Directed Evolution of OgeuIscB With Enhanced Activity in Human Cells

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Biotechnology

Background:

• IscB, an RNA-guided endonuclease and progenitor of Cas9, is a promising tool for in vivo genome editing due to its small size.
• Low editing efficiency of IscB in eukaryotic cells hinders its application in precise human genome editing.

Purpose of the Study:

• To enhance the editing efficiency of IscB in mammalian cells.
• To develop novel base editors for precise gene correction.

Main Methods:

• Structure-guided rational design and protein engineering were used to optimize OgeuIscB.
• An adenine base editor (miABE-F138R) was engineered based on the optimized IscB variant.
• The engineered editor was applied to correct a mutation associated with retinitis pigmentosa.

Main Results:

• The engineered IscB variant, enIscB-F138R, showed up to 3.49-fold increased editing activity in mammalian cells.
• The nickase-based adenine base editor, miABE-F138R, demonstrated enhanced base editing efficiency.
• miABE-F138R effectively corrected the R560C mutation in Pde6β, improving upon existing base editors.

Conclusions:

• Engineered IscB variants, enIscB-F138R and miABE-F138R, provide enhanced platforms for genome editing.
• These novel tools hold significant potential for future biomedical applications, including the treatment of genetic diseases.