THRUST: translesion synthesis-driven hierarchical regulation using a template-activator construct for Cas12a activity

Abstract

The CRISPR/Cas12a system has demonstrated extraordinary capabilities in biosensing and molecular diagnostics, owing to its precise recognition and efficient trans-cleavage ability. However, current crRNA-based Cas12a regulation is relatively crude, requiring costly modifications and multiple components, increasing system complexity. Here, we develop translesion synthesis-driven hierarchical regulation using a template-activator construct for Cas12a activity (THRUST), a powerful and economical Cas12a regulation strategy. This strategy utilizes a bifunctional template-activator construct (TAC) that simultaneously functions as a transcriptional template for T7 RNA polymerase (T7 RNAP) and an activator for Cas12a. T7 RNAP skips the deoxyuridine (dU) lesion while being blocked by the apurinic/apyrimidinic (AP) site. Strategic positioning of transcriptional regulatory units on the TAC allows precise control of crRNA length and simultaneously regulates Cas12a activation at the activator level, thereby achieving hierarchical regulation of Cas12a. Through the construction of “Dim down” and “Light up” biosensing platforms and an aggregation-induced emission lateral flow test, THRUST enriches the CRISPR/Cas12a regulatory toolbox for molecular diagnostics.

Graphical abstract: THRUST: translesion synthesis-driven hierarchical regulation using a template-activator construct for Cas12a activity

Supplementary files

Article information

Article type
Edge Article
Submitted
07 Apr 2025
Accepted
06 Jun 2025
First published
16 Jun 2025
This article is Open Access

All publication charges for this article have been paid for by the Royal Society of Chemistry
Creative Commons BY-NC license

Chem. Sci., 2025, Advance Article

THRUST: translesion synthesis-driven hierarchical regulation using a template-activator construct for Cas12a activity

L. Qin, W. Wang, X. Xia, T. Zuo, Y. Cai, G. Xu, F. Wei, S. Wang, Q. Hu, Z. Zhao, F. Zhang, B. Z. Tang and Y. Cen, Chem. Sci., 2025, Advance Article , DOI: 10.1039/D5SC02575C

This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. You can use material from this article in other publications, without requesting further permission from the RSC, provided that the correct acknowledgement is given and it is not used for commercial purposes.

To request permission to reproduce material from this article in a commercial publication, please go to the Copyright Clearance Center request page.

If you are an author contributing to an RSC publication, you do not need to request permission provided correct acknowledgement is given.

If you are the author of this article, you do not need to request permission to reproduce figures and diagrams provided correct acknowledgement is given. If you want to reproduce the whole article in a third-party commercial publication (excluding your thesis/dissertation for which permission is not required) please go to the Copyright Clearance Center request page.

Read more about how to correctly acknowledge RSC content.

Social activity

Spotlight

Advertisements