APPLICATION OF THE CRISPRA TECHNOLOGY WITH ASGARD IN CHLORELLA SOROKINIANA FZU60

To establish an efficient genetic transformation system for investigating molecular regulatory mechanisms and to increase the yield of its high-value-added metabolites in Chlorella sorokiniana FZU60, a CRISPR/dCas9-based transcriptional activation system (CRISPRa) was developed and combined with Adaptive Single-guide Assistant Regulatory DNA (ASGARD) to enable random gene activation. Resistance screening indicated that 600 μg/mL hygromycin B effectively inhibited algal cell growth. The constructed plasmid pLWR-dzCas9-VP64::sgHG, which carries a sgRNA with high guanine content, was introduced into algal cells via electroporation. After multiple rounds of screening on hygromycin B-resistant plates, transformed algal strains with stable resistance and rapid growth were obtained. The PCR and RT-qPCR verification results showed that the plasmid had been successfully transferred and expressed. Further analysis revealed that all transformed algal strains achieved higher biomass concentration than the wild type strain under heterotrophic conditions. The content and production of protein, lipid, and pigment of the transformed algal strain were significantly increased. Among them, the protein content and production of the transformed algal strain V-6 reached 452.83 mg/g and 2384.32 mg/L, representing increases of 17.97% and 32.82% over the wild type strain, respectively; lipid content and production were as high as 169.38 mg/g and 891.56 mg/L, which were 23.20% and 38.97% higher than those of the wild type respectively. Chlorophyll content remained similar to that of the wild type, whereas chlorophyll production increased by 18.44%; carotenoid content showed no significant difference from the wild type, while carotenoid production rose by 13.57%. Therefore, the transformed algal strain obtained in this study exhibits excellent capabilities for producing protein, lipid, and pigment. These findings provide technical support for establishing gene-editing techniques in C. sorokiniana FZU60 and offer a crucial theoretical basis for enhancing the production of high-value metabolites from this alga.