KNIT editing enables precise DNA insertion without double-strand breaks
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Researchers report a new gene-editing method called KNIT that achieves up to 89% efficiency for inserting DNA fragments up to 10 kb without causing double-strand breaks. The technique reduces unintended mutations and translocations, and was used to engineer CAR-T cells with effective antitumour activity in mice.
The Method
CRISPR kilobase-scale nickase-targeting (KNIT) editing couples a Cas9 nickase with a DNA donor recruiting system to insert fragments from 0.7 kb to over 10 kb. It works across multiple genomic loci and cell types, achieving up to 89% efficiency while markedly reducing indel rates and off-target editing. The enhanced KNIT editor 2 further improves efficiency via a single transfection.
Therapeutic Application
In mutant cells with a pathological mutation, KNIT restored normal gene expression by inserting a therapeutic gene into a safe harbour or native locus. The method enabled non-viral, programmable CAR-T cell engineering without double-strand breaks, achieving clinically relevant efficiencies. Engineered CAR-T cells showed effective antitumour activity in vitro and in mouse models.
What's Next
The researchers plan to further develop KNIT for in vivo applications and explore its potential in personalized medicine. It remains unclear whether the method can be scaled for clinical use and how it compares to other emerging gene-editing technologies.
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KNIT editing enables precise DNA insertion without double-strand breaks
