title: In Vitro Evaluation of CRISPR/Cas9 Function by an Electrochemiluminescent Assay.
abstract: 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.
authors: MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University , Guangzhou, China 510631.
authors: Weipeng
authors: W
authors: Liu
authors: MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University , Guangzhou, China 510631.
authors: Huiheng
authors: H
authors: Yu
authors: MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University , Guangzhou, China 510631.
authors: Xiaoming
authors: X
authors: Zhou
authors: MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University , Guangzhou, China 510631.
authors: Da
authors: D
authors: Xing
mesh_terms: D064113:CRISPR-Cas Systems / Q000235:genetics; D004247:DNA / Q000032:analysis* / Q000235:genetics; D055664:Electrochemical Techniques; D005456:Fluorescent Dyes / Q000737:chemistry; D008163:Luminescent Measurements; D015394:Molecular Structure
chemical_list: D005456:Fluorescent Dyes; D004247:DNA