A qPCR method for genome editing efficiency determination and single-cell clone screening in human cells

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• CRISPR/Cas9 is a powerful tool for genome editing, but evaluating its efficiency and screening clones remains challenging.
• Existing methods for assessing genome editing outcomes are often time-consuming or lack precision.
• There is a need for a rapid and accurate method to quantify genome editing events in various applications.

Purpose of the Study:

• To develop and validate a real-time PCR method for evaluating genome editing efficiency.
• To enable rapid screening of single-cell clones for desired genetic modifications.
• To provide a practical tool for quantifying indel, HDR, and base editing events.

Main Methods:

• Developed a real-time PCR assay leveraging Taq DNA polymerase's sensitivity to 3' end nucleotide mismatches.
• Applied the method to assess CRISPR/Cas9 and base editor (BE4) treatments targeting EMX1, DYRK1A, and HOXB13 genes in Lenti-X 293T cells.
• Utilized genomic DNA from treated cells for one-round qPCR analysis.

Main Results:

• The real-time PCR method accurately and quickly determined genome editing efficiency for indel, HDR, and base editing.
• The assay successfully enabled accurate genotype determination of single-cell clones.
• Demonstrated comprehensive advantages in evaluating genome editing in various gene targets.

Conclusions:

• The developed real-time PCR method offers a rigorous and practical approach for quantifying genome editing events.
• This technique significantly improves the efficiency of assessing CRISPR/Cas9 and base editing outcomes.
• Provides a valuable tool for researchers involved in genome engineering and cell line development.