Chapter title |
All-in-One CRISPR-Cas9/FokI-dCas9 Vector-Mediated Multiplex Genome Engineering in Cultured Cells.
|
---|---|
Chapter number | 4 |
Book title |
In Vitro Mutagenesis
|
Published in |
Methods in molecular biology, January 2017
|
DOI | 10.1007/978-1-4939-6472-7_4 |
Pubmed ID | |
Book ISBNs |
978-1-4939-6470-3, 978-1-4939-6472-7
|
Authors |
Tetsushi Sakuma, Takuya Sakamoto, Takashi Yamamoto |
Editors |
Andrew Reeves |
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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