Reduced off-target effect of NG-BE4max by using NG-HiFi system

Area of Science:

• Genetics and Genomics
• Molecular Biology
• Biotechnology

Background:

• SpCas9-NG variants expand genome editing targeting scope by recognizing a minimal NG protospacer adjacent motif (PAM).
• Previous studies and our findings indicate SpCas9-NG can lead to increased off-target mutations and lower on-target efficiency compared to standard SpCas9.
• The NG-BE4max base editor showed limitations, including reduced base editing frequencies and elevated off-target mutations in Hoxc13-ablated rabbits.

Purpose of the Study:

• To develop and evaluate a high-fidelity SpCas9-NG base editor (NG-HiFi) with improved specificity.
• To compare the efficacy and specificity of NG-HiFi against NG-BE4max in a rabbit disease model.
• To assess the potential of NG-HiFi for precise genome editing in modeling human diseases.

Main Methods:

• Engineering of a high-fidelity SpCas9-NG base editor (NG-HiFi).
• Application of NG-HiFi and NG-BE4max for Hoxc13 gene ablation in rabbits.
• Assessment of on-target editing efficiency and genome-wide off-target mutations.

Main Results:

• NG-HiFi demonstrated retained on-target activity compared to NG-BE4max.
• NG-HiFi exhibited significantly decreased off-target activity in Hoxc13-ablated rabbits.
• The SpCas9-NG system, when utilized with NG-HiFi, showed enhanced specificity and reduced off-target effects.

Conclusions:

• The NG-HiFi base editor offers improved specificity and reduced off-target mutations for SpCas9-NG-assisted cytidine base editing.
• NG-HiFi represents a promising tool for precise genome editing in rabbits, facilitating accurate modeling of human diseases.
• Enhanced specificity of SpCas9-NG with NG-HiFi overcomes previous limitations, enabling more reliable genetic manipulations.