Abstract
CRISPR-Cas9 enables highly convenient multiplex genome engineering in cultured cells, because it utilizes generic Cas9 nuclease and an easily customizable single-guide RNA (sgRNA) for site-specific DNA double-strand break induction. We previously established a multiplex CRISPR-Cas9 assembly system for constructing an all-in-one vector simultaneously expressing multiple sgRNAs and Cas9 nuclease or other Cas9 variants including FokI-dCas9, which supersedes the wild-type Cas9 with regard to high specificity. In this chapter, we describe a streamlined protocol to design and construct multiplex CRISPR-Cas9 or FokI-dCas9 vectors, to introduce them into cultured cells by lipofection or electroporation, to enrich the genomically edited cells with a transient puromycin selection, to validate the mutation efficiency by Surveyor nuclease assay, and to perform off-target analyses. We show that our protocol enables highly efficient multiplex genome engineering even in hard-to-transfect HepG2 cells.
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Acknowledgments
This work was supported by the Uehara Memorial Foundation (to T.S.), the Japan Society for the Promotion of Science (16K18478 to T.S. and 26290070 to T.Y.), and the Research Program on Hepatitis from Japan Agency for Medical Research and Development (AMED) to T.Y. The authors wish to express their thanks to Prof. Kazuaki Chayama, Department of Gastroenterology and Metabolism, Applied Life Science, Institute of Biomedical and Health Science, Hiroshima University, for his helpful suggestions concerning HepG2 cell culture.
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Sakuma, T., Sakamoto, T., Yamamoto, T. (2017). All-in-One CRISPR-Cas9/FokI-dCas9 Vector-Mediated Multiplex Genome Engineering in Cultured Cells. In: Reeves, A. (eds) In Vitro Mutagenesis. Methods in Molecular Biology, vol 1498. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6472-7_4
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DOI: https://doi.org/10.1007/978-1-4939-6472-7_4
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