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Enhancing CRISPR/Cas-Mediated Gene Knockout With Short Non-Homologous Oligonucleotides.

PubMed · 2026-02-22

Scientists discovered a way to make CRISPR gene editing up to 100 times more effective in green algae by adding short synthetic DNA fragments that trick the cell into using a sloppier repair pathway, dramatically improving the ability to knock out genes.

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Co-delivering short double-stranded non-homologous DNA fragments with CRISPR reagents increased gene knockout efficiency up to 100-fold in Chlamydomonas reinhardtii.

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The enhancement effect is mediated primarily through the KU70/80 protein complex — a key sensor of DNA double-strand breaks — and works independently of the specific gene target, Cas nuclease used, or algae strain.

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The synthetic DNA fragments shift cellular DNA repair from precise non-homologous end joining (NHEJ) toward error-prone microhomology-mediated end joining (MMEJ), with effectiveness depending on fragment length, structure, and chemical modifications.

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