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 2

Detection of CFTR mutations in the cloned lambs by PCR/restriction fragment length polymorphism assays and Sanger sequencing.

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Detection of CFTR mutations in the cloned lambs by PCR/restriction fragm...
(A) Cloned CFTR+/– sheep at 13–16 months of age: 4 males (top) and 6 females (bottom). (B) Identification of CFTR–/– and CFTR+/– cloned lambs with mutations in exon 2 (upper and middle panels) or exon 11 (lower panel) by PCR/restriction fragment length polymorphism (RFLP) assays (702–706, 708, 709, 711, 713–715, and 724 are CFTR–/–; 716–721, 723, 725–730, and 733 are CFTR+/–. (C) Representative sequence analysis for CF lambs. In total, 6 CFTR-targeted colonies (4 colonies with mutations in exon 2 and 2 in exon 11) were successfully used for production of CFTR–/– or CFTR+/– cloned sheep (Table 3). Representative images show sequence results for one animal obtained from each of the donor cell colonies used in the study. Exon 2 and 11 WT sequences are also shown. Restriction enzyme recognition sites used for the PCR/RFLP assays are underlined. Arrows indicate the mutation sites. The protospacer-adjacent motif (PAM) sequences used for Cas9 targeting are indicated by boxes.