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TitleScalable modulation of CRISPR‒Cas enzyme activity using photocleavable phosphorothioate DNA.
Journal, issue, pagesNat Commun, Vol. 16, Issue 1, Page 5939, Year 2025
Publish dateJul 1, 2025
AuthorsMenglu Hu / Bingni Zhang / Yuanyue Shan / Feng Cao / Yihui Wang / Weiwei Qi / Xue Wang / Yuting Shen / Xinyi Guo / Mengmeng Zhang / Tian Tian / Wei Xie / Mingfeng Zhang / Fang Liang / Duanqing Pei / Xiaoming Zhou /
PubMed AbstractThe 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 ...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.
External linksNat Commun / PubMed:40593724 / PubMed Central
MethodsEM (single particle)
Resolution2.6 - 2.9 Å
Structure data

62606

9kwb

EMDB-62606, PDB-9kwb:
Cas12a-PCPS-dark
Method: EM (single particle) / Resolution: 2.6 Å

62607

9kwc

EMDB-62607, PDB-9kwc:
Cas12a-PCPS-light
Method: EM (single particle) / Resolution: 2.9 Å

Source
  • lachnospiraceae bacterium nd2006 (bacteria)
  • synthetic construct (others)
KeywordsDNA BINDING PROTEIN/RNA / Cas12a-PCPS-dark / DNA BINDING PROTEIN-RNA complex / header DNA BINDING PROTEIN/RNA / header DNA BINDING PROTEIN-RNA complex

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