Efficient C-to-G Base Editing with Improved Target Compatibility Using Engineered Deaminase-nCas9 Fusions

Area of Science:

• Molecular Biology
• Genome Editing Technologies
• Biotechnology

Background:

• CRISPR-guided DNA base editors (BEs) are powerful tools for genome modification.
• Conventional BEs are limited to base transitions (C-to-T, A-to-G), lacking the ability to induce base transversions.
• The development of C-to-G base editors (CGBEs) is crucial for expanding genome editing capabilities.

Purpose of the Study:

• To develop and characterize novel C-to-G base editors (CGBEs) for efficient induction of C-to-G base transversions.
• To investigate the factors influencing CGBE efficiency, particularly the role of engineered deaminases.
• To expand the genome-targeting scope of CGBEs for broader applications in gene modification.

Main Methods:

• Comparison of different CGBE designs, focusing on engineered deaminases versus base excision repair proteins.
• Rational engineering of eAID deaminase to enhance C-to-G transversion efficiency.
• Utilizing the SpRY Cas9 variant to broaden the targeting range of CGBEs.
• Application of CGBEs in human cells and mouse embryos for gene disruption.

Main Results:

• Engineered deaminases significantly improved C-to-G base editing efficiency compared to additional base excision repair proteins.
• Rationally engineered eAID deaminase demonstrated increased C-to-G transversion efficiency, especially in the GC context.
• The use of SpRY Cas9 expanded the genome-targeting scope of CGBEs.
• Successful induction of a stop codon (TAC to TAG) in the Tyr gene in mouse embryos using CGBEs.

Conclusions:

• Novel C-to-G base editors (CGBEs) with engineered deaminase-nCas9 fusions represent a significant advancement in base editing technology.
• These CGBEs overcome the limitations of conventional base editors by enabling C-to-G transversions.
• The enhanced efficiency and expanded targeting scope of these new CGBEs broaden the toolset for precise gene modification and hold promise for therapeutic applications.