Evolutionguided Protein Design Of Iscb For Persistent Epigenome Editing In Vivo Soumya Kannan Han Altaetran 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 by Soumya 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 101038/S41587025026553 instant download after payment.

Naturally existing enzymes have been adapted for a variety of molecular technologies, with enhancements or modifcations to the enzymes introduced to improve the desired function; however, it is difcult to engineer variants with enhanced activity while maintaining specifcity. 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 specifcity relative to existing IscBs. We further show that NovaIscB can be fused with a methyltransferase to create a programmable transcriptional repressor, OMEGAof, 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.