Abstract
The emergence of CRISPR/Cas9 system as a precise and affordable method for genome editing has prompted its rapid adoption for the targeted integration of transgenes in Chinese hamster ovary (CHO) cells. Targeted gene integration allows the generation of stable cell lines with a controlled and predictable behavior, which is an important feature for the rational design of cell factories aimed at the large-scale production of recombinant proteins. Here we present the protocol for CRISPR/Cas9-mediated integration of a gene expression cassette into a specific genomic locus in CHO cells using homology-directed DNA repair.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Templeton N, Young JD (2018) Biochemical and metabolic engineering approaches to enhance production of therapeutic proteins in animal cell cultures. Biochem Eng J 136:40–50
Lee J-H, Park J-H, Park S-H, Kim S-H, Kim JY, Min J-K, Lee GM, Kim Y-G (2018) Co-amplification of EBNA-1 and PyLT through dhfr-mediated gene amplification for improving foreign protein production in transient gene expression in CHO cells. Appl Microbiol Biotechnol 102(11):4729–4739
Kim JY, Kim Y-G, Lee GM (2012) CHO cells in biotechnology for production of recombinant proteins: current state and further potential. Appl Microbiol Biotechnol 93:917–930
Lombardo A, Cesana D, Genovese P, Di Stefano B, Provasi E, Colombo DF, Neri M, Magnani Z, Cantore A, Lo Riso P, Damo M, Pello OM, Holmes MC, Gregory PD, Gritti A, Broccoli V, Bonini C, Naldini L (2011) Site-specific integration and tailoring of cassette design for sustainable gene transfer. Nat Methods 8:861–869
Lee JS, Kallehauge TB, Pedersen LE, Kildegaard HF (2015) Site-specific integration in CHO cells mediated by CRISPR/Cas9 and homology-directed DNA repair pathway. Sci Rep 5:8572
Carroll D (2014) Genome engineering with targetable nucleases. Annu Rev Biochem 83:409–439
Cristea S, Freyvert Y, Santiago Y, Holmes MC, Urnov FD, Gregory PD, Cost GJ (2013) In vivo cleavage of transgene donors promotes nuclease-mediated targeted integration. Biotechnol Bioeng 110:871–880
Hockemeyer D, Soldner F, Beard C, Gao Q, Mitalipova M, DeKelver RC, Katibah GE, Amora R, Boydston EA, Zeitler B, Meng X, Miller JC, Zhang L, Rebar EJ, Gregory PD, Urnov FD, Jaenisch R (2009) Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases. Nat Biotechnol 27:851–857
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 339:819–823
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 339:823–826
Miller JC, Tan S, Qiao G, Barlow KA, Wang J, Xia DF, Meng X, Paschon DE, Leung E, Hinkley SJ, Dulay GP, Hua KL, Ankoudinova I, Cost GJ, Urnov FD, Zhang HS, Holmes MC, Zhang L, Gregory PD, Rebar EJ (2011) A TALE nuclease architecture for efficient genome editing. Nat Biotechnol 29:143–148
Orlando SJ, Santiago Y, DeKelver RC, Freyvert Y, Boydston EA, Moehle EA, Choi VM, Gopalan SM, Lou JF, Li J, Miller JC, Holmes MC, Gregory PD, Urnov FD, Cost GJ (2010) Zinc-finger nuclease-driven targeted integration into mammalian genomes using donors with limited chromosomal homology. Nucleic Acids Res 38:e152
Lee JS, Grav LM, Pedersen LE, Lee GM, Kildegaard HF (2016) Accelerated homology-directed targeted integration of transgenes in Chinese hamster ovary cells via CRISPR/Cas9 and fluorescent enrichment. Biotechnol Bioeng 113:2518–2523
Gaidukov L, Wroblewska L, Teague B, Nelson T, Zhang X, Liu Y, Jagtap K, Mamo S, Tseng WA, Lowe A, Das J, Bandara K, Baijuraj S, Summers NM, Lu TK, Zhang L, Weiss R (2018) A multi-landing pad DNA integration platform for mammalian cell engineering. Nucleic Acids Res 46:4072–4086
Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821
Ronda C, Pedersen LE, Hansen HG, Kallehauge TB, Betenbaugh MJ, Nielsen AT, Kildegaard HF (2014) Accelerating genome editing in CHO cells using CRISPR Cas9 and CRISPy, a web-based target finding tool. Biotechnol Bioeng 111:1604–1616
Lund AM, Kildegaard HF, Petersen MBK, Rank J, Hansen BG, Andersen MR, Mortensen UH (2014) A versatile system for USER cloning-based assembly of expression vectors for mammalian cell engineering. PLoS One 9:e96693
Grav LM, la Cour Karottki KJ, Lee JS, Kildegaard HF (2017) Application of CRISPR/Cas9 genome editing to improve recombinant protein production in CHO Cells. Methods Mol Biol 1603:101–118
Haeussler M, Schönig K, Eckert H, Eschstruth A, Mianné J, Renaud JB, Schneider-Maunoury S, Shkumatava A, Teboul L, Kent J, Joly JS, Concordet JP (2016) Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR. Genome Biol 17(1):148. https://doi.org/10.1186/s13059-016-1012-2
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Sergeeva, D., Camacho-Zaragoza, J.M., Lee, J.S., Kildegaard, H.F. (2019). CRISPR/Cas9 as a Genome Editing Tool for Targeted Gene Integration in CHO Cells. In: Luo, Y. (eds) CRISPR Gene Editing. Methods in Molecular Biology, vol 1961. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9170-9_13
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9170-9_13
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-9169-3
Online ISBN: 978-1-4939-9170-9
eBook Packages: Springer Protocols