Detection of genome-edited mutant clones by a simple competition-based PCR method

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• CRISPR/Cas9 gene editing is vital for studying gene function.
• Achieving 100% gene disruption efficiency in cell cultures is challenging.
• Cloning is often required to isolate fully mutated cells, slowing research.

Purpose of the Study:

• To develop a rapid and accurate method for discriminating gene-edited (knockout) cells.
• To facilitate faster research utilizing the CRISPR/Cas9 system.
• To enable efficient identification of mutations conferring drug resistance.

Main Methods:

• A competition-based PCR assay utilizing a three-primer mixture.
• One primer is designed to overlap with the Cas9 cleavage site.
• Optimization of primer design for enhanced discrimination effectiveness.

Main Results:

• Successfully discriminated knockout cells from wild-type cells using the developed PCR method.
• Demonstrated high accuracy in detecting mutations associated with drug resistance.
• The method is simple, requires basic laboratory equipment, and is widely applicable.

Conclusions:

• The competition-based PCR method offers a fast, accurate, and accessible approach for identifying CRISPR/Cas9-induced mutations.
• This technique significantly accelerates gene function studies and mutation screening.
• The method's ease of use makes it suitable for diverse laboratory settings.