Engineering Mutation Clones in Mammalian Cells with CRISPR/Cas9

Area of Science:

• Molecular Biology
• Genetics
• Stem Cell Biology

Background:

• CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) technology offers a more efficient and less complex alternative to ZFNs and TALENs for genome engineering.
• The versatility of CRISPR extends beyond gene knockout to precise modifications, gene activation/repression, and genome-wide screening.

Purpose of the Study:

• To describe a protocol for generating stable cell lines with a knock-in mutation using CRISPR.
• To demonstrate the creation of a specific point mutation (R249S) in the TP53 gene within human embryonic stem cells (hESC).

Main Methods:

• Design of a CRISPR system targeting the TP53 genomic region.
• Delivery of the CRISPR system into H9 hESC line and subsequent clone selection.
• Screening and selection of correctly modified clones.

Main Results:

• Successful generation of a stable hESC line with the targeted R249S mutation in TP53 exon 7.
• Demonstration of CRISPR's efficacy in creating precise point mutations in human embryonic stem cells.

Conclusions:

• CRISPR technology provides a robust method for generating specific mutations in human embryonic stem cells.
• This protocol facilitates the creation of genetically modified cell lines for further research into gene function and disease modeling.