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.
This is a preview of subscription content, access via your institution
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Rent or buy this article
Get just this article for as long as you need it
Prices may be subject to local taxes which are calculated during checkout
The raw data used to generate the figures are included as supplementary information. There are no restrictions on data availability.
Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).
Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).
Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).
Ran, F. A. et al. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8, 2281–2308 (2013).
Grav, L. M. et al. One-step generation of triple knockout CHO cell lines using CRISPR/Cas9 and fluorescent enrichment. Biotechnol. J. 10, 1446–1456 (2015).
Wang, F. et al. A comparison of CRISPR/Cas9 and siRNA-mediated ALDH2 gene silencing in human cell lines. Mol. Genet. Genomics 293, 769–783 (2018).
Li, C. et al. Generation of FOS gene knockout lines from a human embryonic stem cell line using CRISPR/Cas9. Stem Cell Res. 39, 101479 (2019).
Gantz, V. M. & Bier, E. Genome editing. The mutagenic chain reaction: a method for converting heterozygous to homozygous mutations. Science 348, 442–444 (2015).
Housden, B. E. et al. Identification of potential drug targets for tuberous sclerosis complex by synthetic screens combining CRISPR-based knockouts with RNAi. Sci. Signal 8, rs9 (2015).
Housden, B. E., Nicholson, H. E. & Perrimon, N. Synthetic lethality screens using RNAi in combination with CRISPR-based knockout in Drosophila cells. Bio. Protoc. https://doi.org/10.21769/BioProtoc.2119 (2017).
Housden, B. E. & Perrimon, N. Detection of indel mutations in Drosophila by high-resolution melt analysis (HRMA). Cold Spring Harb. Protoc. https://doi.org/10.1101/pdb.prot090795 (2016).
Boettcher, M. & McManus, M. T. Choosing the right tool for the job: RNAi, TALEN, or CRISPR. Mol. Cell 58, 575–585 (2015).
Scharf, I. et al. Dynamics of CRISPR/Cas9-mediated genomic editing of the AXL locus in hepatocellular carcinoma cells. Oncol. Lett. 15, 2441–2450 (2018).
Lee, H. et al. DNA copy number evolution in Drosophila cell lines. Genome Biol. 15, R70 (2014).
Friedel, R. H. et al. Gene targeting using a promoterless gene trap vector (“targeted trapping”) is an efficient method to mutate a large fraction of genes. Proc. Natl Acad. Sci. USA 102, 13188–13193 (2005).
Ye, L. et al. Programmable DNA repair with CRISPRa/i enhanced homology-directed repair efficiency with a single Cas9. Cell Discov. 4, 46 (2018).
Maruyama, T. et al. Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. Nat. Biotechnol. 33, 538–542 (2015).
Chu, V. T. et al. Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat. Biotechnol. 33, 543–548 (2015).
Yu, C. et al. Small molecules enhance CRISPR genome editing in pluripotent stem cells. Cell Stem Cell 16, 142–147 (2015).
Kosicki, M., Tomberg, K. & Bradley, A. Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements. Nat. Biotechnol. 36, 765–771 (2018).
Ma, Z. et al. PTC-bearing mRNA elicits a genetic compensation response via Upf3a and COMPASS components. Nature 568, 259–263 (2019).
El-Brolosy, M. A. et al. Genetic compensation triggered by mutant mRNA degradation. Nature 568, 193–197 (2019).
Hu, Y., Comjean, A., Perrimon, N. & Mohr, S. E. The Drosophila gene expression tool (DGET) for expression analyses. BMC Bioinforma. 18, 98 (2017).
Cherbas, L. et al. The transcriptional diversity of 25 Drosophila cell lines. Genome Res. 21, 301–314 (2011).
Sakurai, T., Watanabe, S., Kamiyoshi, A., Sato, M. & Shindo, T. A single blastocyst assay optimized for detecting CRISPR/Cas9 system-induced indel mutations in mice. BMC Biotechnol. 14, 69 (2014).
Port, F., Chen, H. M., Lee, T. & Bullock, S. L. Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila. Proc. Natl Acad. Sci. USA 111, E2967–E2976 (2014).
Port, F. & Bullock, S. L. Augmenting CRISPR applications in Drosophila with tRNA-flanked sgRNAs. Nat. Methods 13, 852–854 (2016).
Viswanatha, R., Li, Z., Hu, Y. & Perrimon, N. Pooled genome-wide CRISPR screening for basal and context-specific fitness gene essentiality in Drosophila cells. eLife https://doi.org/10.7554/eLife.36333 (2018).
Caudron-Herger, M. et al. Identification, quantification and bioinformatic analysis of RNA-dependent proteins by RNase treatment and density gradient ultracentrifugation using R-DeeP. Nat. Protoc. 15, 1338–1370 (2020).
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.
The authors declare no competing interests.
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.
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 Data 1 and 2 and Supplementary Figs. 1–3.
Rights and permissions
About this article
Cite this article
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
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.