Engineering Mutation Clones in Mammalian Cells with CRISPR/Cas9.
Methods Mol Biol
; 2108: 355-369, 2020.
Article
in En
| MEDLINE
| ID: mdl-31939194
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. 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.Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Genetic Engineering
/
Gene Targeting
/
CRISPR-Cas Systems
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Gene Editing
/
Mutation
Limits:
Animals
/
Humans
Language:
En
Journal:
Methods Mol Biol
Journal subject:
BIOLOGIA MOLECULAR
Year:
2020
Document type:
Article
Affiliation country: