A CRISPR-Cas9 System-Mediated Genetic Disruption and Multi-fragment Assembly in .

Gene editing technology plays an extremely significant role in synthetic biology and metabolic engineering. Traditional genetic manipulation methods, such as homologous recombination, however, are inefficient, time-consuming, and barely feasible when disrupting multiple genes simultaneously. , a nonconventional yeast that overproduces sophorolipids, lacks convenient genetic tools for engineering strains. Here, we developed an efficient CRISPR-Cas9 genome editing technology by combining molecular element mining and expression system optimization for . This CRISPR-Cas9 system improved the efficiency of gene-integration/target gene-introducing disruption by homology-directed repair and realized the multi-gene simultaneous disruptions. Based on this CRISPR-Cas9 system, we also further constructed an engineered strain via the assembly of multiple DNA fragments (10 kb) that can produce acid-type sophorolipids. These results showed that the CRISPR-Cas9 system may be an efficient and convenient strategy to perform genetic manipulation in .