A qPCR Method to Assay Endonuclease Activity of Cas9-sgRNA Ribonucleoprotein Complexes

Area of Science:

• Molecular Biology
• Biotechnology
• Genome Editing

Background:

• CRISPR-Cas is a leading genome editing technology.
• Cas9-sgRNA ribonucleoprotein (RNP) complexes are increasingly used for gene editing.
• Quantifying Cas9 RNP activity is crucial for optimizing editing efficiency.

Purpose of the Study:

• To develop a quantitative polymerase chain reaction (qPCR)-based assay.
• To measure the double-strand break (DSB) reaction mediated by Cas9 RNP.
• To compare the endonuclease activities of different Cas9 RNPs.

Main Methods:

• Targeted dsr gene from Leuconostoc citreum and upp gene from Bifidobacterium bifidum were used.
• Cas9 protein produced via recombinant Escherichia coli.
• qPCR was employed to monitor target DNA concentration changes post-Cas9 RNP treatment.

Main Results:

• Optimized in vitro conditions led to specific cleavage of dsr DNA by Cas9 RNPs.
• qPCR assay successfully quantified endonuclease activities: 28.74 unit/μg for dsr365RNP and 34.48 unit/μg for dsr433RNP.
• Method validated for different target genes and demonstrated utility in assessing electroporation effects on Cas9 RNP activity.

Conclusions:

• A novel qPCR-based assay effectively quantifies Cas9 RNP endonuclease activity.
• The assay provides a versatile tool for measuring genome editing efficiency.
• This method aids in optimizing CRISPR-Cas9 RNP delivery and application.