Issue 7, 2022

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

Abstract

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.

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

Supplementary files

Article information

Article type
Edge Article
Submitted
07 Des 2021
Accepted
20 Jan 2022
First published
01 Feb 2022
This article is Open Access

All publication charges for this article have been paid for by the Royal Society of Chemistry
Creative Commons BY-NC license

Chem. Sci., 2022,13, 2050-2061

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

Y. Ke, B. Ghalandari, S. Huang, S. Li, C. Huang, X. Zhi, D. Cui and X. Ding, Chem. Sci., 2022, 13, 2050 DOI: 10.1039/D1SC06832F

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