First Report of CRISPR-Cas9 Ribonucleoprotein Delivery Into Teleost Spermatozoa With Preserved Membrane and Genomic...

Area of Science:

• Reproductive Biotechnology
• Molecular Biology
• Aquaculture

Background:

• Delivering large molecular complexes into sperm, especially teleost sperm with compacted chromatin and small cytoplasm, is difficult.
• Existing methods face challenges in maintaining sperm integrity and functionality.

Purpose of the Study:

• To assess the efficacy of capillary electroporation for delivering CRISPR-Cas9 ribonucleoprotein (RNP) complexes into Nile tilapia spermatozoa.
• To evaluate the impact of electroporation on sperm viability, motility, and DNA integrity.

Main Methods:

• CRISPR-Cas9 RNP complexes were introduced into Nile tilapia sperm using capillary electroporation.
• RNP uptake was measured by EGFP fluorescence.
• Sperm performance was analyzed via motility, membrane integrity, mitochondrial activity, and DNA fragmentation assays.

Main Results:

• Approximately 10%-13% of spermatozoa showed detectable EGFP signal after electroporation.
• A single pulse (1250 V, 40 ms) optimized RNP uptake while preserving sperm function.
• Increased pulse numbers negatively affected motility and mitochondrial activity but not membrane integrity or DNA fragmentation.

Conclusions:

• Capillary electroporation is a viable and minimally disruptive method for delivering CRISPR-RNP complexes into teleost sperm.
• This technique holds promise for advancing genetic applications in fish reproductive biology.