Journal Article

Scalable modulation of CRISPR‒Cas enzyme activity using photocleavable phosphorothioate DNA

Pei, Duanqing, Zhou, Xiaoming, Guo, Xinyi, Wang, Yihui, Hu, Menglu, Liang, Fang, Zhang, Bingni, Shan, Yuanyue, Zhang, Mengmeng, Zhang, Mingfeng, Wang, Xue, Tian, Tian, Qi, Weiwei, Cao, Feng, Shen, Yuting, Xie, Wei

2025

Overview

The regulation of CRISPR‒Cas activity is critical for developing advanced biotechnologies. Optical control of CRISPR‒Cas system activity can be achieved by modulation of Cas proteins or guide RNA (gRNA), but these approaches either require complex protein engineering modifications or customization of the optically modulated gRNAs according to the target. Here, we present a method, termed photocleavable phosphorothioate DNA (PC&PS DNA)-mediated regulation of CRISPR‒Cas activity (DNACas), that is versatile and overcomes the limitations of conventional methods. In DNACas, CRISPR‒Cas activity is silenced by the affinity binding of PC&PS DNA and restored through light-triggered chemical bond breakage of PC&PS DNA. The universality of DNACas is demonstrated by adopting the PC&PS DNA to regulate various CRISPR‒Cas enzymes, achieving robust light-switching performance. DNACas is further adopted to develop a light-controlled one-pot LAMP-BrCas12b detection method and a spatiotemporal gene editing strategy. We anticipate that DNACas could be employed to drive various biotechnological advances. CRISPR systems are powerful tools for gene editing and diagnostics, but their regulation is challenging. Here, the authors present DNACas, a light-controlled method using photocleavable phosphorothioate DNA to modulate CRISPR activity, enabling precise gene editing and one-pot diagnostic detection.

• Publisher: Nature Publishing Group UK,Nature Publishing Group,Nature Portfolio
• Subject: 38/91 / 45/41 / 631/1647/1511 / 631/337/4041/3196 / 631/45/2783 / 631/535/1258/1259 / 631/92/610 / 64/116 / 9/10 / Biotechnology / Chemical bonds / CRISPR / CRISPR-Cas Systems - genetics / Deoxyribonucleic acid / DNA / DNA - chemistry / DNA - genetics / DNA - metabolism / Editing / Engineering / Enzymatic activity / Enzyme activity / Enzymes / Gene Editing - methods / Genetic modification / Genome editing / gRNA / Humanities and Social Sciences / Humans / Light / Modulation / multidisciplinary / Optical control / Phosphorothioate / Phosphorothioate Oligonucleotides - chemistry / Phosphorothioate Oligonucleotides - genetics / Phosphorothioate Oligonucleotides - metabolism / Protein engineering / Proteins / RNA, Guide, CRISPR-Cas Systems - genetics / RNA, Guide, CRISPR-Cas Systems - metabolism / Science / Science (multidisciplinary)