Efficient and high-fidelity base editor with expanded PAM compatibility for cytidine dinucleotide

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Biotechnology

Background:

• Cytidine base editors (CBEs) enable C-to-T conversions but are limited by protospacer adjacent motif (PAM) sequence constraints.
• Existing CBEs primarily target PAMs with G, T, or A bases, restricting their genomic reach.

Purpose of the Study:

• To develop a novel base editor with expanded PAM compatibility for broader genome targeting.
• To enhance the efficiency and fidelity of base editing for gene modification applications.

Main Methods:

• Development of nNme2-CBE by fusing a cytidine deaminase to a Cas9 nickase variant.
• Testing nNme2-CBE editing efficiency and fidelity in human cells and rabbit embryos.
• Combining nNme2Cas9 with engineered cytidine deaminases for improved performance.

Main Results:

• nNme2-CBE demonstrated excellent PAM compatibility for cytidine dinucleotides, expanding targeting scope.
• Achieved targeted editing efficiencies of 29.0%-55.0% in human cells and 17.3%-52.5% in rabbit embryos.
• nNme2-CBE exhibited high fidelity with minimal off-targeting in vivo and enhanced efficiency in GC contexts.
• Successfully generated founder rabbits with precise single-base substitutions in the Fgf5 gene.

Conclusions:

• The novel nNme2-CBE system significantly expands the genome-targeting capabilities of base editors.
• This high-fidelity editor offers a valuable new tool for efficient gene modification and potential therapeutic applications.