nCas9 Engineering for Improved Target Interaction Presents an Effective Strategy to Enhance Base Editing

Area of Science:

• Molecular Biology
• Genome Engineering
• Biotechnology

Background:

• Base editors (BEs) are advanced genome editing tools.
• They enable precise base conversions using a deaminase fused to a catalytically impaired Cas9 (nCas9).
• Ongoing development focuses on enhancing BE efficiency and safety.

Purpose of the Study:

• To explore an nCas9-centric strategy for improving base editor (BE) performance.
• To identify and engineer enhanced variants of cytosine and adenine base editors (CBEs and ABEs).

Main Methods:

• Systematic testing of 20 point mutations in nCas9 for cytosine base editors (CBEs).
• Combinatorial modifications of promising nCas9 variants to create enhanced CBEs (variants 1.1-1.3).
• Application of the nCas9 modification strategy to various CBE and adenine base editor (ABE) systems, including high-fidelity variants.

Main Results:

• Engineered nCas9 variants significantly enhanced the activity of multiple CBE and ABE systems.
• Stabilization of nCas9-substrate interactions was identified as the mechanism for improved BE activity.
• Enhanced CBE and ABE variants demonstrated significantly higher editing rates in primary human T-cells (82% and 25% increase, respectively).

Conclusions:

• A highly adaptable nCas9-centric strategy effectively enhances base editor efficiency.
• This approach optimizes various base editors and other Cas9-derived genome editing tools.
• The engineered variants show strong translational potential for therapeutic applications.