Abstract
The advent of the easily programmable and efficient CRISPR/Cas9 nuclease system has revolutionized genetic engineering. While conventional gene knockout experiments using CRISPR/Cas9 are very valuable, these are not well suited to study stage-specific gene function in dynamic situations such as development or disease. Here we describe a CRISPR/Cas9-based OPTimized inducible gene KnockOut method (OPTiKO) for conditional loss-of-function studies in human cells. This approach relies on an improved tetracycline-inducible system for conditional expression of single guide RNAs (sgRNAs) that drive Cas9 activity. In order to ensure homogeneous and stable expression, the necessary transgenes are expressed following rapid and efficient single-step genetic engineering of the AAVS1 genomic safe harbor. When implemented in human pluripotent stem cells (hPSCs), the approach can be then efficiently applied to virtually any hPSC-derived human cell type at various stages of development or disease.
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References
Cooper GM, Shendure J (2011) Needles in stacks of needles: finding disease-causal variants in a wealth of genomic data. Nat Rev Genet 12:628–640
Zhang F, Lupski JR (2015) Non-coding genetic variants in human disease. Hum Mol Genet 24:R102–R110
Trounson A, DeWitt ND (2016) Pluripotent stem cells progressing to the clinic. Nat Rev Mol Cell Biol 17:194–200
Avior Y, Sagi I, Benvenisty N (2016) Pluripotent stem cells in disease modelling and drug discovery. Nat Rev Mol Cell Biol 17:170–182
Murry CE, Keller G (2008) Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development. Cell 132:661–680
Cong L, Ran FA, Cox D et al (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823
Mali P, Yang L, Esvelt KM et al (2013) RNA-guided human genome engineering via Cas9. Science 339:823–826
Jinek M, Chylinski K, Fonfara I et al (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821
Wright AV, Nuñez JK, Doudna JA (2016) Biology and applications of CRISPR systems: harnessing nature’s toolbox for genome engineering. Cell 164:29–44
Hsu PD, Lander ES, Zhang F (2014) Development and applications of CRISPR-Cas9 for genome engineering. Cell 157:1262–1278
González F, Zhu Z, Shi Z-D et al (2014) An iCRISPR platform for rapid, multiplexable, and inducible genome editing in human pluripotent stem cells. Cell Stem Cell 15:215–226
Chen Y, Cao J, Xiong M et al (2015) Engineering human stem cell lines with inducible gene knockout using CRISPR/Cas9. Cell Stem Cell 17:233–244
Mandegar MA, Huebsch N, Frolov EB et al (2016) CRISPR interference efficiently induces specific and reversible gene silencing in human iPSCs. Cell Stem Cell 18:541–553
Haenebalcke L, Goossens S, Naessens M et al (2013) Efficient ROSA26-based conditional and/or inducible transgenesis using RMCE-compatible F1 hybrid mouse embryonic stem cells. Stem Cell Rev 9:774–785
Ordovas L, Boon R, Pistoni M et al (2015) Efficient recombinase-mediated cassette exchange in hPSCs to study the hepatocyte lineage reveals AAVS1 locus-mediated transgene inhibition. Stem Cell Rep 5:918–931
Bertero A, Pawlowski M, Ortmann D et al (2016) Optimized inducible shRNA and CRISPR/Cas9 platforms for in vitro studies of human development using hPSCs. Development 143:4405–4418
Fogarty NME, McCarthy A, Snijders KE et al (2017) Genome editing reveals a role for OCT4 in human embryogenesis. Nature 550(7674):67–73
Brummelkamp TR, Bernards R, Agami R (2002) A system for stable expression of short interfering RNAs in mammalian cells. Science 296:550–553
Smith JR, Maguire S, Davis LA et al (2008) Robust, persistent transgene expression in human embryonic stem cells is achieved with AAVS1-targeted integration. Stem Cells 26:496–504
Hockemeyer D, Soldner F, Beard C et al (2009) Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases. Nat Biotechnol 27:851–857
DeKelver RC, Choi VM, Moehle EA et al (2010) Functional genomics, proteomics, and regulatory DNA analysis in isogenic settings using zinc finger nuclease-driven transgenesis into a safe harbor locus in the human genome. Genome Res 20:1133–1142
Pawlowski M, Ortmann D, Bertero A et al (2017) Inducible and deterministic forward programming of human pluripotent stem cells into neurons, skeletal myocytes, and oligodendrocytes. Stem Cell Rep 8:803–812
Gaj T, Gersbach CA, Barbas CF (2013) ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol 31:397–405
Bertero A, Yiangou L, Brown S et al (2018) Conditional manipulation of gene function in human cells with optimized inducible shRNA. Curr Protoc Stem Cell Biol 44:5C.4.1–5C.4.49
Santos DP, Kiskinis E, Eggan K et al (2016) Comprehensive protocols for CRISPR/Cas9-based gene editing in human pluripotent stem cells. Curr Protoc Stem Cell Biol 38:5B.6.1–5B.6.60
Fan H, Robetorye RS (2010) Real-time quantitative reverse transcriptase polymerase chain reaction. Methods Mol Biol 630:199–213
Wang L, Gaigalas AK, Yan M (2011) Quantitative fluorescence measurements with multicolor flow cytometry. Methods Mol Biol 699:53–65
Willingham MC (2010) Fluorescence labeling of intracellular antigens of attached or suspended tissue-culture cells. Methods Mol Biol 588:153–164
Willingham MC (2010) Fluorescence labeling of surface antigens of attached or suspended tissue-culture cells. Methods Mol Biol 588:143–151
Komatsu S (2015) Western blotting using PVDF membranes and its downstream applications. Methods Mol Biol 1312:227–236
Tsai SQ, Joung JK (2016) Defining and improving the genome-wide specificities of CRISPR–Cas9 nucleases. Nat Rev Genet 17:300–312
Mohr SE, Hu Y, Ewen-Campen B et al (2016) CRISPR guide RNA design for research applications. FEBS J 283:3232–3238
Graham DB, Root DE (2015) Resources for the design of CRISPR gene editing experiments. Genome Biol 16:260
Perez AR, Pritykin Y, Vidigal JA et al (2017) GuideScan software for improved single and paired CRISPR guide RNA design. Nat Biotechnol 35:347–349
MacPherson CR, Scherf A (2015) Flexible guide-RNA design for CRISPR applications using Protospacer Workbench. Nat Biotechnol 33:805–806
Bloom K, Ely A, Arbuthnot P (2017) A T7 endonuclease I assay to detect talen-mediated targeted mutation of HBV cccDNA. Methods Mol Biol 1540:85–95
Ranganathan V, Wahlin K, Maruotti J, Zack DJ (2014) Expansion of the CRISPR-Cas9 genome targeting space through the use of H1 promoter-expressed guide RNAs. Nat Commun 5:4516
Ma H, Wu Y, Dang Y et al (2014) Pol III promoters to express small RNAs: delineation of transcription initiation. Mol Ther 3:e161
Watanabe K, Ueno M, Kamiya D et al (2007) A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat Biotechnol 25:681–686
Henriksen JR, Løkke C, Hammerø M et al (2007) Comparison of RNAi efficiency mediated by tetracycline-responsive H1 and U6 promoter variants in mammalian cell lines. Nucleic Acids Res 35:e67
Slaymaker IM, Gao L, Zetsche B et al (2015) Rationally engineered Cas9 nucleases with improved specificity. Science 351:84–88
Kleinstiver BP, Pattanayak V, Prew MS et al (2016) High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529:490–495
Chatzispyrou IA, Held NM, Mouchiroud L et al (2015) Tetracycline antibiotics impair mitochondrial function and its experimental use confounds research. Cancer Res 75:4446–4449
Moullan N, Mouchiroud L, Wang X et al (2015) Tetracyclines disturb mitochondrial function across eukaryotic models: a call for caution in biomedical research. Cell Rep 10:1681–1691
Acknowledgments
The authors would like to thank Dr. Mark Kotter, Dr. Sanjay Sinha, and the members of the Vallier, Kotter, and Sinha laboratories for their help with the validation of this methodology. We would also like to thank Dr. Kosuke Yusa for providing the AAVS1 ZFN plasmids. This work was supported by a British Heart Foundation PhD Studentship (FS/11/77/39327; A.B.); a European Research Council starting grant Relieve IMDs (281335; L.V., A.B.); the Cambridge University Hospitals National Institute for Health Research Biomedical Research Centre (L.V., K.E.S.); the British Heart Foundation Oxbridge Centre of Regenerative Medicine (J.D.C.), and a core support grant from the Wellcome Trust and Medical Research Council to the Wellcome Trust—Medical Research Council Cambridge Stem Cell Institute (PSAG028; L.V.).
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Snijders, K.E., Cooper, J.D., Vallier, L., Bertero, A. (2019). Conditional Gene Knockout in Human Cells with Inducible CRISPR/Cas9. In: Luo, Y. (eds) CRISPR Gene Editing. Methods in Molecular Biology, vol 1961. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9170-9_12
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DOI: https://doi.org/10.1007/978-1-4939-9170-9_12
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