CRISPR, Clustered Regularly Interspaced Short Palindromic Repeat, as a powerful genome engineering system has been widely accepted and employed in gene editing of a vast range of cell types. Comparing to zinc finger nucleases (ZFNs) or transcription-activator-like effector nucleases (TALENs), CRISPR shows less complicated process and higher efficiency. With the development of different CRISPR systems, it can be used not only to knock out a gene, but also to make precise modifications, activate or repress target genes with epigenetic modifications, and even for genome-wide screening. Here we will describe the procedure of generating stable cell lines with a knock-in mutation created by CRISPR. Specifically, this protocol demonstrated how to apply CRISPR to create the point mutation of R249 to S249 on TP53 exon 7 in human embryonic stem cells (hESC) H9 line, which includes three major steps: (1) design CRISPR system targeting TP53 genomic region, (2) deliver the system to H9 hESC and clone selection, and (3) examination and selection of positive clones.
CITATION STYLE
Huo, Z., Tu, J., Lee, D. F., & Zhao, R. (2020). Engineering mutation clones in mammalian cells with CRISPR/Cas9. In Methods in Molecular Biology (Vol. 2108, pp. 355–369). Humana Press Inc. https://doi.org/10.1007/978-1-0716-0247-8_29
Mendeley helps you to discover research relevant for your work.