Elimination of cis-cleavage in CRISPR diagnostics for one-pot rapid nucleic acid detection

TL;DR

This paper introduces a new CRISPR-based diagnostic method that eliminates amplicon degradation, enabling faster and more sensitive one-pot nucleic acid detection.

Contribution

A PAM-independent and cis-cleavage-free Cas12a system using a bipartite split-crRNA is developed for improved one-pot diagnostics.

Findings

SCas12a eliminates cis-cleavage while maintaining trans-cleavage activity, improving sensitivity by 100–1000-fold.

The system reduces time-to-signal by >10-fold and detects targets at attomolar levels within 30 minutes.

SCas12a successfully detected HPV16, SARS-CoV-2, and TP53 SNPs in clinical samples with high selectivity.

Abstract

Current one-pot clustered regularly interspaced short palindromic repeats diagnostics are limited by the cis-cleavage activity of Cas nucleases, which leads to amplicon degradation during amplification. Here, we report a streamlined strategy that overcomes this limitation. By integrating a bipartite split-crRNA into Cas12a (SCas12a), we separate target recognition from PAM dependency and completely eliminate cis-cleavage while preserving robust trans-cleavage. This strategy is broadly applicable for one-pot testing, compatible with recombinase polymerase amplification, RT–RPA, and loop-mediated isothermal amplification, as well as multiple Cas12a orthologs, including As, Lb, and Ct Cas12a. Moreover, the SCas12a accelerates one-pot testing with 100–1000-fold improved sensitivity and achieves >10-fold reduction in time-to-signal, enabling detection of targets at attomolar levels within 30 min. Additionally, it provides single-base resolution with up to 91-fold selectivity. The system has been successfully applied to detect HPV16, SARS-CoV-2, and TP53 SNPs in clinical samples. Together, we have developed a PAM-independent and cis-cleavage-free one-pot Cas12a assay, which holds strong potential for point-of-care diagnostics.

Graphical Abstract

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