CATHEPSIN L GENE EDITION IN Penaeus vannamei ZYGOTES

Aquaculture is a growing sector where genetic edition has begun to gain ground. A variety of aquaculture species have been successfully genome-edited via CRISPR/Cas9 system. However, so far transfection methods have been low-throughput techniques, which requires special equipment and demands technical skills. Additionally, in penaeid shrimps, a major group in the aquaculture industry, genetic edition has not been reported (Gratacap et al., 2019). Thus, our aim was tested the efficacy of two transfection reagents, based in cationic molecules, combined with three gene editing cargoes (Table1) in  P. vannamei zygotes; to achieve genetic edition in the cathepsin L gene, a well-described and highly expressed cysteine peptidase, which participates in multiple physiological processes (Le Boulay et al., 1998; Hui et al., 2008).

Three RNA guides were designed to target the catalytic sites of the enzyme. However, just one was functional in vitro and was used for downstream experiments. We collected freshly spawned eggs from one female in a 24 wells plate, the zygotes were incubated with each Cas9 cargo molecule, at different concentrations, and the RNA guide. For Cas9 nucleic acids (DNA or RNA) both transfection reagents were tested, when Cas9 was delivery as protein, in a ribonucleic complex with the RNA guide, only the cationic lipids were used. After hatching, nauplii DNA was extracted by salt precipitation technique and the intended genomic region was analyzed by High-Resolution Melting analysis (HRM), PCR cloning and Sanger sequencing. 

Our results showed that treatments with the higher concentrations of Cas9 cargo molecule resulted in evident variations in the HRM profiles compared to wildtype. In Sanger sequencing variations in the chromatogram was observed in four different treatments. A point mutation within the target site was observed in three different treatments , a substitution from A to G, which results in a non-conservative amino-acid change Lys136Glu in the protein, closed to the active site Cys138. Additionally, mutations in the intron region, 30 to 40 bp downstream the guide, were observed.  This is the first approach to applied gene edition CRISPR/Cas9 technology in a penaeid shrimp, we demonstrated the feasibility for genomic manipulation of multiple zygotes at one time without losing viability of the embryos. Our results could accelerate functional research of biological features relevant for this important species.