Abstract
CRISPR/Cas9 system is a powerful technique for genome editing and engineering but obtaining a sizeable population of edited cells can be challenging for some cell types. CRISPR/Cas9-induced cell cycle arrest is a possible cause of this barrier to efficient editing; thus, it is desirable to know the cell cycle progression profile of any given cell line or type of interest resulting from CRISPR/Cas9 treatment. Here we describe a flow cytometry-based assay that enables the determination of cell cycle progression in the presence of CRISPR/Cas9 treatment, in addition to the transfection and expression efficiencies of Cas9 vectors. This assay can also easily determine the effect of various interventions on obtaining a larger pool of Cas9-treated cells.
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References
Kim YG, Cha J, Chandrasegaran S (1996) Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc Natl Acad Sci 93:1156–1160
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 186:757–761
Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA et al (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823
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 339:823–826
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 337:816–821
Haapaniemi E, Botla S, Persson J, Schmierer B, Taipale J (2018) CRISPR-Cas9 genome editing induces a p53-mediated DNA damage response. Nat Med 24:927–930
Ihry RJ, Worringer KA, Salick MR, Frias E, Ho D, Theriault K, Kommineni S, Chen J, Sondey M, Ye C et al (2018) p53 inhibits CRISPR–Cas9 engineering in human pluripotent stem cells. Nat Med 24:939–946
van den Berg J, Manjón GA, Kielbassa K, Feringa FM, Freire R, Medema RH (2018) A limited number of double-strand DNA breaks is sufficient to delay cell cycle progression. Nucleic Acids Res. https://doi.org/10.1093/nar/gky786
Geisinger JM, Stearns T (2020) CRISPR/Cas9 treatment causes extended TP-53 dependent cell cycle arrest in human cells. Nucleic Acids Res 48(16):9067–9081
Aguirre AJ, Meyers RM, Weir BA, Vazquez F, Zhang C-Z, Ben-David U, Cook A, Ha G, Harrington WF, Doshi MB et al (2016) Genomic copy number dictates a gene-independent cell response to CRISPR/Cas9 targeting. Cancer Discov 6:914–929
Kaulich M, Lee YJ, Lönn P, Springer AD, Meade BR, Dowdy SF (2015) Efficient CRISPR-rAAV engineering of endogenous genes to study protein function by allele-specific RNAi. Nucleic Acids Res 43:e45–e45
Byrne SM, Ortiz L, Mali P, Aach J, Church GM (2015) Multi-kilobase homozygous targeted gene replacement in human induced pluripotent stem cells. Nucleic Acids Res 43:e21–e21
Makino S, Fukumura R, Gondo Y (2016) Illegitimate translation causes unexpected gene expression from on-target out-of-frame alleles created by CRISPR-Cas9. Sci Rep 6:39608
Rodriguez-Rodriguez J-A, Lewis C, McKinley KL, Sikirzhytski V, Corona J, Maciejowski J, Khodjakov A, Cheeseman IM, Jallepalli PV (2018) Distinct roles of RZZ and Bub1-KNL1 in mitotic checkpoint signaling and kinetochore expansion. Curr Biol 28:3422–3429.e5
Brown KR, Mair B, Soste M, Moffat J (2019) CRISPR screens are feasible in TP53 wild-type cells. Mol Syst Biol 15(8):e8679
Haapaniemi E, Botla S, Persson J, Schmierer B, Taipale J (2019) Reply to “CRISPR screens are feasible in TP53 wild-type cells”. Mol Syst Biol 15(8):e9059
Merkle FT, Ghosh S, Kamitaki N, Mitchell J, Avior Y, Mello C, Kashin S, Mekhoubad S, Ilic D, Charlton M et al (2017) Human pluripotent stem cells recurrently acquire and expand dominant negative P53 mutations. Nature 545:229–233
Salic A, Mitchison TJ (2008) A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proc Natl Acad Sci 105:2415–2420
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Geisinger, J.M., Stearns, T. (2021). Assaying Cell Cycle Progression via Flow Cytometry in CRISPR/Cas9-Treated Cells. In: Coutts, A.S., Weston, L. (eds) Cell Cycle Oscillators . Methods in Molecular Biology, vol 2329. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1538-6_14
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DOI: https://doi.org/10.1007/978-1-0716-1538-6_14
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Publisher Name: Humana, New York, NY
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Online ISBN: 978-1-0716-1538-6
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