Abstract
Precise and efficient genome modifications provide powerful tools for biological studies. Previous CRISPR gene knockout methods in cell lines have relied on frameshifts caused by stochastic insertion/deletion in all alleles. However, this method is inefficient for genes with high copy number due to polyploidy or gene amplification because frameshifts in all alleles can be difficult to generate and detect. Here we describe a homology-directed insertion method to knockout genes in the polyploid Drosophila S2R+ cell line. This protocol allows generation of homozygous mutant cell lines using an insertion cassette which autocatalytically generates insertion mutations in all alleles. Knockout cells generated using this method can be directly identified by PCR without a need for DNA sequencing. This protocol takes 2–3 months and can be applied to other polyploid cell lines or high-copy-number genes.
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The raw data used to generate the figures are included as supplementary information. There are no restrictions on data availability.
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Acknowledgements
We thank the Harvard Medical School Immunology Flow Cytometry Facility for cell sorting and Professor Y. Ahmed (Department of Molecular and Systems Biology, Dartmouth Geisel School of Medicine) for the anti-Tnks antibody. This study was supported by NIH NIGMS R01 GM067761, NIH NIGMS P41 GM132087 and NIH ORIP R24 OD019847. S.E.M. is additionally supported in part by the Dana Farber/Harvard Cancer Center, which is supported in part by NCI Cancer Center Support grant number NIH 5 P30 CA06516. N.P. is an investigator of the Howard Hughes Medical Institute.
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B.X. and N.P. designed the experiments. B.X. developed the protocol with G.A., R.V., J.Z. and S.E.M. B.X. and G.A. performed the experiments. B.X., S.E.M. and N.P. wrote the protocol.
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Peer review information Nature Protocols thanks E. Bier, J-L. Liu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Related links
Key references using this protocol:
Housden, B. E. et al. Sci. Signal. 8, rs9 (2015): https://doi.org/10.1126/scisignal.aab3729
Housden, B. E. et al. Bio. Protoc. 7, e2119 (2017): https://doi.org/10.21769/BioProtoc.2119
Nicholson, H. E. et al. Sci. Signal. 12, eaay0482 (2019): https://doi.org/10.1126/scisignal.aay0482
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Supplementary Data 1 and 2 and Supplementary Figs. 1–3.
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Xia, B., Amador, G., Viswanatha, R. et al. CRISPR-based engineering of gene knockout cells by homology-directed insertion in polyploid Drosophila S2R+ cells. Nat Protoc 15, 3478–3498 (2020). https://doi.org/10.1038/s41596-020-0383-8
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DOI: https://doi.org/10.1038/s41596-020-0383-8
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