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TitleEvolution-guided protein design of IscB for persistent epigenome editing in vivo.
Journal, issue, pagesNat Biotechnol, Year 2025
Publish dateMay 7, 2025
AuthorsSoumya Kannan / Han Altae-Tran / Shiyou Zhu / Peiyu Xu / Daniel Strebinger / Rachel Oshiro / Guilhem Faure / Lukas Moeller / Julie Pham / Kepler S Mears / Heyuan M Ni / Rhiannon K Macrae / Feng Zhang /
PubMed AbstractNaturally existing enzymes have been adapted for a variety of molecular technologies, with enhancements or modifications to the enzymes introduced to improve the desired function; however, it is ...Naturally existing enzymes have been adapted for a variety of molecular technologies, with enhancements or modifications to the enzymes introduced to improve the desired function; however, it is difficult to engineer variants with enhanced activity while maintaining specificity. Here we engineer the compact Obligate Mobile Element Guided Activity (OMEGA) RNA-guided endonuclease IscB and its guiding RNA (ωRNA) by combining ortholog screening, structure-guided protein domain design and RNA engineering, and deep learning-based structure prediction to generate an improved variant, NovaIscB. We show that the compact NovaIscB achieves up to 40% indel activity (~100-fold improvement over wild-type OgeuIscB) on the human genome with improved specificity relative to existing IscBs. We further show that NovaIscB can be fused with a methyltransferase to create a programmable transcriptional repressor, OMEGAoff, that is compact enough to be packaged in a single adeno-associated virus vector for persistent in vivo gene repression. This study highlights the power of combining natural diversity with protein engineering to design enhanced enzymes for molecular biology applications.
External linksNat Biotechnol / PubMed:40335752
MethodsEM (single particle)
Resolution2.71 Å
Structure data

49856

9nvu

EMDB-49856, PDB-9nvu:
Engineered OrufIscB-omegaRNA-target DNA complex
Method: EM (single particle) / Resolution: 2.71 Å

Chemicals

ChemComp-MG:
Unknown entry

ChemComp-ZN:
Unknown entry

Source
  • metagenome (others)
  • synthetic construct (others)
KeywordsRNA BINDING PROTEIN/RNA/DNA / Gene editing / IscB / OMEGA / RNA-guided nuclease / RNA BINDING PROTEIN-RNA-DNA complex

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