Mitra, K.; Holmberg, S. R.; Mota, M.; Sabharwal, A.; Ekker, S. C.

The rapid advancement of nuclear and mitochondrial genomic editing tools has created an urgent need for efficient, non-lethal larval genotyping methods in zebrafish Danio rerio research. This study optimizes and validates a non-destructive proteinase K digestion method for mitochondrial DNA genotyping while characterizing its impact on larval survival and gene expression. Using optimized protocol parameters, we demonstrate successful amplification of different mitochondrial genetic loci with consistently high sensitivity. Molecular validation through PCR, restriction fragment length polymorphism analysis, and Sanger sequencing confirmed the specificity and reliability of the extracted DNA, while RNA sequencing analysis revealed no significant transcriptional differences between genotyped and unshaken control larvae, indicating minimal physiological impact. The method successfully detected C to T base edits in the mt-tl1 gene introduced using the FusX TALE Base editor system, demonstrating its applicability to gene editing studies. Both 48-well and optimized 96-well formats were used, enabling this approach to be deployed at scale. It is particularly valuable for investigating early-onset mitochondrial diseases and utilizes standard laboratory equipment and reagents, facilitating widespread adoption in zebrafish research while adhering to ethical principles in reducing animal mortality.