Development of a CRISPRi system in Fusarium fujikuroi and its application in gibberellic acid production

Gene knockdown is a pivotal genetic manipulation technique, particularly when targeting lethal genes or genes involved in product synthesis pathways, where complete gene knockout is not a viable option. This approach is particularly valuable in multinucleate species, such as Fusarium fujikuroi , where generating homogeneous gene knockouts is notoriously difficult. To address these limitations, we first screened a set of repression domains, and then leveraged the optimal candidates to construct a CRISPR/dCas9-mediated knockdown platform for F. fujikuroi . By targeting erg9 , which encodes squalene synthase, the first committed enzyme in the mevalonate pathway for ergosterol biosynthesis, we successfully diverted a portion of the metabolic flux from sterol production to gibberellic acid (GA) biosynthesis. This strategy minimizes carbon loss to competing pathways while retaining phenotypically normal growth. Additionally, CRISPR/dCas9-mediated knockdown of the dehydrogenase gene des enhanced GA 4 production by 2.62-fold and eliminated the intermediate GA 7 , generating a GA 3+4 -producing strain and fine-tuning its metabolic profile. Using our CRISPRi system, we achieved a 70–89% reduction in erg9 mRNA levels and a 67– 84% reduction in des mRNA levels. Our findings establish a tailored CRISPRi platform for effective gene repression in F. fujikuroi .