An efficient genotyping method for genome-modified animals and human cells generated with CRISPR/Cas9 system.

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• CRISPR/Cas9 technology enables rapid generation of genome-modified animals for gene function studies.
• Traditional genotyping methods like T7E1 endonuclease assay face limitations with the increasing demand.
• Efficient and sensitive genotyping is crucial for advancing genetic research.

Purpose of the Study:

• To introduce a novel polyacrylamide gel electrophoresis-based (PAGE) method for genotyping indel mutations.
• To validate the PAGE method's efficacy in various generations of genome-modified mice.
• To assess the PAGE method's utility in detecting on- and off-target mutations in human cell lines.

Main Methods:

• Development of a PAGE-based assay for detecting insertions and deletions (indels).
• Application of the PAGE assay to genotype six strains of CRISPR/Cas9-modified mice across F0-F2 generations.
• Testing the PAGE assay's sensitivity for detecting mosaic DNA, down to 0.5%.
• Utilizing the PAGE assay to analyze CRISPR/Cas9-mediated effects in human 293T cells and induced pluripotent stem cells (iPSCs).

Main Results:

• The PAGE-based method successfully genotyped single and multiplexed genome-modified mice.
• Maximal detection sensitivity for mosaic DNA using PAGE was determined to be 0.5%.
• The PAGE assay effectively detected CRISPR/Cas9-mediated on- and off-target mutations in human cells.

Conclusions:

• The developed PAGE-based genotyping approach is a sensitive and efficient tool for genome-modified organisms.
• This method addresses the growing need for high-throughput genotyping in CRISPR/Cas9 and other nuclease-based research.
• The PAGE assay is applicable to a wide range of genome editing applications in both animal models and cell lines.