Abstract
The revolutionary CRISPR technology opens a new era of cell biology in mammalian cells. The InDel mutation is induced by CRISPR and results in the frameshift mutation of the gene. Owing to the nature of CRISPR induced knockout, the conditional knockout using CRISPR technology is not common. With the recent development of the small molecule-inducible degron system, an analogous system to the classical genetic conditional knockout has become feasible. By integrating CRISPR-knockout, the tetracycline-controlled transcriptional and auxin-induced degradation post-translational control of protein expression, a method imitating the conditional knockout is developed. We herein describe the detailed protocol for the generation of a conditional protein inactivation in human cancer cells. The system is especially useful to study essential gene function in aneuploidy cancer cells where gain in copy number is common.
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References
Verghese J, Abrams J, Wang Y, Morano KA (2012) Biology of the heat shock response and protein chaperones: budding yeast (Saccharomyces cerevisiae) as a model system. Microbiol Mol Biol Rev 76:115–158
Sun W, Zhang W, Zhang C, Mao M, Zhao Y, Chen X et al (2017) Light-induced protein degradation in human-derived cells. Biochem Biophys Res Commun 487:241–246
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 6:917–922
Koduri V, McBrayer SK, Liberzon E, Wang AC, Briggs KJ, Cho H et al (2019) Peptidic degron for IMiD-induced degradation of heterologous proteins. Proc Natl Acad Sci U S A 116:2539–2544
Wilmington SR, Matouschek A (2016) An inducible system for rapid degradation of specific cellular proteins using proteasome adaptors. PLoS One 11(4):e0152679
Holland AJ, Fachinetti D, Han JS, Cleveland DW (2012) Inducible, reversible system for the rapid and complete degradation of proteins in mammalian cells. Proc Natl Acad Sci U S A 109:E3350–E3357
Hayashi KI, Neve J, Hirose M, Kuboki A, Shimada Y, Kepinski S et al (2012) Rational design of an auxin antagonist of the SCF TIR1 auxin receptor complex. ACS Chem Biol 7:590–598
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 15:210–218
Adey A, Burton JN, Kitzman JO, Hiatt JB, Lewis AP, Martin BK et al (2013) The haplotype-resolved genome and epigenome of the aneuploid HeLa cancer cell line. Nature 500:207–211
Li S, Prasanna X, Salo VT, Vattulainen I, Ikonen E (2019) An efficient auxin-inducible degron system with low basal degradation in human cells. Nat Methods 16:866–869
Uchida N, Takahashi K, Iwasaki R, Yamada R, Yoshimura M, Endo TA et al (2018) Chemical hijacking of auxin signaling with an engineered auxin-TIR1 pair. Nat Chem Biol 14:299–305
Ng LY, Ma HT, Liu JCY, Huang X, Lee N, Poon RYC (2019) Conditional gene inactivation by combining tetracycline-mediated transcriptional repression and auxin-inducible degron-mediated degradation. Cell Cycle 18:238–248
Gossen M, Bujard H (1992) Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci U S A 89:5547–5551
Ma HT, Poon RYC (2018) TRIP13 functions in the establishment of the spindle assembly checkpoint by replenishing O-MAD2. Cell Rep 22:1439–1450
Kingston RE, Chen CA, Okayama H (1999) Calcium phosphate transfection. Curr Protoc Immunol 31:10.13.1–10.13.9
Brinkman EK, Chen T, Amendola M, Van Steensel B (2014) Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res 42(22):e168
Brosh R, Hrynyk I, Shen J, Waghray A, Zheng N, Lemischka IR (2016) A dual molecular analogue tuner for dissecting protein function in mammalian cells. Nat Commun 7:11742
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Ma, H.T. (2021). The Conditional Knockout Analogous System: CRISPR-Mediated Knockout Together with Inducible Degron and Transcription-Controlled Expression. In: Coutts, A.S., Weston, L. (eds) Cell Cycle Oscillators . Methods in Molecular Biology, vol 2329. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1538-6_23
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DOI: https://doi.org/10.1007/978-1-0716-1538-6_23
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