Evolution-guided protein design of IscB for persistent epigenome editing in vivo

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

doi:10.1038/s41587-025-02655-3

Evolution-guided protein design of IscB for persistent epigenome editing in vivo

Nat Biotechnol. 2025 May 7. doi: 10.1038/s41587-025-02655-3. Online ahead of print.

Authors

Soumya Kannan^#^{1

2

3

4

5

6}, Han Altae-Tran^#^{1

2

3

4

5

6}, Shiyou Zhu^#^{1

2

3

4

5

6}, Peiyu Xu^{1

2

3

4

5

6}, Daniel Strebinger^{1

2

3

4

5

6}, Rachel Oshiro^{1

2

3

4

5

6}, Guilhem Faure^{1

2

3

4

5

6}, Lukas Moeller^{1

2

3

4

5

6}, Julie Pham^{1

2

3

4

5

6}, Kepler S Mears^{1

2

3

4

5

6}, Heyuan M Ni⁵, Rhiannon K Macrae^{1

2

3

4

5

6}, Feng Zhang^{7

8

9

10

11

12}

Affiliations

¹ Howard Hughes Medical Institute, Cambridge, MA, USA.
² Broad Institute of MIT and Harvard, Cambridge, MA, USA.
³ McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA, USA.
⁴ Department of Brain and Cognitive Science, Massachusetts Institute of Technology, Cambridge, MA, USA.
⁵ Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.
⁶ Yang Tan Collective, Cambridge, MA, USA.
⁷ Howard Hughes Medical Institute, Cambridge, MA, USA. zhang@broadinstitute.org.
⁸ Broad Institute of MIT and Harvard, Cambridge, MA, USA. zhang@broadinstitute.org.
⁹ McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA, USA. zhang@broadinstitute.org.
¹⁰ Department of Brain and Cognitive Science, Massachusetts Institute of Technology, Cambridge, MA, USA. zhang@broadinstitute.org.
¹¹ Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA. zhang@broadinstitute.org.
¹² Yang Tan Collective, Cambridge, MA, USA. zhang@broadinstitute.org.

^# Contributed equally.

PMID: 40335752
DOI: 10.1038/s41587-025-02655-3

Abstract

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.