Genotyping strategy and results for F0 embryos after CRISPR‐Cas9 gene editing. (A) Scheme of the tgds gene and strategy used for detecting the generated large deletions. The figure shows the gene organization with exons and the specific exons targeted by the guide RNAs (sgRNAs), which are indicated in red. The diagram highlights the different primer combinations used in PCR to amplify DNA fragments only if a large deletion occurred between the targeted exons. For example, primers spanning exons 4 and 12 would only produce a smaller amplicon if the intervening exons (5–11) were deleted. This strategy allows the detection of specific deletion events (Del4‐12, Del4‐8, and Del8‐12). (B) Representative genotyping results from nine embryos injected with the target sgRNAs, following the strategy outlined in panel A. The observed amplicon sizes agree with the expected size. Specifically, embryos #1, #3, and #5 presented both Del4‐12 and Del4‐8. Embryo #4 included Del4‐12 and Del8‐12. Notably, embryo #9 displayed all three deletion types. The control C, which was injected with a scrambled sgRNA, and the no‐template control, noT, showed no amplification. (C) Frequencies of deletions in 40 genotyped embryos obtained across three independent microinjections. Approximately 80% of the embryos presented at least one deletion. Del8‐12 was never found as an isolated deletion. Del4‐12 and Del4‐8, either isolated or in combination, accounted for most cases.
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