Generation of lrpap1 knockout zebrafish. A Representative image of a zebrafish embryo (72 hpf) subjected to whole-mount in situ hybridization targeting lrpap1. B The gRNA target and its corresponding position in the lrpap1 gene sequence are shown. C Agarose gel electrophoresis of the lrpap1 DNA amplified fragments. D DNA sequencing results of wild-type, heterozygous, and homozygous zebrafish; the 5 bp deletion/4 bp insertion mutation (c.262_266delinsTCTC) is highlighted by the black arrows. The mutation was predicted to cause a frameshift with premature translation termination, resulting in an abnormal protein (p.Lys35Asnfs*3). E lrpap1 mRNA expression was slightly decreased in the lrpap mutant lines, although this was not statistically significant (p = 0.631). F The protein structure of wild-type and mutant Lrpap1, as predicted by Phyre2. Image colored using the rainbow colors, from the N to C terminus. G Western blot analysis of the levels of Lrpap1 in the eyes of lrpap1 mutant and wild-type zebrafish two months and three months post-fertilization. H Relative quantitative result of Lrpap1 protein revealed that the Lrpap1 protein levels were markedly lower in mutant zebrafish than in the wild-type. n = 10 for wild-type zebrafish and n = 10 for mutants. Statistical significance was determined using the Student’s t-test. ****p < 0.0001. β-Actin was used as the internal control. WT, wild-type; MU, lrpap1 homozygous mutant. hpf, hours post-fertilization; 2 m, two months; 3 m, three months
