Engineering Mutation Clones in Mammalian Cells with CRISPR/Cas9

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) is a powerful genome engineering system.
• CRISPR offers higher efficiency and a less complicated process compared to ZFNs and TALENs.
• CRISPR technology enables gene knockout, precise modifications, gene activation/repression, and genome-wide screening.

Purpose of the Study:

• To describe the procedure for generating a stable cell line with a CRISPR-induced knock-in mutation.
• To demonstrate the application of CRISPR for creating a specific point mutation (R249S) in the TP53 gene.
• To outline the steps for generating a knock-in mutation in human embryonic stem cells (hESCs).

Main Methods:

• Design of a CRISPR system targeting the TP53 genomic region.
• Delivery of the CRISPR system into H9 hESCs.
• Selection and examination of clones with the desired knock-in mutation.

Main Results:

• Successful generation of a stable cell line with the R249S point mutation in TP53 exon 7.
• Demonstration of CRISPR's efficacy in precise gene editing in hESCs.
• Establishment of a protocol for creating specific knock-in mutations.

Conclusions:

• CRISPR technology provides an efficient method for generating precise gene modifications in hESCs.
• The described protocol facilitates the creation of engineered cell lines for studying gene function and disease.
• This approach is valuable for developing stable cell lines with specific genetic alterations.