Homologous knock-in processes facilitated by CRISPR-Cas9 in Aurantiochytrium and disrupting the gene for DNA ligase IV prevents transformation events

Abstract This study explores advanced molecular breeding techniques for Aurantiochytrium limacinum , a eukaryotic microorganism valued for its industrial production of DHA and astaxanthin. We first employed the CRISPR-Cas9 system to simultaneously disrupt two genes: crtIBY , a multifunctional carotenoid synthesis gene used as a visual marker, and lig4 , which is involved in non-homologous end joining (NHEJ). This genetic disruption caused the wild-type orange colonies to turn into a white colony (Δ lig4 -Δ crtIBY -TA#6), indicating the loss of carotenoid production. Subsequently, we successfully demonstrated marker recycling by repairing the crtIBY gene using single-strand oligodeoxynucleotide (ssODN) templates, which restored the orange phenotype (Δ lig4 -TB#18). Further investigation into the Δ lig4 strain (TB#18) revealed that its overall transformation efficiency dropped significantly compared to that of the wild-type strain when using only a bleomycin resistance expression cassette with two crtIBY homologous regions. However, when we combined the bleomycin resistance expression cassette with CRISPR-Cas9 ribonucleoproteins (RNPs) during electroporation, the Δ lig4 strains (TA#6 and TB#18) exhibited higher homologous recombination-type efficiencies through double-crossover-type events than the wild-type. These results indicate that while Lig4 is important for general transformation against zeocin, disrupting the NHEJ pathway enhances precise gene targeting. This study demonstrated the potential of these CRISPR-Cas9 systems to serve as a robust foundation for the functional genomics and metabolic engineering of A. limacinum .