MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University , Guangzhou, China 510631.
Anal Chem. 2016 Sep 6;88(17):8369-74. doi: 10.1021/acs.analchem.6b02338. Epub 2016 Aug 10.
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.
CRISPR/Cas9 系统是一种革命性的基因组编辑工具，可实现靶向和高效的基因敲除。然而，脱靶效应和酶活性依赖于基因座限制了其在研究和治疗领域的应用。在这项研究中，我们设计了一种方便、灵敏的体外检测方法，该方法基于电化学发光（ECL）技术来评估 CRISPR/Cas9 系统的切割活性。研究发现 Cas9 可以容忍其靶 DNA 的一些常见遗传修饰。还发现靶 DNA/sgRNA 存在单个碱基错配和靶 DNA 的 UV 损伤会导致 Cas9 切割效率显著降低。与传统方法相比，该方法将评估时间从数周缩短至 2 小时。因此，本研究为 CRISPR/Cas9 系统的先验分析提供了一种通用的体外方法，并突出了指导体内基因组编辑的潜力。
