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CRISPR Guide RNA Library Screens in Human Induced Pluripotent Stem Cells

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Induced Pluripotent Stem Cells and Human Disease

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2549))

Abstract

High-throughput CRISPR guide RNA (gRNA) library screen, that is, CRISPR/Cas9 screen, enables the unbiased identification of gene functions in a variety of biological processes. Typical pooled CRISPR/Cas9 screen couples a gRNA library and a guided Cas9 or dCas9 endonuclease to target specific gene loci, and then systematically uncover the causal link between candidate genes and observed cellular phenotypes via gRNA depletion or enrichment in screens. Here, we describe a detailed method of puromycin (PURO) concentration titration and lentiviral CRISPR gRNA library titration in Cas9 expressing monoclonal human iPSC line (Cas9+MNhiPSC) prior to performing the screens, conducting pooled CRISPR gRNA library screens in Cas9+MNhiPSC, genomic DNA extraction from the selected cell subpopulation and sequencing library preparation as well as next generation sequencing (NGS) to generate gRNA read counts. In CRISPR/Cas9 screen, we aim for 30% transduction efficiency (i.e., multiplicity of infection = 0.3) to ensure most of infected cells receive only one gRNA. The principles in this method can be applied to CRISPR perturbation (knockout, activation, repression or base editing) screens with other CRISPR gRNA libraries across many other cell models and other species.

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References

  1. Nakamura M, Gao Y, Dominguez AA, Qi LS (2021) CRISPR technologies for precise epigenome editing. Nat Cell Biol 23(1):11–22. https://doi.org/10.1038/s41556-020-00620-7

    Article  CAS  PubMed  Google Scholar 

  2. Chen JR, Tang ZH, Zheng J, Shi HS, Ding J, Qian XD, Zhang C, Chen JL, Wang CC, Li L, Chen JZ, Yin SK, Shao JZ, Huang TS, Chen P, Guan MX, Wang JF (2016) Effects of genetic correction on the differentiation of hair cell-like cells from iPSCs with MYO15A mutation. Cell Death Differ 23(8):1347–1357. https://doi.org/10.1038/cdd.2016.16

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Yang Y, Zhang X, Yi L, Hou Z, Chen J, Kou X, Zhao Y, Wang H, Sun XF, Jiang C, Wang Y, Gao S (2016) Naive induced pluripotent stem cells generated from beta-thalassemia fibroblasts allow efficient gene correction with CRISPR/Cas9. Stem Cells Transl Med 5(1):8–19. https://doi.org/10.5966/sctm.2015-0157

    Article  CAS  PubMed  Google Scholar 

  4. 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. https://doi.org/10.1126/science.1231143

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM (2013) RNA-guided human genome engineering via Cas9. Science 339(6121):823–826. https://doi.org/10.1126/science.1232033

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelson T, Heckl D, Ebert BL, Root DE, Doench JG, Zhang F (2014) Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 343(6166):84–87. https://doi.org/10.1126/science.1247005

    Article  CAS  PubMed  Google Scholar 

  7. Pickar-Oliver A, Gersbach CA (2019) The next generation of CRISPR-Cas technologies and applications. Nat Rev Mol Cell Biol 20(8):490–507. https://doi.org/10.1038/s41580-019-0131-5

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Kiani S, Chavez A, Tuttle M, Hall RN, Chari R, Ter-Ovanesyan D, Qian J, Pruitt BW, Beal J, Vora S, Buchthal J, Kowal EJ, Ebrahimkhani MR, Collins JJ, Weiss R, Church G (2015) Cas9 gRNA engineering for genome editing, activation and repression. Nat Methods 12(11):1051–1054. https://doi.org/10.1038/nmeth.3580

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Gilbert LA, Horlbeck MA, Adamson B, Villalta JE, Chen Y, Whitehead EH, Guimaraes C, Panning B, Ploegh HL, Bassik MC, Qi LS, Kampmann M, Weissman JS (2014) Genome-scale CRISPR-mediated control of gene repression and activation. Cell 159(3):647–661. https://doi.org/10.1016/j.cell.2014.09.029

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Konermann S, Brigham MD, Trevino AE, Joung J, Abudayyeh OO, Barcena C, Hsu PD, Habib N, Gootenberg JS, Nishimasu H, Nureki O, Zhang F (2015) Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature 517(7536):583–588. https://doi.org/10.1038/nature14136

    Article  CAS  PubMed  Google Scholar 

  11. Porto EM, Komor AC, Slaymaker IM, Yeo GW (2020) Base editing: advances and therapeutic opportunities. Nat Rev Drug Discov 19(12):839–859. https://doi.org/10.1038/s41573-020-0084-6

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Wang T, Wei JJ, Sabatini DM, Lander ES (2014) Genetic screens in human cells using the CRISPR-Cas9 system. Science 343(6166):80–84. https://doi.org/10.1126/science.1246981

    Article  CAS  PubMed  Google Scholar 

  13. Koike-Yusa H, Li Y, Tan EP, Velasco-Herrera Mdel C, Yusa K (2014) Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library. Nat Biotechnol 32(3):267–273. https://doi.org/10.1038/nbt.2800

    Article  CAS  PubMed  Google Scholar 

  14. Sanson KR, Hanna RE, Hegde M, Donovan KF, Strand C, Sullender ME, Vaimberg EW, Goodale A, Root DE, Piccioni F, Doench JG (2018) Optimized libraries for CRISPR-Cas9 genetic screens with multiple modalities. Nat Commun 9(1):5416. https://doi.org/10.1038/s41467-018-07901-8

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Doench JG (2018) Am I ready for CRISPR? A user's guide to genetic screens. Nat Rev Genet 19(2):67–80. https://doi.org/10.1038/nrg.2017.97

    Article  CAS  PubMed  Google Scholar 

  16. Tzelepis K, Koike-Yusa H, De Braekeleer E, Li Y, Metzakopian E, Dovey OM, Mupo A, Grinkevich V, Li M, Mazan M, Gozdecka M, Ohnishi S, Cooper J, Patel M, McKerrell T, Chen B, Domingues AF, Gallipoli P, Teichmann S, Ponstingl H, McDermott U, Saez-Rodriguez J, Huntly BJP, Iorio F, Pina C, Vassiliou GS, Yusa K (2016) A CRISPR dropout screen identifies genetic vulnerabilities and therapeutic targets in acute myeloid leukemia. Cell Rep 17(4):1193–1205. https://doi.org/10.1016/j.celrep.2016.09.079

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Shi J, Wang E, Milazzo JP, Wang Z, Kinney JB, Vakoc CR (2015) Discovery of cancer drug targets by CRISPR-Cas9 screening of protein domains. Nat Biotechnol 33(6):661–667. https://doi.org/10.1038/nbt.3235

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Xue VW, Wong SCC, Cho WCS (2020) Genome-wide CRISPR screens for the identification of therapeutic targets for cancer treatment. Expert Opin Ther Targets 24(11):1147–1158. https://doi.org/10.1080/14728222.2020.1820986

    Article  CAS  PubMed  Google Scholar 

  19. Wang T, Birsoy K, Hughes NW, Krupczak KM, Post Y, Wei JJ, Lander ES, Sabatini DM (2015) Identification and characterization of essential genes in the human genome. Science 350(6264):1096–1101. https://doi.org/10.1126/science.aac7041

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Chen S, Sanjana NE, Zheng K, Shalem O, Lee K, Shi X, Scott DA, Song J, Pan JQ, Weissleder R, Lee H, Zhang F, Sharp PA (2015) Genome-wide CRISPR screen in a mouse model of tumor growth and metastasis. Cell 160(6):1246–1260. https://doi.org/10.1016/j.cell.2015.02.038

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. van der Weyden L, Harle V, Turner G, Offord V, Iyer V, Droop A, Swiatkowska A, Rabbie R, Campbell AD, Sansom OJ, Pardo M, Choudhary JS, Ferreira I, Tullett M, Arends MJ, Speak AO, Adams DJ (2021) CRISPR activation screen in mice identifies novel membrane proteins enhancing pulmonary metastatic colonisation. Commun Biol 4(1):395. https://doi.org/10.1038/s42003-021-01912-w

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Behan FM, Iorio F, Picco G, Goncalves E, Beaver CM, Migliardi G, Santos R, Rao Y, Sassi F, Pinnelli M, Ansari R, Harper S, Jackson DA, McRae R, Pooley R, Wilkinson P, van der Meer D, Dow D, Buser-Doepner C, Bertotti A, Trusolino L, Stronach EA, Saez-Rodriguez J, Yusa K, Garnett MJ (2019) Prioritization of cancer therapeutic targets using CRISPR-Cas9 screens. Nature 568(7753):511–516. https://doi.org/10.1038/s41586-019-1103-9

    Article  CAS  PubMed  Google Scholar 

  23. Ihry RJ, Salick MR, Ho DJ, Sondey M, Kommineni S, Paula S, Raymond J, Henry B, Frias E, Wang Q, Worringer KA, Ye C, Russ C, Reece-Hoyes JS, Altshuler RC, Randhawa R, Yang Z, McAllister G, Hoffman GR, Dolmetsch R, Kaykas A (2019) Genome-scale CRISPR screens identify human pluripotency-specific genes. Cell Rep 27(2):616–630. e616. https://doi.org/10.1016/j.celrep.2019.03.043

    Article  CAS  PubMed  Google Scholar 

  24. Navarro-Guerrero E, Tay C, Whalley JP, Cowley SA, Davies B, Knight JC, Ebner D (2021) Genome-wide CRISPR/Cas9-knockout in human induced pluripotent stem cell (iPSC)-derived macrophages. Sci Rep 11(1):4245. https://doi.org/10.1038/s41598-021-82137-z

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Tian R, Gachechiladze MA, Ludwig CH, Laurie MT, Hong JY, Nathaniel D, Prabhu AV, Fernandopulle MS, Patel R, Abshari M, Ward ME, Kampmann M (2019) CRISPR interference-based platform for multimodal genetic screens in human iPSC-derived neurons. Neuron 104(2):239–255. e212. https://doi.org/10.1016/j.neuron.2019.07.014

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Xue HY, Ji LJ, Gao AM, Liu P, He JD, Lu XJ (2016) CRISPR-Cas9 for medical genetic screens: applications and future perspectives. J Med Genet 53(2):91–97. https://doi.org/10.1136/jmedgenet-2015-103409

    Article  CAS  PubMed  Google Scholar 

  27. Hanna RE, Hegde M, Fagre CR, DeWeirdt PC, Sangree AK, Szegletes Z, Griffith A, Feeley MN, Sanson KR, Baidi Y, Koblan LW, Liu DR, Neal JT, Doench JG (2021) Massively parallel assessment of human variants with base editor screens. Cell 184(4):1064–1080. e1020. https://doi.org/10.1016/j.cell.2021.01.012

    Article  CAS  PubMed  Google Scholar 

  28. Liao JQ, Zhou G, Zhou Y (2020) Generation of monoclonal iPSC lines with stable Cas9 expression and high Cas9 activity. Methods Mol Biol. https://doi.org/10.1007/7651_2020_304

  29. Zhou Y, Liao J, Fang C, Mo C, Zhou G, Luo Y (2018) One-step derivation of functional mesenchymal stem cells from human pluripotent stem cells. Bio Protoc 8(22):e3080. https://doi.org/10.21769/BioProtoc.3080

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Sanjana NE, Shalem O, Zhang F (2014) Improved vectors and genome-wide libraries for CRISPR screening. Nat Methods 11(8):783–784. https://doi.org/10.1038/nmeth.3047

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Zhou Y, Zhu S, Cai C, Yuan P, Li C, Huang Y, Wei W (2014) High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells. Nature 509(7501):487–491. https://doi.org/10.1038/nature13166

    Article  CAS  PubMed  Google Scholar 

  32. Li W, Xu H, Xiao T, Cong L, Love MI, Zhang F, Irizarry RA, Liu JS, Brown M, Liu XS (2014) MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens. Genome Biol 15(12):554. https://doi.org/10.1186/s13059-014-0554-4

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Allen F, Behan F, Khodak A, Iorio F, Yusa K, Garnett M, Parts L (2019) JACKS: joint analysis of CRISPR/Cas9 knockout screens. Genome Res 29(3):464–471. https://doi.org/10.1101/gr.238923.118

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

This work was supported by the National Natural Science Foundation of China (32100603, 31760742, 32060042, 81701195 and 81472126) and International Science and Technology Cooperation Program of Xinjiang Uygur Autonomous Region (2020E01006). We also acknowledged FACS core facility in Shenzhen University.

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Author notes

  1. Yan Zhou and Qiang Fu contributed equally to this work.

Authors and Affiliations

  1. Department of Medical Cell Biology and Genetics, Guangdong Key Laboratory of Genomic Stability and Disease Prevention, Shenzhen Key Laboratory of Anti-aging and Regenerative Medicine, and Shenzhen Engineering Laboratory of Regenerative Technologies for Orthopaedic Diseases, Health Science Center, Shenzhen University, Shenzhen, China

    Yan Zhou & Guangqian Zhou

  2. Lungene Technologies Co., Ltd, Shenzhen, China

    Yan Zhou

  3. College of Veterinary Medicine, Xinjiang Agricultural University, Xinjiang, China

    Qiang Fu & Huijun Shi

Authors
  1. Yan Zhou
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  2. Qiang Fu
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  3. Huijun Shi
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  4. Guangqian Zhou
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Editors and Affiliations

  1. Ottawa, ON, Canada

    Kursad Turksen

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Zhou, Y., Fu, Q., Shi, H., Zhou, G. (2022). CRISPR Guide RNA Library Screens in Human Induced Pluripotent Stem Cells. In: Turksen, K. (eds) Induced Pluripotent Stem Cells and Human Disease. Methods in Molecular Biology, vol 2549. Humana, New York, NY. https://doi.org/10.1007/7651_2021_455

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  • DOI: https://doi.org/10.1007/7651_2021_455

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2584-2

  • Online ISBN: 978-1-0716-2585-9

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