In Vitro Evaluation of CRISPR/Cas9 Function by an Electrochemiluminescent Assay

The CRISPR/Cas9 system is a revolutionary genome-editing tool that enables targeted and efficient gene knockouts. However, the off-target effects and loci-dependent enzyme activity limit its uses on the field of research and treatment. In this study, we designed a convenient and sensitive in vitro test method, which was based on electrochemiluminescence (ECL) technology for evaluating cleavage activity of the CRISPR/Cas9 system. It was find that Cas9 can tolerate some common genetic modifications to its target DNA. It was also find that target DNA/sgRNA with single-base mismatch and UV damages of target DNA resulted in significantly reduction of Cas9 cleavage efficiency. Comparing with traditional method, the proposed method reduced the evaluation time from weeks to 2 h. Therefore, our study provides a versatile in vitro method for a priori analysis of CRISPR/Cas9 system and highlights the potential to guide in vivo genome editing.