Hairpin-Spacer crRNA-Enhanced CRISPR/Cas13a System Promotes the Specificity of Single Nucleotide Polymorphism (SNP) Identification

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.

Automated summary

This study demonstrates the potential of hairpin-spacer crRNAs in improving specificity and sensitivity of the CRISPR/Cas13a system. The high-specificity RNA editing tool establishes a new approach for rational crRNA design, offering significant improvements in the Cas13a system. Theoretical simulations elucidate the mechanism of Cas13a/hs-crRNA/target RNA interaction, further enhancing our understanding of the system's activation.