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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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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Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-0615-5
Online ISBN: 978-1-0716-0616-2
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