Light-controlled genome editing by activation of Cas9-mRNA translation

Schepers, Helena; Dahm, Greta Charlotte ; Sumser, Martin ; Hüwel, Sabine und Rentmeister, Andrea (2025): Light-controlled genome editing by activation of Cas9-mRNA translation. In: Chemical Science, Nr. 30: S. 13916-13922

Abstract

Genome editing by the nuclease Cas9 and guide RNAs enables precise inactivation of genes and presents the basis for numerous research tools and emerging therapies. A critical aspect is the nuclease activity causing off-target effects. Approaches to control where and when active Cas9 is present are therefore desirable. Using Cas9-mRNA already presents a viable way to limit nuclease activity temporally but does not permit controlled induction. Here, we show that Cas9 activity is readily obtained by irradiation of cells transfected with a translationally muted Cas9-mRNA. Using a dual reporter system, we confirm light-mediated knockout of the eGFP-gene by flow cytometry, fluorescence microscopy, Western blotting and sequencing. This system does not involve photocaged proteins nor photocaged guide RNAs but relies on mRNA with a single photocleavable protecting group at the 5′ cap produced by in vitro transcription. This is the first demonstration of using light to activate muted Cas9-mRNA, leading to permanent alterations on the DNA level, despite the messenger itself being transient in nature.

• Dokumententyp: Zeitschriftenartikel
• EU Funded Grant Agreement Number: 772280
• Fakultät: Chemie und Pharmazie > Department Chemie
• Themengebiete: 500 Naturwissenschaften und Mathematik > 540 Chemie
• ISSN: 2041-6520
• Sprache: Englisch