A cleavage-based surrogate reporter for the evaluation of CRISPR-Cas9 cleavage efficiency

CRISPR-Cas9 is a powerful tool for genome engineering, but its efficiency largely depends on guide RNA (gRNA). There are multiple methods available to evaluate the efficiency of gRNAs, including the T7E1 assay, surveyor nuclease assay, deep sequencing, and surrogate reporter systems. In the present study, we developed a cleavage-based surrogate that we have named the LacI-reporter to evaluate gRNA cleavage efficiency. The LacI repressor, under the control of the EF-1 alpha promoter, represses luciferase or EGFP reporter expression by binding to the lac operator. Upon CRISPR-Cas9 cleavage at a target site located between the EF-1 alpha promoter and the lacI gene, repressor expression is disrupted, thereby triggering luciferase or EGFP expression. Using this system, we can quantitate gRNA cleavage efficiency by assessing luciferase activity or EGFP expression. We found a strong positive correlation between the cleavage efficiency of gRNAs measured using this reporter and mutation frequency, measured using surveyor and deep sequencing. The genome-editing efficiency of gRNAs was validated in human liver organoids. Our LacI-reporter system provides a useful tool to select efficient gRNAs for genome editing.

Automated summary

CRISPR-Cas9 is a powerful tool for genome engineering, and a LacI-reporter system was developed in the study to evaluate gRNA cleavage efficiency by quantitating luciferase activity or EGFP expression. The study found a strong positive correlation between the efficiency of gRNAs measured using this system and mutation frequency measured using surveyor and deep sequencing, with validation in human liver organoids. The LacI-reporter system provides a useful tool for selecting efficient gRNAs for genome editing.