Abstract
CRISPR/Cas9 is an effective and easy-to-use tool for editing the genome of many human cancer cell lines. However, in some hard-to-transfect cell lines and primary cells, gene editing is more challenging. This protocol details an electroporation-based protocol for the delivery of Cas9 protein from Streptococcus pyogenes complexed with chemically modified sgRNAs. We have found this protocol to work very efficiently in numerous cell lines and primary cells that are difficult to transfect by conventional chemical-based transfection methods.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Barrangou R, May AP (2015) Unraveling the potential of CRISPR-Cas9 for gene therapy. Expert Opin Biol Ther 15(3):311–314. https://doi.org/10.1517/14712598.2015.994501
Mandal PK, Ferreira LM, Collins R, Meissner TB, Boutwell CL, Friesen M, Vrbanac V, Garrison BS, Stortchevoi A, Bryder D, Musunuru K, Brand H, Tager AM, Allen TM, Talkowski ME, Rossi DJ, Cowan CA (2014) Efficient ablation of genes in human hematopoietic stem and effector cells using CRISPR/Cas9. Cell Stem Cell 15(5):643–652. https://doi.org/10.1016/j.stem.2014.10.004
Burdette DL, Monroe KM, Sotelo-Troha K, Iwig JS, Eckert B, Hyodo M, Hayakawa Y, Vance RE (2011) STING is a direct innate immune sensor of cyclic di-GMP. Nature 478(7370):515–518. https://doi.org/10.1038/nature10429
Hornung V, Rothenfusser S, Britsch S, Krug A, Jahrsdorfer B, Giese T, Endres S, Hartmann G (2002) Quantitative expression of toll-like receptor 1–10 mRNA in cellular subsets of human peripheral blood mononuclear cells and sensitivity to CpG oligodeoxynucleotides. J Immunol 168(9):4531–4537
Hendel A, Bak RO, Clark JT, Kennedy AB, Ryan DE, Roy S, Steinfeld I, Lunstad BD, Kaiser RJ, Wilkens AB, Bacchetta R, Tsalenko A, Dellinger D, Bruhn L, Porteus MH (2015) Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nat Biotechnol 33(9):985–989. https://doi.org/10.1038/nbt.3290
Kim S, Kim D, Cho SW, Kim J, Kim JS (2014) Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res 24(6):1012–1019. https://doi.org/10.1101/gr.171322.113
Yin H, Song CQ, Suresh S, Wu Q, Walsh S, Rhym LH, Mintzer E, Bolukbasi MF, Zhu LJ, Kauffman K, Mou H, Oberholzer A, Ding J, Kwan SY, Bogorad RL, Zatsepin T, Koteliansky V, Wolfe SA, Xue W, Langer R, Anderson DG (2017) Structure-guided chemical modification of guide RNA enables potent non-viral in vivo genome editing. Nat Biotechnol 35(12):1179–1187. https://doi.org/10.1038/nbt.4005
Lee K, Mackley VA, Rao A, Chong AT, Dewitt MA, Corn JE, Murthy N (2017) Synthetically modified guide RNA and donor DNA are a versatile platform for CRISPR-Cas9 engineering. elife 6:e25312. https://doi.org/10.7554/eLife.25312
Wong TK, Neumann E (1982) Electric field mediated gene transfer. Biochem Biophys Res Commun 107(2):584–587
Chicaybam L, Sodre AL, Curzio BA, Bonamino MH (2013) An efficient low cost method for gene transfer to T lymphocytes. PLoS One 8(3):e60298. https://doi.org/10.1371/journal.pone.0060298
Bak RO, Porteus MH (2017) CRISPR-mediated integration of large gene cassettes using AAV donor vectors. Cell Rep 20(3):750–756. https://doi.org/10.1016/j.celrep.2017.06.064
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. https://doi.org/10.1038/nprot.2013.143
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. https://doi.org/10.1093/nar/gku936
Bak RO, Dever DP, Porteus MH (2018) CRISPR/Cas9 genome editing in human hematopoietic stem cells. Nat Protoc 13(2):358–376. https://doi.org/10.1038/nprot.2017.143
Acknowledgments
R.O.B. gratefully acknowledges the support from a Lundbeck Foundation Fellowship (R238-2016-3349), an AIAS-COFUND (Marie Curie) fellowship from Aarhus Institute of Advanced Studies (AIAS) co-funded by Aarhus University’s Research Foundation and the European Union’s seventh Framework Program under grant agreement no 609033, the Novo Nordisk Foundation (NNF17OC0028894), Innovation Fund Denmark (8056-00010B), the Carlsberg Foundation (CF17-0129), Slagtermester Max Wørzner og Hustru Inger Wørzners Mindelegat, The AP Møller Foundation, and the Riisfort Foundation.
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
Laustsen, A., Bak, R.O. (2019). Electroporation-Based CRISPR/Cas9 Gene Editing Using Cas9 Protein and Chemically Modified sgRNAs. 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_9
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9170-9_9
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