Abstract
Genome engineering provides a powerful tool to explore TGF-β/SMAD signaling by enabling the deletion and modification of critical components of the pathway. Over the past years, CRISPR-Cas9 technology has matured and can now be used to routinely generate knockout cell lines. Here, we describe a method to design and generate deletions of genes from the SMAD pathway in somatic human cell lines based on homologous recombination.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Fuchs O (2011) Inhibition of TGF- signaling for the treatment of tumor metastasis and fibrotic diseases. Curr Signal Transduct Ther 6(1):29–43
Derynck R, Zhang YE (2003) Smad-dependent and Smad-independent pathways in TGF-beta family signalling. Nature 425(6958):577–584
Novina CD, Sharp PA (2004) The RNAi revolution. Nature 430(6996):161–164
Aagaard L, Rossi JJ (2007) RNAi therapeutics: principles, prospects and challenges. Adv Drug Deliv Rev 59(2–3):75–86
Rago C, Vogelstein B, Bunz F (2007) Genetic knockouts and knockins in human somatic cells. Nat Protoc 2(11):2734–2746
Strasen J, Sarma U, Jentsch M, Bohn S, Sheng C, Horbelt D, Knaus P, Legewie S, Loewer A (2018) Cell-specific responses to the cytokine TGFbeta are determined by variability in protein levels. Mol Syst Biol 14(1):e7733
Horvath P, Barrangou R (2010) CRISPR/Cas, the immune system of bacteria and archaea. Science 327(5962):167–170
Hsu PD, Lander ES, Zhang F (2014) Development and applications of CRISPR-Cas9 for genome engineering. Cell 157(6):1262–1278
Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F (2013) Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8(11):2281–2308
Sheng C, Mendler IH, Rieke S, Snyder P, Jentsch M, Friedrich D, Drossel B, Loewer A (2019) PCNA-mediated degradation of p21 coordinates the DNA damage response and cell cycle regulation in individual cells. Cell Rep 27(1):48–58
Doench JG, Hartenian E, Graham DB, Tothova Z, Hegde M, Smith I, Sullender M, Ebert BL, Xavier RJ, Root DE (2014) Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation. Nat Biotechnol 32(12):1262–1267
Doench JG, Fusi N, Sullender M, Hegde M, Vaimberg EW, Donovan KF, Smith I, Tothova Z, Wilen C, Orchard R, Virgin HW, Listgarten J, Root DE (2016) Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol 34(2):184–191
Heigwer F, Kerr G, Boutros M (2014) E-CRISP: fast CRISPR target site identification. Nat Methods 11(2):122–123
Guo D, Li X, Zhu P, Feng Y, Yang J, Zheng Z, Yang W, Zhang E, Zhou S, Wang H (2015) Online high-throughput mutagenesis designer using scoring matrix of sequence-specific endonucleases. J Integr Bioinform 12(1):35–48
Moreno-Mateos MA, Vejnar CE, Beaudoin JD, Fernandez JP, Mis EK, Khokha MK, Giraldez AJ (2015) CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo. Nat Methods 12(10):982–988
Labun K, Montague TG, Krause M, Torres CY, Tjeldnes H, Valen E (2019) CHOPCHOP v3: expanding the CRISPR web toolbox beyond genome editing. Nucleic Acids Res 47(W1):W171–W174
Wong N, Liu W, Wang X (2015) WU-CRISPR: characteristics of functional guide RNAs for the CRISPR/Cas9 system. Genome Biol 16:218
Naito Y, Hino K, Bono H, Ui-Tei K (2015) CRISPRdirect: software for designing CRISPR/Cas guide RNA with reduced off-target sites. Bioinformatics 31(7):1120–1123
Ran FA, Hsu PD, Lin CY, Gootenberg JS, Konermann S, Trevino AE, Scott DA, Inoue A, Matoba S, Zhang Y, Zhang F (2013) Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154(6):1380–1389
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
Acknowledgments
This work was supported by grants and fellowships from the China Scholarship Council (to Z.H.) and the Morbus Osler Foundation (to A.L.)
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Huang, Z., Loewer, A. (2022). Generating Somatic Knockout Cell Lines with CRISPR-Cas9 Technology to Investigate SMAD Signaling. In: Zi, Z., Liu, X. (eds) TGF-Beta Signaling. Methods in Molecular Biology, vol 2488. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2277-3_7
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2277-3_7
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2276-6
Online ISBN: 978-1-0716-2277-3
eBook Packages: Springer Protocols