Targeted indel mutation induced by engineered Cas9/gRNA at the cftr gene in zebrafish. (A) The target sites highlighted by yellow and the PAM sequence marked by red underlined text. Deletions of cftr mutant are shown as dashes. Boxes show the start codon of WT and destroyed start codon of cftr mutant. (B) Gel shows T7E1 digestion of PCR products amplified from adult tail genomic DNA of F1 heterozygous generation. The uncleaved and cleaved PCR products are indicated. After digestion with T7E1, the cleaved PCR product of the adult tail represents the fragments containing the mutation. (C) Sequencing results show that F1 heterozygous generation fish carrying cftr mutant produces overlapping peaks marked by dashed box. (D) Western blot assay indicates the significant reduced Cftr protein level in offspring embryos from mutant line. (E) The got genotyped homozygous cftr mutant embryo also demonstrated the absent of Kupffer’s vesicle (KV) lumen (pointed by arrow) at 8-somite stage.

cftr mutant induces nanos1/vasa-marked PGCs disorder in early zebrafish embryo. Analysis of localization of nanos1/vasa positive cells in offspring embryos from WT and mutant line by WISH at 4-cell stage (A), Dome stage (B), 50% Epiboly stage (C), 8-somite stage (D) and Prim-5 stage (E). Embryo orientations: 4-cell, Dome stage and 50% Epiboly stage, top view; 8-somite, dorsal view with anterior oriented at the top; Prim-5 stage, lateral views with anterior oriented toward the left. Arrows show the normal location of PGCs, arrowheads demonstrate the aberrant position of PGCs in cftr mutant. Region between two dotted circles on embryo shows normal location of PGCs. The numbers indicated in each picture are the number (left) of affected embryos with phenotype similar to what is shown in the picture and the total number (right) of observed embryos. The same number labeling was used thereafter.

CFTR inhibitor CFTR_inh172 leads to PGCs disorder in early zebrafish embryo. Analysis of localization of nanos1/vasa positive cells in embryos by WISH at 50% Epiboly stage with top view.

CFTR mutants with loss of ion channel function fail to recover PGCs development. Analysis of localization of nanos1/vasa positive cells in embryos by WISH at 50% Epiboly stage with top view.

Aberrant expression of key factors (A: cxcr4b; B: cxcl12a; C: rgs14a; D: ca15b) is detected in offspring embryos from mutant line. 50%-epiboly stage embryos were used in these assays. All of the factors were detected by qPCR; ca15b was also detected by WISH.

Genotype identification of cftr mutant. Sequencing result indicate that embryos with absent KV lumen and disordered PGCs carry the cftr mutant. Embryos at 8-somite stage were analyzed, and vasa probe was used in whole-mount in situ hybridization(WISH) assay.