The Hurdles Of Delivery Crispr-Cas9 Components For Gene Editing In Penaeid Shrimps

Gene edition using the CRISPR/Cas9 system has been achieved in few crustaceans, but in penaeid shrimps, several hurdles exist, restraining the potential of CRISPR-Cas9 technology in shrimp Aquaculture. A key step for gene editing is the efficient introduction of the CRISPR-Cas9 system into the cells. Although microinjection is an effective method for CRISPR-Cas9 system delivery into zygotes, the successful gene edition in this commercially important taxon is hindered due to the challenges posed by this technique. Therefore, experimental approaches based on microinjection-free protocols are of scientific interest for Aquaculture researchers. In this work, we performed physical (electroporation) and chemical (polyethylenimine and cationic lipids) transfection methods in P. vannamei zygotes. For Cas9 delivery, we used three different cargoes: DNA plasmid, mRNA and a recombinant protein. Different ratios of sgRNA designed to recognize the PvCatL gene were used to prepare the CRISPR-Cas9 complexes. Treated shrimp zygotes were genotyped by HRM analysis and Sanger sequencing. Although high hatching rates were observed for most treatments, no irrefutable evidence of typical CRISPR-Cas9-induced gene edition was found; instead, an enrichment of gene variants was observed in treated organisms, which was detectable by HRM. Our effort is of interest to Aquaculture researchers working on this challenging topic, helping to improve their experimental design or as a reference to evaluate additional conditions to achieve the gene editing in penaeid shrimps.