4.8 Article

2′-O-Methyl modified guide RNA promotes the single nucleotide polymorphism (SNP) discrimination ability of CRISPR-Cas12a systems

Journal

CHEMICAL SCIENCE
Volume 13, Issue 7, Pages 2050-2061

Publisher

ROYAL SOC CHEMISTRY
DOI: 10.1039/d1sc06832f

Keywords

-

Funding

  1. Nature Scientific Foundation of China [81871448, 81971736]
  2. National Key Research and Development Program of China [2017FYA0205301]
  3. Nature Scientific Foundation of Shanghai [21ZR1435000, 22N31900400]
  4. Interdisciplinary Program of Shanghai Jiao Tong University [ZH2018QNA51, YG2021QN58, Agri-X20200101]
  5. Shanghai Clinical Medical Research Center Project [19MC1910800]
  6. Shanghai Municipal Health Commission Project [2019CXJQ03]
  7. Center of Advanced Electronics Materials and Devices (AEMD)
  8. Shanghai Jiao Tong University

Ask authors/readers for more resources

In this study, we propose a strategy to enhance the specificity of the Cas12a system by using 2'-O-methyl modified guide RNA (gRNA), and we validate the effectiveness of this strategy through experiments. Our results show that the 2'-OMe modified gRNAs can significantly improve the specificity of Cas12a in discriminating single-base mutations, and molecular docking simulations reveal the mechanism of this enhancement.
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 '-O-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.

Authors

I am an author on this paper
Click your name to claim this paper and add it to your profile.

Reviews

Primary Rating

4.8
Not enough ratings

Secondary Ratings

Novelty
-
Significance
-
Scientific rigor
-
Rate this paper

Recommended

No Data Available
No Data Available