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CRISPR-based engineering of gene knockout cells by homology-directed insertion in polyploid Drosophila S2R+ cells

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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Fig. 1: Overview of two homology-directed gene knockout methods.
Fig. 2: Construction of a four-sgRNA cassette using two pCFD4 vectors.
Fig. 3: Representative sequencing results of knockout loci in knockout cells after two rounds of homology-directed insertion (procedure A).
Fig. 4: Tnks nested PCR results for single-cell clones using procedure B.
Fig. 5: Tnks knockout cell lines and Apc knockout cell lines established using procedure B.

Data availability

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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Authors and Affiliations

Authors

Contributions

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.

Corresponding author

Correspondence to Norbert Perrimon.

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Competing interests

The authors declare no competing interests.

Additional information

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.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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

Supplementary information

Supplementary Information

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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