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Updated: Nov 12, 2025

Author Spotlight: Development of Simplified CRISPR-Based Tests for Rapid Detection of Infectious Diseases
Published on: August 16, 2024
Hairpin-Spacer crRNA-Enhanced CRISPR/Cas13a System Promotes the Specificity of Single Nucleotide Polymorphism (SNP)
Yuqing Ke1, Shiyi Huang1, Behafarid Ghalandari1
1State Key Laboratory of Oncogenes and Related Genes Institute for Personalized Medicine School of Biomedical Engineering Shanghai Jiao Tong University Shanghai 200030 China.
Abstract:
The Cas13a system has great potential in RNA interference and molecular diagnostic fields. However, lacking guidelines for crRNA design hinders practical applications of the Cas13a system in RNA editing and single nucleotide polymorphism identification. This study posits that crRNAs with hairpin spacers improve the specificity of CRISPR/Cas13a system (termed hs-CRISPR). Gibbs free energy analysis suggests that the hairpin-spacer crRNAs (hs-crRNAs) suppress Cas13a's affinity to off-target RNA. A hepatitis B virus DNA genotyping platform is established to further validate the high-specificity of hs-CRISPR/Cas13a system. Compared to ordinary crRNA, hs-crRNAs increase the specificity by threefold without sacrificing the sensitivity of the CRISPR/Cas13a system. Furthermore, the mechanism of the Cas13a/hs-crRNA/target RNA composition is elucidated with theoretical simulations. This work builds on the fundamental understanding of Cas13a activation and offers significant improvements for the rational design of crRNA for the CRISPR/Cas13a system.
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