Establishment of CRISPR-Cas9-Mediated Gene Editing in the Swimming Crab Portunus trituberculatus

<i>Portunus trituberculatus</i> is an economically important marine crustacean in East Asia's aquaculture industry. Nevertheless, precise genome modification has not yet been established. In this study, we evaluated the applicability of the CRISPR-Cas9 gene editing system in <i>P. trituberculatus</i> using electroporation for efficient delivery of the Cas9-sgRNA complex into zygotes. We systematically investigated electroporation parameters, including buffer composition, voltage, capacitance, and pulse times. Our results showed that artificial seawater was a superior buffer to phosphate-buffered saline (PBS) and identified an effective electroporation condition of 600 V, 1 μF capacitance, and two pulses, resulting in approximately 72.7% fluorescent zygotes. Under these electroporated conditions, we detected gene indels and putative insertion events at the targeted locus of myostatin (<i>mstn</i>) gene. These results demonstrate the feasibility of Cas9-based genome editing in <i>P. trituberculatus</i> and provide a proof-of-concept for functional genomics studies and future genetic improvement of this species.