Sec24C CRISPR-edited zebrafish larvae present with lens damage, motility impairment, and smaller cerebellum. (A) Zebrafish sec24C gene overview and localization of single guide RNAs a and b (sgRNA a and sgRNA b), targeting exons 2 and 10, respectively. (B) In situ images of lenses in wild-type, sgRNA a sec24C(–), and sgRNA b sec24C(–) zebrafish larvae at 4 dpf showing lens artifacts. Arrowheads point to droplet-like crystal inclusions in insets. Deep ridges are present along lens circumference in CRISPR-edited lenses. (C) Quantification of lens artifacts in single eye per analyzed animal. Graph represents number of artifacts identified per lens/animal sorted by WT (n = 13), sgRNA a (n = 22), WT (n = 9), and sgRNA b (n = 16). (D) Movement tracks of WT, sec24C(–), sec24C(–) sec24D(–), and sec24D(–) zebrafish larvae at 5 dpf. Each colored line represents an individual animal track (n = 10 per group). (E) Quantification of maximum velocity reached during recorded travel, and (F) total distance traveled by each larva. (G) Whole-mount immunostaining of craniofacial muscles at 5 dpf in WT, sec24C(–), sec24C(–) sec24D(–), and sec24D(–). Scale bar: 100 μm. ima, intermandibularis anterior; imp, intermandibularis posterior; ih, interhyoideus; hh, hyohyoideus; sh, sternohyoideus; h, heart muscle. (H) Quantification of craniofacial muscles area in WT (n = 7), sec24C(–) (n = 6), sec24C(–) sec24D(–) (n = 7), and sec24D(–) (n = 6). (I) Whole-mount immunostaining (5 dpf) for acetylated tubulin (axonal tracks), maximum-intensity projection confocal image of a wild-type cerebellum. Double-headed arrow (yellow) marks cerebellum width. Scale bar: 50 μm. EG, eminentia granularis; LCa, lobus caudalis cerebelli; CCe, corpus cerebelli. (J) Graph of cerebellum width in WT (n = 13), sec24C(–) (n = 13), sec24C(–) sec24D(–) (n = 16), and sec24D(–) (n = 12). Statistics: Graphs show mean ± SEM. (C, H, and J) Student’s t test. (E and F) Mann-Whitney U test. Significance thresholds: *P < 0.05 or Bonferroni-corrected threshold (E, F, H, and J; see Methods), **P < 0.01, ***P < 0.001, ****P < 0.001.
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