Generation of dual-gRNA library for combinatorial CRISPR screening of synthetic lethal gene pairs

doi:10.1016/j.xpro.2022.101556

. 2022 Aug 24;3(3):101556.

doi: 10.1016/j.xpro.2022.101556. eCollection 2022 Sep 16.

Generation of dual-gRNA library for combinatorial CRISPR screening of synthetic lethal gene pairs

Shan Tang ¹, Xue Wu ², Jinghui Liu ³, Qiongsi Zhang ³, Xinyi Wang ³, Shuai Shao ⁴, Birkan Gokbag ², Kunjie Fan ², Xiaoqi Liu ³, Fuhai Li ⁵, Lijun Cheng ², Lang Li ⁶

Affiliations

¹ College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA. Electronic address: shan.tang@osumc.edu.
² Department of Biomedical Informatics, College of Medicine, The Ohio State University, Columbus, OH 43210, USA.
³ Department of Toxicology and Cancer Biology, University of Kentucky, Lexington, KY 40536, USA.
⁴ College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA.
⁵ Institute for Informatics and Department of Pediatrics, Washington University in St. Louis, St. Louis, MO 63110, USA.
⁶ Department of Biomedical Informatics, College of Medicine, The Ohio State University, Columbus, OH 43210, USA. Electronic address: lang.li@osumc.edu.

PMID: 36060092
PMCID: PMC9428847
DOI: 10.1016/j.xpro.2022.101556

Generation of dual-gRNA library for combinatorial CRISPR screening of synthetic lethal gene pairs

Shan Tang et al. STAR Protoc. 2022.

. 2022 Aug 24;3(3):101556.

doi: 10.1016/j.xpro.2022.101556. eCollection 2022 Sep 16.

Authors

Shan Tang ¹, Xue Wu ², Jinghui Liu ³, Qiongsi Zhang ³, Xinyi Wang ³, Shuai Shao ⁴, Birkan Gokbag ², Kunjie Fan ², Xiaoqi Liu ³, Fuhai Li ⁵, Lijun Cheng ², Lang Li ⁶

Affiliations

¹ College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA. Electronic address: shan.tang@osumc.edu.
² Department of Biomedical Informatics, College of Medicine, The Ohio State University, Columbus, OH 43210, USA.
³ Department of Toxicology and Cancer Biology, University of Kentucky, Lexington, KY 40536, USA.
⁴ College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA.
⁵ Institute for Informatics and Department of Pediatrics, Washington University in St. Louis, St. Louis, MO 63110, USA.
⁶ Department of Biomedical Informatics, College of Medicine, The Ohio State University, Columbus, OH 43210, USA. Electronic address: lang.li@osumc.edu.

PMID: 36060092
PMCID: PMC9428847
DOI: 10.1016/j.xpro.2022.101556

Abstract

Combinatorial CRISPR screening is useful for investigating synthetic lethality (SL) gene pairs. Here, we detail the steps for dual-gRNA library construction, with the introduction of two backbones, LentiGuide_DKO and LentiCRISPR_DKO. We describe steps for in vitro screening with 22Rv1-Cas9 and SaOS2-Cas9 cells followed by sequencing and data analysis. By introducing two backbones, we optimized the library construction process, facilitated standard pair-end sequencing, and provided options of screening on cells with or without modification of Cas9 expression.

Keywords: Biotechnology and bioengineering; CRISPR; High Throughput Screening; Molecular Biology.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None

Graphical abstract

Figure 1

Figure 1

Protocol flow chart

Figure 2

Figure 2

Backbone design and KO efficiency validation

Figure 3

Figure 3

Overall library construction process and gRNA pools/primer design

Figure 4

Figure 4

Expected sequencing outcomes Expected sequencing outcomes: sequencing statistic summary with (A) mapping ratio and mapped reads; (B) number of missed constructions; (C) pairwise sample correlation.

See this image and copyright information in PMC

References

1. Broad DepMap. 2021. https://depmap.org/portal/
1. Cui Y., Xu J., Cheng M., Liao X., Peng S. Review of CRISPR/Cas9 sgRNA design tools. Interdiscip. Sci. 2018;10:455–465. doi: 10.1007/s12539-018-0298-z. - DOI - PubMed
1. Diehl V., Wegner M., Grumati P., Husnjak K., Schaubeck S., Gubas A., Shah V.J., Polat I.H., Langschied F., Prieto-Garcia C. Minimized combinatorial CRISPR screens identify genetic interactions in autophagy. Nucleic Acids Res. 2021;49:5684–5704. - PMC - PubMed
1. Doench J.G., Fusi N., Sullender M., Hegde M., Vaimberg E.W., Donovan K.F., Smith I., Tothova Z., Wilen C., Orchard R., et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat. Biotechnol. 2016;34:184–191. doi: 10.1038/nbt.3437. - DOI - PMC - PubMed
1. Gilbert L.A., Horlbeck M.A., Adamson B., Villalta J.E., Chen Y., Whitehead E.H., Guimaraes C., Panning B., Ploegh H.L., Bassik M.C., et al. Genome-scale CRISPR-mediated control of gene repression and activation. Cell. 2014;159:647–661. doi: 10.1016/j.cell.2014年09月02日9. - DOI - PMC - PubMed

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[1] Broad DepMap. 2021. https://depmap.org/portal/

[2] Broad DepMap. 2021. https://depmap.org/portal/

[3] Cui Y., Xu J., Cheng M., Liao X., Peng S. Review of CRISPR/Cas9 sgRNA design tools. Interdiscip. Sci. 2018;10:455–465. doi: 10.1007/s12539-018-0298-z. - DOI - PubMed

[4] Cui Y., Xu J., Cheng M., Liao X., Peng S. Review of CRISPR/Cas9 sgRNA design tools. Interdiscip. Sci. 2018;10:455–465. doi: 10.1007/s12539-018-0298-z. - DOI - PubMed

[5] Diehl V., Wegner M., Grumati P., Husnjak K., Schaubeck S., Gubas A., Shah V.J., Polat I.H., Langschied F., Prieto-Garcia C. Minimized combinatorial CRISPR screens identify genetic interactions in autophagy. Nucleic Acids Res. 2021;49:5684–5704. - PMC - PubMed

[6] Diehl V., Wegner M., Grumati P., Husnjak K., Schaubeck S., Gubas A., Shah V.J., Polat I.H., Langschied F., Prieto-Garcia C. Minimized combinatorial CRISPR screens identify genetic interactions in autophagy. Nucleic Acids Res. 2021;49:5684–5704. - PMC - PubMed

[7] Doench J.G., Fusi N., Sullender M., Hegde M., Vaimberg E.W., Donovan K.F., Smith I., Tothova Z., Wilen C., Orchard R., et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat. Biotechnol. 2016;34:184–191. doi: 10.1038/nbt.3437. - DOI - PMC - PubMed

[8] Doench J.G., Fusi N., Sullender M., Hegde M., Vaimberg E.W., Donovan K.F., Smith I., Tothova Z., Wilen C., Orchard R., et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat. Biotechnol. 2016;34:184–191. doi: 10.1038/nbt.3437. - DOI - PMC - PubMed

[9] Gilbert L.A., Horlbeck M.A., Adamson B., Villalta J.E., Chen Y., Whitehead E.H., Guimaraes C., Panning B., Ploegh H.L., Bassik M.C., et al. Genome-scale CRISPR-mediated control of gene repression and activation. Cell. 2014;159:647–661. doi: 10.1016/j.cell.2014年09月02日9. - DOI - PMC - PubMed

[10] Gilbert L.A., Horlbeck M.A., Adamson B., Villalta J.E., Chen Y., Whitehead E.H., Guimaraes C., Panning B., Ploegh H.L., Bassik M.C., et al. Genome-scale CRISPR-mediated control of gene repression and activation. Cell. 2014;159:647–661. doi: 10.1016/j.cell.2014年09月02日9. - DOI - PMC - PubMed

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Generation of dual-gRNA library for combinatorial CRISPR screening of synthetic lethal gene pairs

Affiliations

Generation of dual-gRNA library for combinatorial CRISPR screening of synthetic lethal gene pairs

Authors

Affiliations

Abstract

Conflict of interest statement

Figures

References

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources

Full Text Sources

Research Materials