Enhancing CRISPR/Cas-Mediated Gene Knockout With Short Non-Homologous Oligonucleotides.
Chew YP, Ferenczi A, Dannay M, Ponce-Lilly C, Kovac A
Crispr
Algae engineered with sharper gene-editing tools could soon produce the sustainable biofuels and plant-based compounds that reduce how much petroleum goes into your fertilizer bag and how far your food travels.
When scientists edit plant-like algae using molecular scissors (CRISPR), the cells usually fix the cut too cleanly, leaving the target gene intact. Researchers found that adding tiny snippets of synthetic DNA confuses the cell's repair crew, causing it to patch the cut sloppily and permanently disabling the gene — boosting success rates up to 100-fold. This trick works across different genes and algae strains, opening a faster path to engineering algae for medicines, fuels, and other useful compounds.
Key Findings
Co-delivering short double-stranded non-homologous DNA fragments with CRISPR reagents increased gene knockout efficiency up to 100-fold in Chlamydomonas reinhardtii.
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.
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.
chevron_right Technical Summary
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.
Abstract Preview
Chlamydomonas reinhardtii is a model green microalga that has great industrial potential as a sustainable bio-factory for recombinant protein and high-value chemical production. Efficient genome ed...
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