Precise, specific gene editing via a compact GoCas12m-FokI chimeric nuclease

Area of Science:

• Biotechnology
• Genomics
• Molecular Biology

Background:

• CRISPR gene editing advanced functional genomics but faces challenges in delivery and specificity.
• Therapeutic translation of gene editing is hindered by limited delivery capacity and off-target mutations.

Purpose of the Study:

• To develop a novel, compact, and highly specific gene editing platform for therapeutic applications.
• To engineer a chimeric system combining a guiding module with a nuclease for precise genome modification.

Main Methods:

• Fused a catalytically inactive Cas12m guiding module (GoCas12m) with the FokI nuclease domain to create the GoCas12m-FokI editor.
• Tested the editor's activity on surrogate reporters and endogenous human loci (CLTA1, HBB, AIFM1, ABL).
• Confirmed specificity using targeted deep sequencing to detect off-target activity.

Main Results:

• The GoCas12m-FokI editor demonstrated robust and high-efficiency editing at clinically relevant targets.
• No detectable off-target activity was observed at in silico-predicted sites.
• The editor's compact size (nearly half of Cas9/Cas12a) facilitates AAV-mediated delivery.

Conclusions:

• The GoCas12m-FokI system provides a precise and specific genome editing tool with a compact architecture.
• This editor is a promising alternative for therapeutic genome editing and in vivo gene therapy.
• The modularity and specificity of GoCas12m-FokI enhance its potential for clinical translation.