubMedCentralPubMedCrossRefGoogle Scholar
  21. Gratz SJ, Cummings AM, Nguyen JN, Hamm DC, Donohue LK, Harrison MM, Wildonger J, O’Connor-Giles KM (2013) Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease. Genetics 194:1029–1035PubMedCentralPubMedCrossRefGoogle Scholar
  22. Güell M, Yang L, Church GM (2014) Genome editing assessment using CRISPR genome analyzer (CRISPR–GA). Bioinformatics 30:2968–2970PubMedCrossRefGoogle Scholar
  23. Guilinger JP, Thompson DB, Liu DR (2014) Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nat Biotechnol 32:577–582PubMedCentralPubMedCrossRefGoogle Scholar
  24. Gupta RM, Musunuru K (2014) Expanding the genetic editing tool kit: ZFNs, TALENs, and CRISPR–Cas9. J Clin Investig 124:4154–4161PubMedCrossRefGoogle Scholar
  25. Heckl D, Kowalczyk MS, Yudovich D, Belizaire R, Puram RV, McConkey ME, Thielke A, Aster JC, Regev A, Ebert BL (2014) Generation of mouse models of myeloid malignancy with combinatorial genetic lesions using CRISPR–Cas9 genome editing. Nat Biotechnol 32:941–946PubMedCentralPubMedCrossRefGoogle Scholar
  26. Horii T, Tamura D, Morita S, Kimura M, Hatada I (2013) Generation of an ICF syndrome model by efficient genome editing of human induced pluripotent stem cells using the CRISPR system. Int J Mol Sci 14:19774–19781PubMedCentralPubMedCrossRefGoogle Scholar
  27. Hsu Patrick D, Lander Eric S, Zhang F (2014) Development and applications of CRISPR–Cas9 for genome engineering. Cell 157:1262–1278PubMedCentralPubMedCrossRefGoogle Scholar
  28. Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, Li Y, Fine EJ, Wu X, Shalem O, Cradick TJ, Marraffini LA, Bao G, Zhang F (2013) DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol 31:827–832PubMedCentralPubMedCrossRefGoogle Scholar
  29. Hwang WY, Fu Y, Reyon D, Maeder ML, Tsai SQ, Sander JD, Peterson RT, Yeh JRJ, Joung JK (2013) Efficient genome editing in zebrafish using a CRISPR–Cas system. Nat Biotechnol 31:227–229PubMedCentralPubMedCrossRefGoogle Scholar
  30. Ishino Y, Shinagawa H, Makino K, Amemura M, Nakata A (1987) Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. J Bacteriol 169:5429–5433PubMedCentralPubMedGoogle Scholar
  31. Jiang W, Bikard D, Cox D, Zhang F, Marraffini LA (2013) RNA-guided editing of bacterial genomes using CRISPR–Cas systems. Nat Biotechnol 31:233–239PubMedCentralPubMedCrossRefGoogle Scholar
  32. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821PubMedCrossRefGoogle Scholar
  33. Jinek M, Jiang F, Taylor DW, Sternberg SH, Kaya E, Ma E, Anders C, Hauer M, Zhou K, Lin S, Kaplan M, Iavarone AT, Charpentier E, Nogales E, Doudna JA (2014) Structures of Cas9 endonucleases reveal RNA-mediated conformational activation. Science. doi: 10.1126/science.1247997 PubMedCentralPubMedGoogle Scholar
  34. Katic I, Großhans H (2013) Targeted heritable mutation and gene conversion by Cas9-CRISPR in caenorhabditis elegans. Genetics 195:1173–1176PubMedCentralPubMedCrossRefGoogle Scholar
  35. Kim S, Kim D, Cho SW, Kim J, Kim J-S (2014) Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res 24:1012–1019PubMedCentralPubMedCrossRefGoogle Scholar
  36. Koike-Yusa H, Li Y, Tan EP, Velasco-Herrera MDC, Yusa K (2014) Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR–guide RNA library. Nat Biotechnol 32:267–273PubMedCrossRefGoogle Scholar
  37. Li D, Qiu Z, Shao Y, Chen Y, Guan Y, Liu M, Li Y, Gao N, Wang L, Lu X, Zhao Y, Liu M (2013a) Heritable gene targeting in the mouse and rat using a CRISPR–Cas system. Nat Biotechnol 31:681–683PubMedCrossRefGoogle Scholar
  38. Li W, Teng F, Li T, Zhou Q (2013b) Simultaneous generation and germline transmission of multiple gene mutations in rat using CRISPR–Cas systems. Nat Biotechnol 31:684–686PubMedCrossRefGoogle Scholar
  39. Li HL, Fujimoto N, Sasakawa N, Shirai S, Ohkame T, Sakuma T, Tanaka M, Amano N, Watanabe A, Sakurai H, Yamamoto T, Yamanaka S, Hotta A (2015) Precise correction of the dystrophin gene in duchenne muscular dystrophy patient induced pluripotent stem cells by TALEN and CRISPR–Cas9. Stem Cell Rep 4:143–154CrossRefGoogle Scholar
  40. Lin Y, Cradick TJ, Brown MT, Deshmukh H, Ranjan P, Sarode N, Wile BM, Vertino PM, Stewart FJ, Bao G (2014) CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences. Nucleic Acids Res 42:7473–7485PubMedCentralPubMedCrossRefGoogle Scholar
  41. Long C, McAnally JR, Shelton JM, Mireault AA, Bassel-Duby R, Olson EN (2014) Prevention of muscular dystrophy in mice by CRISPR/Cas9-mediated editing of germline DNA. Science 345:1184–1188PubMedCentralPubMedCrossRefGoogle Scholar
  42. Maddalo D, Manchado E, Concepcion CP, Bonetti C, Vidigal JA, Han Y-C, Ogrodowski P, Crippa A, Rekhtman N, de Stanchina E, Lowe SW, Ventura A (2014) In vivo engineering of oncogenic chromosomal rearrangements with the CRISPR/Cas9 system. Nature 516:423–427PubMedCrossRefGoogle Scholar
  43. Maggio I, Holkers M, Liu J, Janssen JM, Chen X, Goncalves MAFV (2014) Adenoviral vector delivery of RNA-guided CRISPR/Cas9 nuclease complexes induces targeted mutagenesis in a diverse array of human cells. Sci Rep. doi: 10.1038/srep05105 PubMedCentralPubMedGoogle Scholar
  44. Makarova KS, Aravind L, Grishin NV, Rogozin IB, Koonin EV (2002) A DNA repair system specific for thermophilic Archaea and bacteria predicted by genomic context analysis. Nucleic Acids Res 30:482–496PubMedCentralPubMedCrossRefGoogle Scholar
  45. Makarova KS, Aravind L, Wolf Y, Koonin E (2011a) Unification of Cas protein families and a simple scenario for the origin and evolution of CRISPR–Cas systems. Biol Direct 6:38PubMedCentralPubMedCrossRefGoogle Scholar
  46. Makarova KS, Haft DH, Barrangou R, Brouns SJJ, Charpentier E, Horvath P, Moineau S, Mojica FJM, Wolf YI, Yakunin AF, van der Oost J, Koonin EV (2011b) Evolution and classification of the CRISPR–Cas systems. Nat Rev Microbiol 9:467–477PubMedCrossRefGoogle Scholar
  47. Mali P, Aach J, Stranges PB, Esvelt KM, Moosburner M, Kosuri S, Yang L, Church GM (2013a) CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat Biotechnol 31:833–838PubMedCrossRefGoogle Scholar
  48. Mali P, Esvelt KM, Church GM (2013b) Cas9 as a versatile tool for engineering biology. Nat Methods 10:957–963PubMedCentralPubMedCrossRefGoogle Scholar
  49. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM (2013c) RNA-guided human genome engineering via Cas9. Science 339:823–826PubMedCentralPubMedCrossRefGoogle Scholar
  50. Mladenov E, Iliakis G (2011) Induction and repair of DNA double strand breaks: the increasing spectrum of non-homologous end joining pathways. Mutat Res 711:61–72PubMedCrossRefGoogle Scholar
  51. Montague TG, Cruz JM, Gagnon JA, Church GM, Valen E (2014) CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing. Nucleic Acids Res 42:W401–W407PubMedCentralPubMedCrossRefGoogle Scholar
  52. Nishimasu H, Ran FA, Hsu PD, Konermann S, Shehata SI, Dohmae N, Ishitani R, Zhang F, Nureki O (2014) Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 156:935–949PubMedCentralPubMedCrossRefGoogle Scholar
  53. Niu Y, Shen B, Cui Y, Chen Y, Wang J, Wang L, Kang Y, Zhao X, Si W, Li W, Xiang AP, Zhou J, Guo X, Bi Y, Si C, Hu B, Dong G, Wang H, Zhou Z, Li T, Tan T, Pu X, Wang F, Ji S, Zhou Q, Huang X, Ji W, Sha J (2014) Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos. Cell 156:836–843PubMedCrossRefGoogle Scholar
  54. Pattanayak V, Lin S, Guilinger JP, Ma E, Doudna JA, Liu DR (2013) High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat Biotechnol 31:839–843PubMedCentralPubMedCrossRefGoogle Scholar
  55. Ramakrishna S, Kwaku Dad A-B, Beloor J, Gopalappa R, Lee S-K, Kim H (2014) Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA. Genome Res 24:1020–1027PubMedCentralPubMedCrossRefGoogle Scholar
  56. Ran FA, Hsu PD, Lin C-Y, Gootenberg JS, Konermann S, Trevino A, Scott DA, Inoue A, Matoba S, Zhang Y, Zhang F (2013) Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell. doi: 10.1016/j.cell.2013.08.021 PubMedCentralPubMedGoogle Scholar
  57. Sanchez-Rivera FJ, Papagiannakopoulos T, Romero R, Tammela T, Bauer MR, Bhutkar A, Joshi NS, Subbaraj L, Bronson RT, Xue W, Jacks T (2014) Rapid modelling of cooperating genetic events in cancer through somatic genome editing. Nature 516:428–431PubMedCrossRefGoogle Scholar
  58. Sander JD, Joung JK (2014) CRISPR–Cas systems for editing, regulating and targeting genomes. Nat Biotechnol 32:347–355PubMedCentralPubMedCrossRefGoogle Scholar
  59. Sapranauskas R, Gasiunas G, Fremaux C, Barrangou R, Horvath P, Siksnys V (2011) The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli. Nucleic Acids Res 39:9275–9282PubMedCentralPubMedCrossRefGoogle Scholar
  60. Schwank G, Koo B-K, Sasselli V, Dekkers JF, Heo I, Demircan T, Sasaki N, Boymans S, Cuppen E, van der Ent CK, Nieuwenhuis EE, Beekman JM, Clevers H (2013) Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients. Cell Stem Cell 13:653–658PubMedCrossRefGoogle Scholar
  61. Semenova E, Jore MM, Datsenko KA, Semenova A, Westra ER, Wanner B, van der Oost J, Brouns SJJ, Severinov K (2011) Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence. Proc Natl Acad Sci 108:10098–10103PubMedCentralPubMedCrossRefGoogle Scholar
  62. Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelsen TS, Heckl D, Ebert BL, Root DE, Doench JG, Zhang F (2014) Genome-scale CRISPR–Cas9 knockout screening in human cells. Science 343:84–87PubMedCentralPubMedCrossRefGoogle Scholar
  63. Shen B, Zhang W, Zhang J, Zhou J, Wang J, Chen L, Wang L, Hodgkins A, Iyer V, Huang X, Skarnes WC (2014) Efficient genome modification by CRISPR–Cas9 nickase with minimal off-target effects. Nat Methods 11:399–402PubMedCrossRefGoogle Scholar
  64. Smith C, Gore A, Yan W, Abalde-Atristain L, Li Z, He C, Wang Y, Brodsky RA, Zhang K, Cheng L, Ye Z (2014) Whole-genome sequencing analysis reveals high specificity of CRISPR/Cas9 and TALEN-based genome editing in human iPSCs. Cell Stem Cell 15:12–13PubMedCentralPubMedCrossRefGoogle Scholar
  65. Sternberg SH, Redding S, Jinek M, Greene EC, Doudna JA (2014) DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature 507:62–67PubMedCentralPubMedCrossRefGoogle Scholar
  66. Suzuki K, Yu C, Qu J, Li M, Yao X, Yuan T, Goebl A, Tang S, Ren R, Aizawa E, Zhang F, Xu X, Soligalla RD, Chen F, Kim J, Kim NY, Liao H-K, Benner C, Esteban CR, Jin Y, Liu G-H, Li Y, Izpisua Belmonte JC (2014) Targeted gene correction minimally impacts whole-genome mutational load in human-disease-specific induced pluripotent stem cell clones. Cell Stem Cell 15:31–36PubMedCrossRefGoogle Scholar
  67. Torres R, Martin MC, Garcia A, Cigudosa JC, Ramirez JC, Rodriguez-Perales S (2014) Engineering human tumour-associated chromosomal translocations with the RNA-guided CRISPR–Cas9 system. Nat Commun 5:3964PubMedCrossRefGoogle Scholar
  68. Tsai SQ, Wyvekens N, Khayter C, Foden JA, Thapar V, Reyon D, Goodwin MJ, Aryee MJ, Joung JK (2014) Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nat Biotechnol 32:569–576PubMedCentralPubMedCrossRefGoogle Scholar
  69. van der Oost J, Westra ER, Jackson RN, Wiedenheft B (2014) Unravelling the structural and mechanistic basis of CRISPR–Cas systems. Nat Rev Microbiol 12:479–492PubMedCrossRefGoogle Scholar
  70. Veres A, Gosis Bridget S, Ding Q, Collins R, Ragavendran A, Brand H, Erdin S, Cowan CA, Talkowski Michael E, Musunuru K (2014) Low incidence of off-target mutations in individual CRISPR–Cas9 and TALEN targeted human stem cell clones detected by whole-genome sequencing. Cell Stem Cell 15:27–30PubMedCrossRefGoogle Scholar
  71. Waaijers S, Portegijs V, Kerver J, Lemmens BBLG, Tijsterman M, van den Heuvel S, Boxem M (2013) CRISPR/Cas9-targeted mutagenesis in caenorhabditis elegans. Genetics 195:1187–1191PubMedCentralPubMedCrossRefGoogle Scholar
  72. Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch R (2013) One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153:910–918PubMedCentralPubMedCrossRefGoogle Scholar
  73. Wang T, Wei JJ, Sabatini DM, Lander ES (2014) Genetic screens in human cells using the CRISPR–Cas9 system. Science 343:80–84PubMedCentralPubMedCrossRefGoogle Scholar
  74. Wu Y, Liang D, Wang Y, Bai M, Tang W, Bao S, Yan Z, Li D, Li J (2013) Correction of a genetic disease in mouse via use of CRISPR–Cas9. Cell Stem Cell 13:659–662PubMedCrossRefGoogle Scholar
  75. Wu Y, Zhou H, Fan X, Zhang Y, Zhang M, Wang Y, Xie Z, Bai M, Yin Q, Liang D, Tang W, Liao J, Zhou C, Liu W, Zhu P, Guo H, Pan H, Wu C, Shi H, Wu L, Tang F, Li J (2015) Correction of a genetic disease by CRISPR–Cas9-mediated gene editing in mouse spermatogonial stem cells. Cell Res 25:67–79PubMedCrossRefGoogle Scholar
  76. Xiao A, Cheng Z, Kong L, Zhu Z, Lin S, Gao G, Zhang B (2014) CasOT: a genome-wide Cas9/gRNA off-target searching tool. Bioinformatics 30:1180–1182CrossRefGoogle Scholar
  77. Xie F, Ye L, Chang JC, Beyer AI, Wang J, Muench MO, Kan YW (2014a) Seamless gene correction of β-thalassemia mutations in patient-specific iPSCs using CRISPR/Cas9 and piggyBac. Genome Res 24:1526–1533PubMedCentralPubMedCrossRefGoogle Scholar
  78. Xie S, Shen B, Zhang C, Huang X, Zhang Y (2014b) sgRNAcas9: a software package for designing CRISPR sgRNA and evaluating potential off-target cleavage sites. PLoS ONE 9:e100448PubMedCentralPubMedCrossRefGoogle Scholar
  79. Yin H, Xue W, Chen S, Bogorad RL, Benedetti E, Grompe M, Koteliansky V, Sharp PA, Jacks T, Anderson DG (2014) Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nat Biotechnol 32:551–553PubMedCentralPubMedCrossRefGoogle Scholar
  80. Yu Z, Ren M, Wang Z, Zhang B, Rong YS, Jiao R, Gao G (2013) Highly efficient genome modifications mediated by CRISPR/Cas9 in drosophila. Genetics 195:289–291PubMedCentralPubMedCrossRefGoogle Scholar
  81. Yui S, Nakamura T, Sato T, Nemoto Y, Mizutani T, Zheng X, Ichinose S, Nagaishi T, Okamoto R, Tsuchiya K, Clevers H, Watanabe M (2012) Functional engraftment of colon epithelium expanded in vitro from a single adult Lgr5+ stem cell. Nat Med 18:618–623PubMedCrossRefGoogle Scholar
  82. Zhang F, Wen Y, Guo X (2014) CRISPR/Cas9 for genome editing: progress, implications and challenges. Hum Mol Genet 23:R40–R46PubMedCrossRefGoogle Scholar
  83. Zhou J, Shen B, Zhang W, Wang J, Yang J, Chen L, Zhang N, Zhu K, Xu J, Hu B, Leng Q, Huang X (2014a) One-step generation of different immunodeficient mice with multiple gene modifications by CRISPR/Cas9 mediated genome engineering. Int J Biochem Cell Biol 46:49–55PubMedCrossRefGoogle Scholar
  84. Zhou Y, Zhu S, Cai C, Yuan P, Li C, Huang Y, Wei W (2014b) High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells. Nature 509:487–491PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Andrea Pellagatti
    • 1
  • Hamid Dolatshad
    • 1
  • Simona Valletta
    • 1
  • Jacqueline Boultwood
    • 1
    Email author
  1. 1.Leukaemia and Lymphoma Research Molecular Haematology Unit, Nuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, and NIHR Biomedical Research Centre, Oxford University HospitalsUniversity of OxfordOxfordUK

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