A Reproducible Electroporation Strategy for CRISPR-Cas9 RNP and Mrna Delivery in Fish Embryos.

This study presents a streamlined electroporation-based method for efficient macromolecular delivery into zebrafish embryos. We first characterized the physical barrier posed by the chorion using TEM (Transmission Electron Microscope) and established that its removal is prerequisite for effective delivery. A systematic optimization of electroporation parameters for dechorionated embryos identified optimal conditions (e.g., poring pulse: 25 V, 20 ms; transfer pulse: 5 V, 50 ms) that ensured high embryo survival and robust eGFP mRNA transfection. Applying this protocol, we achieved targeted gene knockout by electroporation-mediated delivery of Cas9 ribonucleoproteins (RNPs) against multiple loci. Targeting the tyr locus resulted in a phenotypic albinism rate of (38.6 ± 3.30)