First Report of CRISPR‐Cas9 Ribonucleoprotein Delivery Into Teleost Spermatozoa With Preserved Membrane and Genomic Integrity

Delivery of large molecular complexes into spermatozoa remains a challenge in reproductive biotechnology. Teleost sperm possess highly compacted chromatin and minimal cytoplasmic volume, limiting intracellular access of ribonucleoprotein assemblies. Here, we evaluated whether CRISPR-Cas9 ribonucleoprotein (RNP) complexes can be introduced into Nile tilapia spermatozoa using capillary electroporation while preserving cellular integrity. RNP uptake was quantified via EGFP fluorescence, and sperm performance was assessed through motility, membrane integrity, mitochondrial activity, and DNA fragmentation analyses. Approximately 10%-13% of spermatozoa exhibited detectable EGFP signal following electroporation. Increased pulse numbers reduced motility and mitochondrial activity, whereas membrane integrity was preserved and no increase in DNA fragmentation was detected. A single pulse (1250 V, 40 ms) provided the best balance between RNP uptake and functional preservation. These findings support capillary electroporation as a minimally disruptive strategy for CRISPR-RNP delivery in teleost reproductive biology.