mCBM mice. Animal protocols were approved by the Marshall University Institutional Animal Care and Use Committee (IACUC) according to National Institutes of Health (NIH) guidelines. All mCBM heterozygous mice were backcrossed to C57BL6 for at least six generations. Female mCBM heterozygous mice (12 to 16 weeks old) were crossed with male mCBM heterozygous mice. Pregnant female mice at the indicated stage were humanely euthanized. Embryos at different ages were collected for morphology and gene analyses.

NKA α+/− mice. Animal protocols were approved by the Marshall University IACUC according to NIH guidelines. Female NKA α+/− mice were crossed with male NKA α+/− mice. Pregnant female mice at the indicated stage were humanely euthanized. Embryos at E9.5 were collected for morphology and gene analyses.

Cell lines. The parental LLC-PK1 cells were purchased from the American Type Culture Collection (ATCC). LLC-PK1, NKA knockdown (PY-17), α1 NKA–rescued (AAC-19), α1 NKA–mutant cell lines (LW-mCBM), and LX-α2 were all cultured in Dulbecco’s modified Eagle’s medium (DMEM) plus 10% fetal bovine serum (FBS) and 10% penicillin/streptomycin. After cells reached 95 to 100% confluence, they were serum-starved overnight and used for experiments unless indicated otherwise.

iPSC culture and generation of mutant human iPSCs by CRISPR-Cas9 genome editing human iPSCs were purchased from iXCells (catalog no. 30HU-002) and cultured with the TeSR-E8 Kit (STEMCELL Technologies Inc., catalog no. 05940). The DNA plasmids for Cas9–green fluorescent protein (GFP) (Addgene, no. 44719) and guide RNA (gRNA) cloning vector (Addgene, no. 41824) were ordered from Addgene. sgDNA (single-guide DNA) and ssODN (single stranded oligodeoxynucleotides) were designed according to the published protocol. The DNA sequences of primer oligos and their locations in the genome were included in fig. S9. The cloned Cas9-GFP vector inserted with sgDNA, along with ssODN, was transfected into human iPSCs with a 4D nucleofector device. Single-cell sorting and plating were later performed with fluorescence-activated cell sorting (FACS) flow cytometry, and the clones were selected and validated via genotyping PCR and DNA sequencing (fig. S9) (Table 1).

Generation of CBM mutation C. elegans and rescue of the mutant with wild-type eat-6. We used CRISPR-Cas9 to knock in the equivalent double mutations of the NKA α1 CBM mutation F75A and F78A into the C. elegans α1 gene eat-6 (syb575 allele) (fig. S8). Comparable to the impact of CBM mutant α1 NKA expression in mice, no live homozygous adults were obtained, whereas the heterozygous worms hatched normally. Moreover, by using the gene balancer nT1 [qIs51], we confirmed that the larva arrest occurred in syb575 homozygous animals because of L1 arrest (Fig. 5A). The defect of larval arrest by CBM mutation was rescued by a transgene expressing a wild-type eat-6 cDNA through an extrachromosomal array. The transgenic construct Peat-6::eat-6::unc-54 3′ untranslated region (SunyBiotech) was generated by fusion of 1963 base pairs (bp) upstream of the translation start of eat-6 with the open reading sequence eat-6 cDNA in the vector pPD49.78.

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