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.

Keywords: Development; Genetic diseases; Genetics.

Figure 1. Generation of CFTR^–/– and CFTR^+/– sheep fetal fibroblast colonies for somatic cell nuclear transfer by CRISPR/Cas9.

(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.

Figure 2. Detection of CFTR mutations in the cloned lambs by PCR/restriction fragment length polymorphism assays and Sanger sequencing.

(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.

Figure 3. Intestinal pathology of newborn CFTR^–/– lamb.

(A) One-day-old control lamb intestinal tract. (B) Meconium ileus in intestinal tract of a 1-day-old CFTR^–/– lamb. Small intestine is dilated with meconium (arrow). Colon is plugged with mucus (arrowhead). (C) Control lamb small intestine histology. (D) CFTR^–/– lamb small intestine is dilated and filled with meconium. (E) Control lamb colon histology. (F) CFTR^–/– colon is filled with large amount of mucus. (G) Control lamb colon histology. (H) CFTR^–/– colon with colonic glands filled and distended with large amount of mucus. C–F: H&E staining. G and H: Periodic acid–Schiff (PAS) staining. C and D: ×20; scale bars: 1 mm. E and F: ×40; scale bars: 500 μm. G and H: ×400; scale bars: 50 μm.

Figure 4. Pancreatic pathology of newborn CFTR^–/– lambs.

(A) Histology of the pancreas of a control lamb. Note the closely packed exocrine pancreatic acini. The asterisk indicates islet of Langerhans. (B) Histology of a CFTR^–/– lamb pancreas with mild pancreatic hypoplasia or atrophy and interstitial fibrosis. Note the decreased density and increased space between the exocrine pancreatic acini. (C) Histology of a CFTR^–/– lamb pancreas with severe pancreatic hypoplasia or atrophy and interstitial fibrosis. Only pancreatic ducts separated by fibrous and adipose tissues are present. (D) Higher magnification of the pancreas of the control lamb in A. (E) Higher magnification of the CFTR^–/– lamb pancreas in B. (F) Higher magnification of the CFTR^–/– lamb pancreas in C. H&E staining. A–C: ×100, scale bars: 100 μm. D–F: ×400; scale bars: 50 μm.

Figure 5. Liver pathology of newborn CFTR^–/– lambs.

(A and D) One-day-old control lamb liver. (B and C) Biliary fibrosis in a 1-day-old CFTR^–/– lamb liver. Prominent hepatocellular glycogen accumulation was present in some lambs (C). (E and F) Close up of the liver with biliary fibrosis. Severe intrahepatic cholestasis is present (arrows). H&E staining. A–C: ×40; scale bars: 500 μm. D–F: ×400; scale bars: 50 μm.

Figure 6. Representative traces of short-circuit current in WT and CFTR^–/– sheep tracheal epithelial cell cultures.

Primary cultures of sheep tracheal epithelial (STE) cells (passage 2) were seeded onto permeable filter supports and maintained at the air-liquid interface for 6 weeks. At the time of measurement, the filter inserts were placed in Ussing chambers and bathed on both sides with Krebs-Ringer bicarbonate solution bubbled with 95% O₂/5% CO₂ and maintained at 37°C. At the indicated times, amiloride (Amil, 100 μM, apical), forskolin (For, 10 μM, basolateral), and GlyH-101 (20 μM, apical) were added. CF, CFTR^–/–.

Figure 7. Summary of transepithelial electrophysiology data for WT and CFTR^–/– sheep tracheal epithelial cell cultures.

Air-liquid interface cultures were mounted in Ussing chambers and bathed on both sides with Krebs-Ringer bicarbonate solution bubbled with 95% O₂/5% CO₂ and maintained at 37°C. Cultures were maintained under short-circuit conditions and treated sequentially with amiloride (Amil, 100 μM, apical), forskolin (For, 10 μM, basolateral), and GlyH-101 (GlyH, 20 μM, apical). Symbols represent values for individual animals (mean of 6–8 replicates from each animal). Mean ± SEM for each group is indicated by the crosshair and error bars. *P < 0.05, between WT and CFTR^–/– cultures; unpaired t test.