Cas12a tolerates different lengths of split scaffold RNA for trans-cleavage activity
AsCas12a is an ortholog of Cas12a that originates from Acidaminococcus sp. BVL36. Leveraging a split crRNA, the SCas12a assay was established for direct RNA detection, addressing limitations in analyzing long RNA molecules with intricate secondary structures. The study optimized the split Cas12a system by relocating the target RNA downstream of the PAM sequence, creating SCas12aV2.
To activate trans-cleavage activity, scaffold and spacer RNAs with varied sequences were assessed. The optimal scaffold length was determined through synthesizing scaffold RNAs of different lengths. The S6 scaffold showed high detection sensitivity, leading to its selection for SCas12aV2.
In vitro trans-cleavage assays targeting hepatitis C virus (HCV) RNA segments demonstrated SCas12aV2's ability to detect highly structured RNA substrates. Structural modeling revealed the mechanism behind SCas12aV2's enhanced sensitivity.
The hybrid dsDNA-ssDNA activator, S6.1, significantly improved detection efficiency over traditional activators, enabling detection of complex RNA structures. SCas12aV2 assay's universality and efficiency in detecting various RNA substrates were highlighted.
Assessing different Cas12a orthologs in SCas12aV2 assay showcased CtCas12a's enhanced sensitivity, suggesting optimization based on specific requirements. The assay demonstrated high sensitivity in detecting RNA targets without amplification.
SCas12aV2's efficacy in SNP detection within long RNA molecules was evaluated, showcasing enhanced specificity and broad applicability. The assay's ability to quantify RNA and detect pathogen-induced infections was validated, emphasizing its clinical utility.
Reference from News: Precise amplification-free detection of highly structured RNA with an enhanced SCas12a assay - Communications Biology