# A tailored phosphorothioate coordinator enables CRISPR/Cas in-situ amplification

## TL;DR

A new CRISPR-based system uses phosphorothioate modifications to boost sensitivity and enable precise in situ imaging of viral mRNA in cancer cells.

## Contribution

A tailored phosphorothioate modification strategy enables exponential CRISPR amplification without external enzymes.

## Key findings

- Phosphorothioate modifications modulate Cas enzyme conformation and trans-cleavage resistance.
- The SACA system achieves 50,000-fold and 10,000-fold sensitivity enhancement for Cas12a and Cas13a, respectively.
- SACA enables precise in situ imaging of HPV16 and HPV18 mRNA in cervical cancer cells.

## Abstract

The CRISPR/Cas system is a powerful tool for molecular diagnostics, but its reliance on linear amplification constrains sensitivity, particularly for in situ imaging. Here, we discovered that phosphorothioate (PS)-modified activators can modulate Cas enzyme conformation via hydrophobic anchoring. By adjusting the PS modification sites, we achieved precise control over Cas activation and trans-cleavage resistance. Guided by this mechanism, we proposed a tailored design strategy featuring a “scattered” PS modification to engineer a linear “Coordinator” probe. This design effectively decouples Cas enzyme activation from substrate trans-cleavage resistance, enabling the construction of a Scattered PS Nucleic Acid-driven Cas Autocatalytic system (SACA). SACA achieves exponential amplification without external enzymes, enhancing Cas12a and Cas13a sensitivity by 50 000-fold and 10 000-fold, respectively. Furthermore, the superior biostability and structural simplicity of these linear probes endow SACA with excellent compatibility, facilitating precise in situ imaging of HPV16 and HPV18 mRNA in cervical cancer cells. This study not only advances the understanding of Cas enzyme regulation by chemically modified nucleic acids but also establishes a new paradigm for precise and efficient molecular diagnostics.

Graphical Abstract