Towards optimizing diversifying base editors for high-throughput studies of single- nucleotide variants

Area of Science:

• Genetics
• Molecular Biology
• Bioengineering

Background:

• Single nucleotide variants (SNVs) impact cellular function and disease.
• Base editors (BEs) are tools for creating specific point mutations to study SNVs.
• Understanding how BE design affects editing efficiency is crucial for their application.

Purpose of the Study:

• To investigate how different design features of base editors influence their editing efficiency and outcomes.
• To compare the performance of various base editor configurations, including different deaminase fusions and delivery methods.
• To identify optimal base editor designs for generating diverse mutations for functional genomics studies.

Main Methods:

• Assessed editing efficiency across ~200 synthetic target sites using various base editor designs.
• Compared N-terminal versus C-terminal fusion of a hyperactive deaminase (DivA-BE) to dCas9.
• Evaluated delivery methods including electroporation and lentiviral transduction.
• Analyzed the types of mutations generated (C>N and G>N) based on editor architecture.

Main Results:

• Direct fusion of hyperactive deaminase to the N-terminus of dCas9 (DivA-BE) yielded the highest editing efficiency, ~4-fold greater than CRISPR-X.
• DivA-BE created G>N mutations by targeting the DNA strand annealing to the sgRNA, unlike C-terminal fusions.
• Base editor design significantly impacts editing outcomes, including mutation type and efficiency.
• The study provides a comprehensive analysis of base editor design features and their influence on activity.

Conclusions:

• DivA-BE represents a highly efficient base editor for installing point mutations.
• The N-terminal fusion strategy and deaminase choice are critical for maximizing editing efficiency and diversifying mutation types.
• These findings offer valuable insights for selecting and engineering base editors for genetic studies and variant discovery.