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Updated: Jun 22, 2026

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening
Published on: April 1, 2016
Directed Evolution of OgeuIscB With Enhanced Activity in Human Cells
Jineng Lv1, Jiang Jin2, Liujun Ding1
1State Key Laboratory of Ophthalmology, Optometry and Vision Science, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China.
Abstract:
The miniature RNA-guided endonuclease IscB, as the evolutionary progenitor of Cas9, is attracting increased attention for genome editing due to its compact size and suitability for in vivo delivery. However, the poor editing efficiency of IscB in eukaryotic cells presents a significant challenge to its widespread application in precise site-specific human genome editing. In this study, we employed structure-guided rational design and protein engineering to optimize OgeuIscB, resulting in the identification of enIscB-F138R, which further enhanced editing activity up to 3.49-fold in mammalian cells compared to the high-activity OgeuIscB variant enIscB. Furthermore, we engineered an enIscB-F138R nickase-based adenine base editor, termed miABE-F138R, exhibiting enhanced base editing efficiency relative to miABE. To illustrate the practical applications of miABE-F138R, we applied it to rectify the prevalent R560C mutation in Pde6β associated with autosomal recessive retinitis pigmentosa, resulting in a significant improvement in activity compared to miABE. In conclusion, enIscB-F138R and miABE-F138R offer adaptable platforms for genome editing with potential significance in future biomedical applications.
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