The CRISPR/Cas9 complex, which is composed of a guide RNA (gRNA) and the Cas9 nuclease, is useful for carrying out genome modifications in various organisms. Recently, the CRISPR/Cas9-mediated locus-specific integration of a reporter, which contains the Mbait sequence targeted using Mbait-gRNA, the hsp70 promoter and the eGFP gene, has allowed the visualization of the target gene expression. However, it has not been ascertained whether the reporter integrations at both targeted alleles cause loss-of-function phenotypes in zebrafish. In this study, we have inserted the Mbait-hs-eGFP reporter into the pax2a gene because the disruption of pax2a causes the loss of the midbrain-hindbrain boundary (MHB) in zebrafish. In the heterozygous Tg[pax2a-hs:eGFP] embryos, MHB formed normally and the eGFP expression recapitulated the endogenous pax2a expression, including the MHB. We observed the loss of the MHB in homozygous Tg[pax2a-hs:eGFP] embryos. Furthermore, we succeeded in integrating the Mbait-hs-eGFP reporter into an uncharacterized gene epdr1. The eGFP expression in heterozygous Tg[epdr1-hs:eGFP] embryos overlapped the epdr1 expression, whereas the distribution of eGFP-positive cells was disorganized in the MHB of homozygous Tg[epdr1-hs:eGFP] embryos. We propose that the locus-specific integration of the Mbait-hs-eGFP reporter is a powerful method to investigate both gene expression profiles and loss-of-function phenotypes.

Krüpple-like factors (Klfs) are highly conserved zinc-finger transcription factors that regulate various developmental processes, such as haematopoiesis and cardiovascular development. In zebrafish, transient knockdown analysis of biklf/klf17 using antisense morpholino suggests the involvement of biklf/klf17 in primitive erythropoiesis and hatching gland development; however, the continuous physiological importance of klf17 remains uncharacterized under the genetic ablation of the klf17 gene among vertebrates. We established the klf17-disrupted zebrafish lines using the CRISPR/Cas9 technology and performed phenotypic analysis throughout early embryogenesis. We found that the klf17-deficient embryos exhibited abnormal lateral line neuromast deposition, whereas the production of primitive erythrocytes and haemoglobin production were observed in the klf17-deficient embryos. The expression of lateral line neuromast genes, klf17 and s100t, in the klf17-deficient embryos was detected in posterior lateral line neuromasts abnormally positioned at short intervals. Furthermore, the klf17-deficient embryos failed to hatch and died without hatching around 15 days post-fertilization (dpf), whereas the dechorionated klf17-deficient embryos and wild-type embryos were alive at 15 dpf. The klf17-deficient embryos abolished hatching gland cells and Ctsl1b protein expression, and eliminated the expression of polster and hatching gland marker genes, he1.1, ctsl1b and cd63. Thus, the klf17 gene plays important roles in posterior lateral line neuromast and hatching gland development.

Genome editing technologies, such as transcription activator-like effector nuclease (TALEN) and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) systems, can induce DNA double-strand breaks (DSBs) at the targeted genomic locus, leading to frameshift-mediated gene disruption in the process of DSB repair. Recently, the technology-induced DSBs followed by DSB repairs are applied to integrate exogenous genes into the targeted genomic locus in various model organisms. In addition to a conventional knock-in technology mediated by homology-directed repair (HDR), novel knock-in technologies using refined donor vectors have also been developed with the genome editing technologies based on other DSB repair mechanisms, including non-homologous end joining (NHEJ) and microhomology-mediated end joining (MMEJ). Therefore, the improved knock-in technologies would contribute to freely modify the genome of model organisms.

Mammalian CEP55 (centrosomal protein 55 kDa) is a coiled‐coil protein localized to the centrosome in interphase cells and is required for cytokinesis. A homozygous non‐sense mutation in human CEP55 has been recently identified in perinatal lethal MARCH (multinucleated neurons, anhydramnios, renal dysplasia, cerebellar hypoplasia and hydranencephaly) syndrome. We have isolated zebrafish cep55 mutants defective in head morphology. The zebrafish cep55 gene was expressed in the head including the retina and the pectoral fin at 1 day post‐fertilization (dpf), and extensive cell death was widely observed in the head and tail of the cep55 mutant. In the cep55 mutant, the anterior–posterior distance of the ventral pharyngeal arches was short, and retinal lamination was disorganized. Neural cells, such as islet1‐positive cells and pax2‐positive cells, and fli1b‐positive vascular cells were reduced in the head of the cep55 mutant. Thus, we propose that the zebrafish cep55 mutant is a model organism for human MARCH syndrome.

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