Abstract
Recent advances in the CRISPR/Cas9 system have dramatically facilitated genome engineering in various cell systems. Among the protocols, the direct delivery of the Cas9-sgRNA ribonucleoprotein (RNP) complex into cells is an efficient approach to increase genome editing efficiency. This method uses purified Cas9 protein and in vitro transcribed sgRNA to edit the target gene without vector DNA. We have applied the RNP complex to CHO cell engineering to obtain desirable phenotypes and to reduce unintended insertional mutagenesis and off-target effects. Here, we describe our routine methods for RNP complex-mediated gene deletion including the protocols to prepare the purified Cas9 protein and the in vitro transcribed sgRNA. Subsequently, we also describe a protocol to confirm the edited genomic positions using the T7E1 enzymatic assay and next-generation sequencing.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Horvath P, Barrangou R (2010) CRISPR/Cas, the immune system of bacteria and archaea. Science 327(5962):167–170
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
Cho SW, Kim S, Kim JM, Kim J-S (2013) Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol 31(3):230–232
Li J-F, Norville JE, Aach J, McCormack M, Zhang D, Bush J, Church GM, Sheen J (2013) Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nat Biotechnol 31(8):688–691
Jiang W, Bikard D, Cox D, Zhang F, Marraffini LA (2013) RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol 31(3):233–239
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(6096):816–821
Sander JD, Joung JK (2014) CRISPR-Cas systems for editing, regulating and targeting genomes. Nat Biotechnol 32(4):347–355
Gabriel R, Lombardo A, Arens A, Miller JC, Genovese P, Kaeppel C, Nowrouzi A, Bartholomae CC, Wang J, Friedman G, Holmes MC, Gregory PD, Glimm H, Schmidt M, Naldini L, von Kalle C (2011) An unbiased genome-wide analysis of zinc-finger nuclease specificity. Nat Biotechnol 29(9):816–823
Kim S, Kim D, Cho SW, Kim J, Kim J-S (2014) Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res 24(6):1012–1019
Ramakrishna S, Kwaku Dad A-B, Beloor J, Gopalappa R, Lee S-K, Kim H (2014) Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA. Genome Res 24(6):1020–1027
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(3):917–930
Lee N, Shin J, Park JH, Lee GM, Cho S, Cho B-K (2016) Targeted gene deletion using DNA-free RNA-guided Cas9 nuclease accelerates adaptation of CHO cells to suspension culture. ACS Synth Biol 5(11):1211–1219
Kim Y, Kweon J, Kim A, Chon JK, Yoo JY, Kim HJ, Kim S, Lee C, Jeong E, Chung E, Kim D, Lee MS, Go EM, Song HJ, Kim H, Cho N, Bang D, Kim S, Kim J-S (2013) A library of TAL effector nucleases spanning the human genome. Nat Biotechnol 31(3):251–258
Qiu P, Shandilya H, D’Alessio JM, O’Connor K, Durocher J, Gerard GF (2004) Mutation detection using Surveyor nuclease. Biotechniques 36(4):702–707
Ramakrishna S, Cho SW, Kim S, Song M, Gopalappa R, Kim J-S, Kim H (2014) Surrogate reporter-based enrichment of cells containing RNA-guided Cas9 nuclease-induced mutations. Nat Commun 5:3378
Güell M, Yang L, Church GM (2014) Genome editing assessment using CRISPR Genome Analyzer (CRISPR-GA). Bioinformatics 30(20):2968–2970
Pinello L, Canver MC, Hoban MD, Orkin SH, Kohn DB, Bauer DE, Yuan G-C (2016) Analyzing CRISPR genome-editing experiments with CRISPResso. Nat Biotechnol 34(7):695–697
Sanjana NE, Shalem O, Zhang F (2014) Improved vectors and genome-wide libraries for CRISPR screening. Nat Methods 11(8):783–784
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
Zuris JA, Thompson DB, Shu Y, Guilinger JP, Bessen JL, Hu JH, Maeder ML, Joung JK, Chen Z-Y, Liu DR (2015) Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat Biotechnol 33(1):73–80
Acknowledgments
This work was supported by the Intelligent Synthetic Biology Center of Global Frontier Project (2011-0031957 to B.-K.C) and Basic Science Research Program (2015R1A2A2A01008006 to B.-K.C., 2015R1C1A2A01053505 to S.C.) through the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT, and Future Planning (MISP).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Shin, J., Lee, N., Cho, S., Cho, BK. (2018). Targeted Genome Editing Using DNA-Free RNA-Guided Cas9 Ribonucleoprotein for CHO Cell Engineering. In: Braman, J. (eds) Synthetic Biology. Methods in Molecular Biology, vol 1772. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7795-6_8
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7795-6_8
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7794-9
Online ISBN: 978-1-4939-7795-6
eBook Packages: Springer Protocols