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)%. Furthermore, we incorporated polyglutamic acid (PGA) to modify the RNP complexes (target tyr), which inhibited aggregation and enhanced editing efficiency to (44.45 ± 1.41)%, outperforming a commercial. electroporation system, while targeting the pigmentation-related gene slc24a5 yielded an albinism or hypopigmentation rate of (38.33 ± 2.62)%. In addition, targeting the development-associated gene ddx19b produced developmental defect phenotypes in (38.33 ± 1.88)% of embryos. The successful introduction of indels at the target site was confirmed by sequencing. Our work establishes a highly effective electroporation strategy, augmented by nanotechnology, for the delivery of mRNA and RNP complexes, enabling high-efficiency protein expression and gene editing in zebrafish embryos, with broad potential applications in aquatic biotechnology.