# Enhanced SfaTnpB enables single-base-specific, one-pot nucleic acid detection for high-sensitivity diagnostics

## TL;DR

A new CRISPR-based system called TOPIC improves single-base nucleic acid detection for highly sensitive diagnostics, especially for viruses like HPV and African swine fever.

## Contribution

The development of enSfaTnpB and TOPIC platform enables ultrasensitive, single-base-specific, one-pot nucleic acid detection with high fidelity.

## Key findings

- enSfaTnpB shows improved trans-cleavage efficiency and single-base mismatch discrimination.
- TOPIC detects HPV subtypes and African swine fever virus DNA at extremely low concentrations (∼3-4 copies/μl).
- RAA-TOPIC achieves high-fidelity subtype discrimination and matches PCR-based clinical diagnostics.

## Abstract

CRISPR/Cas12-based nucleic acid detection has revolutionized molecular diagnostics but shows limited single-nucleotide specificity, limited high-fidelity subtype discrimination, and limited compatibility with one-pot assays, restricting its broader clinical application. Here, we report a transposon-associated transposase B (TnpB) ortholog, SfaTnpB, with high trans-cleavage activity, robust single-base mismatch discrimination, and broad temperature tolerance. By stepwise engineering of its guide RNA (ωRNA), we developed an enhanced SfaTnpB (enSfaTnpB) system with markedly improved trans-cleavage efficiency. In combination with a TAM-independent split-activator strategy, this system enables precise detection of single-nucleotide polymorphisms. We further developed TOPIC (TnpB-based One-Pot nucleIC acid detection), a one-pot detection platform coupling enSfaTnpB with recombinase-aided amplification (RAA) or loop-mediated isothermal amplification that enables ultrasensitive detection of human papillomavirus (HPV) subtypes 16 and 18 (∼4 copies/μl) and African swine fever virus DNA (∼3 copies/μl). Finally, RAA-TOPIC accurately detected and genotyped 14 high-risk HPV subtypes with high-fidelity subtype discrimination, showing complete concordance with quantitative real-time PCR-based clinical diagnostics. These findings establish TOPIC as a compact, programmable, and scalable molecular detection tool with broad potential for precision diagnostics and point-of-care testing, particularly in resource-limited settings.

Graphical Abstract