A Novel Dual-Guide CRISPR-Cas13 Strategy Improves Specificity for Single-Nucleotide Variant Detection

ABSTRACT The emergence of CRISPR-Cas systems has transformed nucleic acid detection and manipulation. Cas13, a type VI CRISPR effector, targets RNA with high sensitivity through both cis (target RNA) and trans (collateral RNA) cleavage. This property enables the use of fluorescent reporters for sensitive diagnostics. However, Cas13’s heightened sensitivity also leads to reduced specificity due to its susceptibility to single-nucleotide mismatches, potentially causing off-target effects. To overcome this limitation, we developed the first dual-guide RNA system for Cas13 that enhances mismatch discrimination and improves target specificity. This system employs two distinct RNAs—dcrRNA and dtracrRNA—which hybridise to refine target recognition and activation. In vitro experiments demonstrated robust cis- and trans-RNase activity, indicating efficient and specific cleavage. The system accurately detected SARS-CoV-2 RNA, demonstrating its potential for pathogen diagnostics, and successfully discriminated between KRAS G12D and G12C mutations—clinically relevant single-nucleotide variants in cancer diagnosis. These results highlight the dual-guide Cas13 platform’s potential for precise, rapid, and reliable RNA detection. Overall, this approach represents a significant advance over conventional Cas13 systems, offering improved specificity without compromising sensitivity. Its versatility makes it a promising tool for next-generation molecular diagnostics and precision gene editing applications. Competing Interest Statement Conflict of interest: F.M., A.A.G., J.J.D.M., and R.M.S.M are inventors of the patent Number: EP4414452A1, DUAL-GUIDE RNA COMPOSITION FOR EXECUTING A SINGLE-GUIDE RNA CRISPR-ASSOCIATED SYSTEM. Funding Statement This work was supported by the Spanish Ministry of Science and Innovation (MCIN)/AEI/10.13039/501100011033 and the European Union Next Generation EU/PRTR (Grant PID2022-141065OB-I00) as well as, by FEDER/Junta de Andalucia-Consejeria de Economia y Conocimiento/Project CV20-77741. Additional support was provided by the Instituto de Salud Carlos III (ISCIII) through research projects PI21/00298 and PI24/00888, a TerAv (RD21/0017/0004) and TerAv+ (RD24/0014/0005]. By the Consejeria de Salud y Familias (Junta de Andalucia) (PI-0236-2024). By the European Cooperation in Science and Technology (COST) [GeneHumdi-CA21113]. IMJ was supported by a predoctoral fellowship from the Spanish Ministry of Science, Innovation and Universities (FPU22/03455). Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The Research Ethics Committee of the Portal de Etica de la Investigacion Biomedica de Andalucia (PEIBA), Junta de Andalucia gave ethical approval for this work (Acta 6/2020, 29 June 2020). All saliva samples were provided by the Biobank of the Public Health System of Andalusia (reference S2000262), and informed consent was obtained from all participants. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes DATA AVAILABILITY The data underlying this article will be shared on reasonable request to the corresponding author.