Engineering Mutation Clones in Mammalian Cells with CRISPR/Cas9

Authors

Zijun Huo
Jian Tu
Rachel Shoemaker
Dung-Fang Lee
Ruiying Zhao

Publication Date

1-1-2026

Journal

Methods in Molecular Biology

DOI

10.1007/978-1-0716-4901-5_28

PMID

41478989

Abstract

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. Compared to zinc finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs), CRISPR shows a 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 a stable cell line 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.

Keywords

CRISPR-Cas Systems, Humans, Gene Editing, Cell Line, Tumor Suppressor Protein p53, Mutation, RNA, Guide, CRISPR-Cas Systems, Human Embryonic Stem Cells, Exons, Gene Knock-In Techniques, Genetic Engineering, CRISPR; Donor Vector; Genome Editing; Precision Gene Editing; Southern Blot; Stable Cell Line Validation; gRNA Design

Published Open-Access

yes

Recommended Citation

Huo, Zijun; Tu, Jian; Shoemaker, Rachel; et al., "Engineering Mutation Clones in Mammalian Cells with CRISPR/Cas9" (2026). Faculty, Staff and Student Publications. 6072.
https://digitalcommons.library.tmc.edu/uthgsbs_docs/6072