Design and Validation of CRISPR/Cas9 Systems for Targeted Gene Modification in Induced Pluripotent Stem Cells

  • Ciaran M. Lee
  • Haibao Zhu
  • Timothy H. Davis
  • Harshahardhan Deshmukh
  • Gang Bao
Part of the Methods in Molecular Biology book series (MIMB, volume 1498)


The CRISPR/Cas9 system is a powerful tool for precision genome editing. The ability to accurately modify genomic DNA in situ with single nucleotide precision opens up new possibilities for not only basic research but also biotechnology applications and clinical translation. In this chapter, we outline the procedures for design, screening, and validation of CRISPR/Cas9 systems for targeted modification of coding sequences in the human genome and how to perform genome editing in induced pluripotent stem cells with high efficiency and specificity.

Key words

CRISPR Genome editing Targeted gene knockout 



This work was supported by the National Institutes of Health as an NIH Nanomedicine Development Center Award (PN2EY018244 to GB) and by the Cancer Prevention and Research Institute of Texas (RR140081 to GB).


  1. 1.
    Urnov FD, Miller JC, Lee YL, Beausejour CM, Rock JM, Augustus S, Jamieson AC, Porteus MH, Gregory PD, Holmes MC (2005) Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 435(7042):646–651. doi: 10.1038/nature03556, PubMed PMID: WOS:000229476200043CrossRefPubMedGoogle Scholar
  2. 2.
    Kim YG, Cha J, Chandrasegaran S (1996) Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc Natl Acad Sci U S A 93(3):1156–1160, PubMed PMID: 8577732; PMCID: 40048CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Christian M, Cermak T, Doyle EL, Schmidt C, Zhang F, Hummel A, Bogdanove AJ, Voytas DF (2010) Targeting DNA double-strand breaks with TAL effector nucleases. Genetics 186(2):757–761. doi: 10.1534/genetics.110.120717, PubMed PMID: 20660643; PMCID: PMC2942870CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Miller JC, Tan S, Qiao G, Barlow KA, Wang J, Xia DF, Meng X, Paschon DE, Leung E, Hinkley SJ, Dulay GP, Hua KL, Ankoudinova I, Cost GJ, Urnov FD, Zhang HS, Holmes MC, Zhang L, Gregory PD, Rebar EJ (2011) A TALE nuclease architecture for efficient genome editing. Nat Biotechnol 29(2):143–148. doi: 10.1038/nbt.1755 CrossRefPubMedGoogle Scholar
  5. 5.
    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(6096):816–821. doi: 10.1126/science.1225829 CrossRefPubMedGoogle Scholar
  6. 6.
    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 U S A 109(39):E2579–E2586. doi: 10.1073/pnas.1208507109, PubMed PMID: 22949671; PMCID: 3465414CrossRefPubMedPubMedCentralGoogle Scholar
  7. 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(6121):819–823. doi: 10.1126/science.1231143, PubMed PMID: 23287718; PMCID: 3795411CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Cradick TJ, Fine EJ, Antico CJ, Bao G (2013) CRISPR/Cas9 systems targeting beta-globin and CCR5 genes have substantial off-target activity. Nucleic Acids Res 41(20):9584–9592. doi: 10.1093/nar/gkt714, PubMed PMID: 23939622; PMCID: 3814385CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    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(9):827–832. doi: 10.1038/nbt.2647, PubMed PMID: 23873081; PMCID: 3969858CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    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(9):822–826. doi: 10.1038/nbt.2623, PubMed PMID: 23792628; PMCID: 3773023CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Tsai SQ, Zheng Z, Nguyen NT, Liebers M, Topkar VV, Thapar V, Wyvekens N, Khayter C, Iafrate AJ, Le LP, Aryee MJ, Joung JK (2015) GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat Biotechnol 33(2):187–197. doi: 10.1038/nbt.3117, PubMed PMID: 25513782; PMCID: 4320685CrossRefPubMedGoogle Scholar
  12. 12.
    Lee CM, Cradick TJ, Fine EJ, Bao G (2016) Nuclease target site selection for maximizing on-target activity and minimizing off-target effects in genome editing. Mol Ther. doi: 10.1038/mt.2016.1 Google Scholar
  13. 13.
    Lee CM, Cradick TJ, Bao G (2016) The Neisseria meningitidis CRISPR-Cas9 system enables specific genome editing in mammalian cells. Mol Ther. doi: 10.1038/mt.2016.8 Google Scholar
  14. 14.
    Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz AJ, Zetsche B, Shalem O, Wu X, Makarova KS, Koonin EV, Sharp PA, Zhang F (2015) In vivo genome editing using Staphylococcus aureus Cas9. Nature. doi: 10.1038/nature14299 Google Scholar
  15. 15.
    Muller M, Lee CM, Gasiunas G, Davis TH, Cradick TJ, Siksnys V, Bao G, Cathomen T, Mussolino C (2015) Streptococcus thermophilus CRISPR-Cas9 systems enable specific editing of the human genome. Mol Ther. doi: 10.1038/mt.2015.218 Google Scholar
  16. 16.
    Cradick TJ, Qiu P, Lee CM, Fine EJ, Bao G (2014) COSMID: a web-based tool for identifying and validating CRISPR/Cas off-target sites. Mol Ther Nucleic Acids 3:e214. doi: 10.1038/mtna.64, PubMed PMID: 25462530; PMCID: 4272406CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9(7):671–675CrossRefPubMedGoogle Scholar
  18. 18.
    Guschin DY, Waite AJ, Katibah GE, Miller JC, Holmes MC, Rebar EJ (2010) A rapid and general assay for monitoring endogenous gene modification. Methods Mol Biol 649:247–256. doi: 10.1007/978-1-60761-753-2_15 CrossRefPubMedGoogle Scholar
  19. 19.
    Brinkman EK, Chen T, Amendola M, van Steensel B (2014) Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res 42(22):e168. doi: 10.1093/nar/gku936, PubMed PMID: 25300484; PMCID: 4267669CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Ciaran M. Lee
    • 1
  • Haibao Zhu
    • 1
  • Timothy H. Davis
    • 1
  • Harshahardhan Deshmukh
    • 1
  • Gang Bao
    • 1
  1. 1.Department of BioengineeringRice UniversityHoustonUSA

Personalised recommendations