A sheep model of cystic fibrosis generated by CRISPR/Cas9 disruption of the CFTR gene
Zhiqiang Fan, Iuri Viotti Perisse, Calvin U. Cotton, Misha Regouski, Qinggang Meng, Chaim Domb, Arnaud J. Van Wettere, Zhongde Wang, Ann Harris, Kenneth L. White, Irina A. Polejaeva
Zhiqiang Fan, Iuri Viotti Perisse, Calvin U. Cotton, Misha Regouski, Qinggang Meng, Chaim Domb, Arnaud J. Van Wettere, Zhongde Wang, Ann Harris, Kenneth L. White, Irina A. Polejaeva
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Research Article Development Genetics

A sheep model of cystic fibrosis generated by CRISPR/Cas9 disruption of the CFTR gene

Abstract

Cystic fibrosis (CF) is a genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The major cause of limited life span in CF patients is progressive lung disease. CF models have been generated in 4 species (mice, rats, ferrets, and pigs) to enhance our understanding of the CF pathogenesis. Sheep may be a particularly relevant animal to model CF in humans due to the similarities in lung anatomy and development in the two species. Here, we describe the generation of a sheep model for CF using CRISPR/Cas9 genome editing and somatic cell nuclear transfer (SCNT) techniques. We generated cells with CFTR gene disruption and used them for production of CFTR–/– and CFTR+/– lambs. The newborn CFTR–/– sheep developed severe disease consistent with CF pathology in humans. Of particular relevance were pancreatic fibrosis, intestinal obstruction, and absence of the vas deferens. Also, substantial liver and gallbladder disease may reflect CF liver disease that is evident in humans. The phenotype of CFTR–/– sheep suggests this large animal model will be a useful resource to advance the development of new CF therapeutics. Moreover, the generation of specific human CF disease–associated mutations in sheep may advance personalized medicine for this common genetic disorder.

Authors

Zhiqiang Fan, Iuri Viotti Perisse, Calvin U. Cotton, Misha Regouski, Qinggang Meng, Chaim Domb, Arnaud J. Van Wettere, Zhongde Wang, Ann Harris, Kenneth L. White, Irina A. Polejaeva

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Figure 1

Generation of CFTR–/– and CFTR+/– sheep fetal fibroblast colonies for somatic cell nuclear transfer by CRISPR/Cas9.

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Generation of CFTR–/– and CFTR+/– sheep fetal fibroblast colonies for so...
(A) Schematic diagram of the CFTR targeting sites. The single-guide RNA target sequences for each locus are depicted, with the restriction enzyme recognition sites used for the PCR/restriction fragment length polymorphism (RFLP) assays underlined. Letters in red indicate the protospacer-adjacent motifs (PAMs). Arrows indicate locations of PCR primers. (B) Gene targeting efficiency analysis of 2 targeting vectors at CFTR loci in sheep fetal fibroblasts (SFFs) detected by PCR/RFLP assays. M, 1-kb DNA ladder; Con, control (WT SFFs); Tar, SFFs transfected with each targeting vector. The targeted alleles lost restriction sites through error-prone non-homologous end joining (NHEJ) following Cas9-mediated double-stranded DNA breaks. The mutation efficiency (indels) for target 1 was 19% and for target 2, 41%. (C) PCR/RFLP assays for detection of CFTR–/– and CFTR+/– single-cell-derived SFF colonies with mutations at exon 2 (upper panel) or exon 11 (lower panel) of the CFTR gene. (D) Sequencing analysis of CFTR–/– and CFTR+/– colonies. Letters in yellow indicate the nucleotides inserted at cleavage sites. M, male; F, female; –/–, both alleles targeted: +/–, single allele targeted; –1nt, 1-nucleotide deletion; +1nt, 1-nucleotide insertion.