Ke Yuqing, Ghalandari Behafarid, Huang Shiyi, Li Sijie, Huang Chengjie, Zhi Xiao, Cui Daxiang, Ding Xianting
State Key Laboratory of Oncogenes and Related Genes, Institute for Personalized Medicine, School of Biomedical Engineering, Shanghai Jiao Tong University Shanghai 200030 China
Shanghai Engineering Centre for Intelligent Diagnosis and Treatment Instrument, School of Electronic Information and Electrical Engineering, Shanghai Jiao Tong University Shanghai 200240 China.
Chem Sci. 2022 Feb 1;13(7):2050-2061. doi: 10.1039/d1sc06832f. eCollection 2022 Feb 16.
The CRISPR-Cas12a system has been widely applied to genome editing and molecular diagnostics. However, off-target cleavages and false-positive results remain as major concerns in Cas12a practical applications. Herein, we propose a strategy by utilizing the 2'--methyl (2'-OMe) modified guide RNA (gRNA) to promote the Cas12a's specificity. Gibbs free energy analysis demonstrates that the 2'-OMe modifications at the 3'-end of gRNA effectively suppress the Cas12a's overall non-specific affinity while maintaining high on-target affinity. For general application illustrations, HBV genotyping and SARS-CoV-2 D614G mutant biosensing platforms are developed to validate the enhanced Cas12a's specificity. Our results indicate that the 2'-OMe modified gRNAs could discriminate single-base mutations with at least two-fold enhanced specificity compared to unmodified gRNAs. Furthermore, we investigate the enhancing mechanisms of the 2'-OMe modified Cas12a systems by molecular docking simulations and the results suggest that the 2'-OMe modifications at the 3'-end of gRNA reduce the Cas12a's binding activity to off-target DNA. This work offers a versatile and universal gRNA design strategy for highly specific Cas12a system development.
CRISPR-Cas12a系统已被广泛应用于基因组编辑和分子诊断。然而，脱靶切割和假阳性结果仍然是Cas12a实际应用中的主要问题。在此，我们提出一种策略，即利用2'-O-甲基（2'-OMe）修饰的引导RNA（gRNA）来提高Cas12a的特异性。吉布斯自由能分析表明，gRNA 3'端的2'-OMe修饰有效抑制了Cas12a的整体非特异性亲和力，同时保持了高靶向亲和力。为了进行一般应用说明，开发了HBV基因分型和SARS-CoV-2 D614G突变体生物传感平台，以验证增强的Cas12a特异性。我们的结果表明，与未修饰的gRNA相比，2'-OMe修饰的gRNA能够以至少两倍增强的特异性区分单碱基突变。此外，我们通过分子对接模拟研究了2'-OMe修饰的Cas12a系统的增强机制，结果表明gRNA 3'端的2'-OMe修饰降低了Cas12a与脱靶DNA的结合活性。这项工作为高度特异性的Cas12a系统开发提供了一种通用的gRNA设计策略。
