Enhancing <scp>CRISPR</scp> /Cas‐Mediated Gene Knockout With Short Non‐Homologous Oligonucleotides

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 editing tools are required to redesign this organism for synthetic biology applications. CRISPR-Cas editing technologies have already been adapted for gene knockout, transgene knock-in, and precise gene editing in C. reinhardtii. However, the efficacy of CRISPR/Cas-mediated gene knockout (KO) is low, which hampers pathway engineering and functional genomic studies. Here we report that co-delivery of CRISPR-Cas gene editing reagents with short double-stranded non-homologous oligodeoxynucleotides (dsNHO) increases gene knockout efficacy up to 100-fold in C. reinhardtii. This phenomenon, referred to as non-homologous oligonucleotide enhancement (NOE), is heavily affected by the length, structure, and chemical modifications of dsNHO, and is largely mediated by the DNA double-stranded break sensor KU70/80 (KU) heterodimer in a Cas nuclease-, locus-, and strain-independent manner. Our data suggest that dsNHOs disrupt the cell's double-stranded break (DSB) sensing pathways, consequently shifting the balance of DNA repair from canonical non-homologous end joining (c-NHEJ) towards the more error-prone, microhomology-mediated end joining (MMEJ), which could be harnessed as a strategy for improving gene KO efficiency in Chlamydomonas and beyond.