Highly efficient genome editing using CRISPR/Cas9 ribonucleoprotein in the marine oleaginous diatom Fistulifera solaris

Microalgae are promising hosts for sustainable aviation fuel production due to their rapid growth and their advantage of not competing with agricultural crops. The oleaginous diatom Fistulifera solaris is notable for its exceptionally high lipid content. However, genetic engineering tools for this species remain limited, partly due to its allodiploid genome structure. Here, we report the successful development of a CRISPR/Cas9 genome editing method using ribonucleoprotein (RNP) complexes in F. solaris. We first targeted the adenine phosphoribosyl transferase (apt) genes as a selectable marker. Delivery of RNPs via particle bombardment to conserved regions of the two homoeologous apt genes resulted in 87% biallelic editing efficiency. Next, we demonstrated multiplex genome editing by co-targeting the apt genes and diadinoxanthin de-epoxidase (dde) genes as representative targets for improving valuable compound production in diatoms. Among 27 co-edited clones, 85% showed mutations in at least one dde homoeolog, and 63% exhibited biallelic mutations in both genes. This study demonstrates the applicability of RNP-mediated genome editing in diatoms. Furthermore, the success of this method suggests its broader applicability to non-model diatom species, providing a valuable tool for genome engineering in diatoms.