Development of a PEI-coated SWNTs Nanocarrier for efficient delivery of CRISPR/Cas9 in early embryos of Litopenaeus vannamei

Cultivation of superior varieties is the key to maintaining the sustainable development of the Litopenaeus van-namei (L.vannamei) industry. CRISPR/Cas9 technology represents a generation of genetic breeding technology based on gene editing. However, the conventional delivery strategy of CRISPR/Cas9 components could not be used due to the particular physiological traits and practical difficulties of L. vannamei embryos. We designed and established polyethylenimine (PEI)-coated nanoparticles with carboxylated SNWTs core to safely deliver CRISPR/Cas9 plasmids into early embryos for target gene editing. The results showed that the transfection ef-ficiency of this strategy was 36%, which was approximately 4-fold higher than the efficiency of the classical lipid transfection method. The transcription factor Pax6, which has notable effects on early embryonic eye develop-ment, provides clear phenotypic proof for this strategy. Unnatural base alterations were found in up to 20% of transfected embryos. This study establishes a foundation for the application of CRISPR technology in L. vannamei and provides an innovative approach for large-scale gene function studies in aquaculture.