From RNA to DNA: CRISPR/LbuCas13a Demonstrates Exceptional Single-Nucleotide Specificity

Abstract

Traditionally perceived as an RNA-specific nuclease, Cas13a has garnered extensive utilization in RNA detection. This paradigm is challenged by our discovery of LbuCas13a's ability to directly target DNA without the restrictions of (Protospacer Flanking Sequence) FPS and (Protospacer Adjacent Motif) PAM sequences, coupled with robust trans-cleavage activity, a breakthrough in CRISPR-based diagnostics. Contrary to conventional understanding, LbuCas13a does not degrade DNA targets, thereby enabling retesting. Remarkably, our study reveals a striking enhancement in LbuCas13a's single nucleotide specificity against DNA (a 98-fold increase compared to RNA). This heightened specificity is attributed to the lower affinity of crRNA towards DNA, raising the crRNA-DNA binding energy barrier. Leveraging this discovery, we introduce a pioneering molecular diagnostic platform: Advanced LbuCas13a-Strong-Specificity DNA Universal Rapid Enhanced Detection (ASSURED), which achieves high-resolution genotyping, exemplified by the accurate discrimination of the CYP2C19*3 gene variant. ASSURED exhibits exceptional sensitivity, capable of detecting DNA concentrations as minute as 0.3 aM (0.18 cps/µL). ASSURED represents a significant advancement in real-time nucleic acid detection, with its unparalleled specificity and sensitivity, making it an ideal tool for pathogen identification and mutation analysis in clinical diagnostics.