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

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Genomics

Background:

• Precise kilobase-scale DNA integration is crucial for gene therapy and cell engineering but remains technically challenging.
• Existing gene editing methods often involve double-strand DNA breaks, leading to unwanted mutations and rearrangements.

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 was employed, coupling a Cas9 nickase with a DNA donor recruiting system.
• The system was tested for DNA fragment integration ranging from 0.7 kb to over 10 kb in different cell types and genomic locations.
• Enhanced versions (KNIT editor 2) and applications in therapeutic gene insertion and CAR-T cell engineering were evaluated.

Main Results:

• KNIT editing achieved high efficiency (up to 89%) for kilobase-scale DNA insertions without double-strand breaks.
• The method significantly reduced unintended insertion-deletion mutations (indels), translocations, and off-target editing compared to conventional methods.
• KNIT editing successfully restored normal gene expression in mutant cells and enabled efficient, non-viral CAR-T cell engineering with demonstrated anti-tumor activity.

Conclusions:

• KNIT editing offers a versatile and precise platform for programmable, site-specific kilobase-scale DNA insertions.
• The system minimizes unintended genetic alterations, making it a promising tool for advancing personalized medicine, gene therapies, and cell-based therapeutics.
• KNIT editing facilitates efficient engineering of CAR-T cells without double-strand breaks, paving the way for safer and more effective cell therapies.