Abstract
Cells with a loss-of-function mutation in a gene (knockout cells) are powerful tools for characterizing the function of such gene product. However, for essential genes, conditional knockout cell lines must be generated. The auxin-inducible degron (AID) technique enables us to conditionally and rapidly deplete a target protein from various eukaryotic cell lines. A combination of CRISPR-/Cas9-based gene editing and AID technique allows us to generate AID-based conditional knockout cell lines. Using these two techniques, we recently proposed a simple and quick way to generate conditional knockout cells for essential genes. In this chapter, we introduce the reader to the experimental procedures to generate these AID-based conditional knockout cell lines.
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References
Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339(6121):819–823. https://doi.org/10.1126/science.1231143
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(6121):823–826. https://doi.org/10.1126/science.1232033
Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelsen TS, Heckl D, Ebert BL, Root DE, Doench JG, Zhang F (2014) Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 343(6166):84–87. https://doi.org/10.1126/science.1247005
Wang T, Wei JJ, Sabatini DM, Lander ES (2014) Genetic screens in human cells using the CRISPR-Cas9 system. Science 343(6166):80–84. https://doi.org/10.1126/science.1246981
Gu H, Marth JD, Orban PC, Mossmann H, Rajewsky K (1994) Deletion of a DNA polymerase beta gene segment in T cells using cell type-specific gene targeting. Science 265(5168):103–106. https://doi.org/10.1126/science.8016642
Lakso M, Pichel JG, Gorman JR, Sauer B, Okamoto Y, Lee E, Alt FW, Westphal H (1996) Efficient in vivo manipulation of mouse genomic sequences at the zygote stage. Proc Natl Acad Sci U S A 93(12):5860–5865. https://doi.org/10.1073/pnas.93.12.5860
Gossen M, Bonin AL, Freundlieb S, Bujard H (1994) Inducible gene expression systems for higher eukaryotic cells. Curr Opin Biotechnol 5(5):516–520. https://doi.org/10.1016/0958-1669(94)90067-1
Sonoda E, Sasaki MS, Buerstedde JM, Bezzubova O, Shinohara A, Ogawa H, Takata M, Yamaguchi-Iwai Y, Takeda S (1998) Rad51-deficient vertebrate cells accumulate chromosomal breaks prior to cell death. EMBO J 17(2):598–608. https://doi.org/10.1093/emboj/17.2.598
Fukagawa T, Mikami Y, Nishihashi A, Regnier V, Haraguchi T, Hiraoka Y, Sugata N, Todokoro K, Brown W, Ikemura T (2001) CENP-H, a constitutive centromere component, is required for centromere targeting of CENP-C in vertebrate cells. EMBO J 20(16):4603–4617. https://doi.org/10.1093/emboj/20.16.4603
Nishimura K, Fukagawa T, Takisawa H, Kakimoto T, Kanemaki M (2009) An auxin-based degron system for the rapid depletion of proteins in nonplant cells. Nat Methods 6(12):917–922. https://doi.org/10.1038/nmeth.1401
Natsume T, Kiyomitsu T, Saga Y, Kanemaki MT (2016) Rapid protein depletion in human cells by auxin-inducible Degron tagging with short homology donors. Cell Rep 15(1):210–218. https://doi.org/10.1016/j.celrep.2016.03.001
Nishimura K, Fukagawa T (2017) An efficient method to generate conditional knockout cell lines for essential genes by combination of auxin-inducible degron tag and CRISPR/Cas9. Chromosome Res 25(3-4):253–260. https://doi.org/10.1007/s10577-017-9559-7
Pyzocha NK, Ran FA, Hsu PD, Zhang F (2014) RNA-guided genome editing of mammalian cells. Methods Mol Biol 1114:269–277. https://doi.org/10.1007/978-1-62703-761-7_17
Fukagawa T, Earnshaw WC (2014) The centromere: chromatin foundation for the kinetochore machinery. Dev Cell 30(5):496–508. https://doi.org/10.1016/j.devcel.2014.08.016
Acknowledgments
We are very grateful to R. Fukuoka and Y. Fukagawa for technical assistance. OsTIR1 cDNA was given from Prof. Masato Kanemaki (National Institute of Genetics). This work was supported by JSPS KAKENHI Grant Numbers 25221106, 15H05972, and 17H06167 to TF and JSPS KAKENHI Grant Number 17K15041 and 19K06611 to KN.
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Nishimura, K., Fukagawa, T. (2022). A Simple Method that Combines CRISPR and AID to Quickly Generate Conditional Knockouts for Essential Genes in Various Vertebrate Cell Lines. In: Zhang, R. (eds) Essential Genes and Genomes. Methods in Molecular Biology, vol 2377. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1720-5_6
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DOI: https://doi.org/10.1007/978-1-0716-1720-5_6
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