Efficient and precise programmable DNA knock-in without double-strand breaks

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Genomic Engineering

Background:

• Precise kilobase-scale DNA integration is crucial for genetic disease treatment and cell therapy development.
• Existing gene editing methods face challenges in achieving efficient and accurate large DNA fragment insertion.

Purpose of the Study:

• To develop a novel gene editing system for efficient and precise kilobase-scale DNA insertion without double-strand DNA cleavage.
• To demonstrate the versatility and safety of this new system across various genomic loci and cell types.

Main Methods:

• CRISPR kilobase-scale nickase-targeting (KNIT) editing, coupling a Cas9 nickase with a DNA donor recruiting system.
• Testing KNIT editing for DNA fragments ranging from 0.7 kb to over 10 kb in diverse cell types and genomic locations.
• Evaluating KNIT editor 2 for enhanced efficiency and single transfection capabilities.
• Assessing the system's performance in restoring gene expression in mutant cells and engineering chimeric antigen receptor T cells (CAR-T cells).

Main Results:

• KNIT editing achieved efficient and programmable integration of large DNA fragments (0.7-10+ kb) with up to 89% efficiency.
• The system significantly reduced unintended insertion-deletion mutation (indel) rates, translocations, and off-target editing.
• KNIT editing enabled precise gene insertion for therapeutic purposes and facilitated non-viral CAR-T cell engineering with clinically relevant efficiencies.
• Engineered CAR-T cells demonstrated effective in vitro and in vivo anti-tumor activity.

Conclusions:

• KNIT editing provides a versatile and efficient platform for programmable, site-specific kilobase-scale DNA insertions without double-strand breaks.
• This technology minimizes unintended genetic modifications, paving the way for safer and more effective gene therapies and cell-based treatments.
• KNIT editing advances personalized medicine by enabling precise genomic engineering for therapeutic applications.