Abstract
The clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9) system is a powerful tool for precise genome editing. The recent efforts of scientists allow to improve current genome engineering techniques, which are used to generate modified cell lines for functional studies. Here we describe, in detail, the protocol for DNA-free CRISPR-Cas9-mediated gene editing in a human liver cell line. Briefly, we used lipofection to deliver a ribonucleoprotein (RNP) complex consisting of Cas9 protein, crRNA, and tracrRNA to the cells. The crRNA quality was validated before transfection by performing an in vitro cleavage assay and visualized using an agarose gel. Afterward, we checked the efficiency of the target gene modification by Sanger sequencing and T7 endonuclease I digestion. Our goal is to provide a simple protocol for CRISPR-Cas9-mediated gene modification.
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References
Rohn TT, Kim N, Isho NF, Mack JM (2018) The potential of CRISPR/Cas9 gene editing as a treatment strategy for Alzheimer’s Disease. J Alzheimers Dis Parkinsonism 8:439. https://doi.org/10.4172/2161-0460.1000439
Kolli N, Lu M, Maiti P, Rossignol J, Dunbar GL (2018) Application of the gene editing tool, CRISPR-Cas9, for treating neurodegenerative diseases. Neurochem Int 112:187–196. https://doi.org/10.1016/j.neuint.2017.07.007
Wong TWY, Cohn RD (2017) Therapeutic applications of CRISPR/Cas for Duchenne muscular dystrophy. Curr Gene Ther 17(4):301–308. https://doi.org/10.2174/1566523217666171121165046
Zhang F, Wen Y, Guo X (2014) CRISPR/Cas9 for genome editing: progress, implications and challenges. Hum Mol Genet 23(R1):R40–R46. https://doi.org/10.1093/hmg/ddu125
Hsu PD, Lander ES, Zhang F (2014) Development and applications of CRISPR-Cas9 for genome engineering. Cell 157(6):1262–1278. https://doi.org/10.1016/j.cell.2014.05.010
Mali P, Yang L, Esvelt KM et al (2013) RNA-guided human genome engineering via Cas9. Science 339(6121):823–826. https://doi.org/10.1126/science.1232033
Sander JD, Joung JK (2014) CRISPR-Cas Systems for editing, regulating and targeting genomes. Nat Biotechnol 32(4):347–355. https://doi.org/10.1038/nbt.2842
Kouranova E, Forbes K, Zhao G et al (2016) CRISPRs for optimal targeting: delivery of CRISPR components as DNA, RNA, and protein into cultured cells and single-cell embryos. Hum Gene Ther 27(6):464–475. https://doi.org/10.1089/hum.2016.009
Liang X, Potter J, Kumar S et al (2015) Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. J Biotechnol 208:44–53. https://doi.org/10.1016/j.jbiotec.2015.04.024
Shao LW, Niu R, Liu Y (2016) Neuropeptide signals cell non-autonomous mitochondrial unfolded protein response. Cell Res 26(11):1182–1196. https://doi.org/10.1038/cr.2016.118
Desktop Genetics. https://www.deskgen.com/landing/cloud. Accessed January 9, 2019
Image LabTM Software—Life Science Research by Bio-Rad. http://www.bio-rad.com/en-pl/product/image-lab-software?ID=KRE6P5E8Z. Accessed January 9, 2019
ImageJ. https://imagej.nih.gov/ij/. Accessed January 9, 2019
Sunset Notice—GeneSifter Lab Edition. http://informatics.perkinelmer.com/Support/SupportNews/details/?SupportNews=124. Accessed January 9, 2019
Sentmanat MF, Peters ST, Florian CP et al (2018) A survey of validation strategies for CRISPR-Cas9 editing. Sci Rep 8(1)
Acknowledgment
This study was financially supported by the National Science Centre, Poland (grant no. 2017/25/N/NZ5/01885; 2017/25/B/NZ5/02762).
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Śmiech, M., Leszczyński, P., Haque, E., Taniguchi, H. (2020). Cloning-Free (DNA-Free) CRISPR-Cas9-Mediated Gene Editing in Human Liver Cell Line and Its Detection. In: Islam, M.T., Bhowmik, P.K., Molla, K.A. (eds) CRISPR-Cas Methods . Springer Protocols Handbooks. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0616-2_10
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DOI: https://doi.org/10.1007/978-1-0716-0616-2_10
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Online ISBN: 978-1-0716-0616-2
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