Cystic fibrosis (CF) is an autosomal recessive genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. Mutations in this gene result in either a lack of CFTR protein or a defective CFTR protein that cannot perform its key function in the cell. CFTR protein is an anion-selective channel that allows primarily chloride ion but also bicarbonate movement across epithelial cell membranes. Aberrant CFTR thus results in dysregulation of epithelial fluid transport in the lung, pancreas, and other organs 1. Common pathologies of CF include recurrent lung infection and inflammation, pancreatic insufficiency, which if untreated gives rise to malnutrition, intestinal obstruction, and reduced growth 2. There are approximately 70,000 individuals living with CF worldwide, with a frequency of 1 in 2,000–3,000 among Caucasian newborns.
To date, over 2000 CFTR mutations have been identified and categorized into 6 types based on their functional effects including protein translation, cellular processing, and channel gating 3. G542X is a primary mutation in the nonsense mutation category, the second most common mutation type, accounting for 4.6% of CF patients. Nonsense mutations generate a premature stop codon, leading to early termination of CFTR translation, mRNA decay through the surveillance pathway, and ultimately, lack of CFTR function 4. Some drugs, such as gentamicin (G418) and ataluren (PTC124) has been developed as potential treatments for CF patients with nonsense mutations, which can promote ribosomal readthrough of premature termination codons. However, the use of G418 was limited due to its severe renal toxicity and ototoxicity 5 and ataluren showed no statistically significant difference compared to placebo in phase 3 clinical trials 6,7. In addition, it has been proved that inhibiting the nonsense-mediated mRNA decay pathway with antisense oligonucleotides can upregulate target RNA and functional protein expression in cells homozygous for the CFTR/W1282X mutation, which is the second most common nonsense mutation that causes CF 8. However, no further clinical trials using this strategy have been reported. Therefore, development of a safe and effective treatment for CF with nonsense mutations remains elusive.
Genome editing technologies, especially those based on CRISRP/Cas9, have been successfully applied in genome manipulation in different animals 9,10. Recent successes in precise genome editing trials in early-stage or cloned human embryos have suggested a potentially true cure for genetic diseases 11,12. However, genome editing of human embryos causes huge concerns because of ethical issues and technical uncertainties regarding the efficiency, mosaicism, off-target effect, and unintended homologous recombination. Genome editing by CRISPR/Cas9 generates double-strand breaks (DSBs) that evoke the error-prone non-homologous end joining (NHEJ) DNA repair pathway, which might cause off-target mutagenesis 13. During the S to G2 phases of cell circle, DSBs can also activate an alternative DNA repair pathway, the homology-directed repair (HDR), that repairs the DSB sites using a template from the homologous chromosome or an introduced exogenous DNA molecule, leading to the introduction of desired mutations 14. Presently, CRISPR/Cas9 is predominantly employed to generate gene knockouts through NHEJ repair pathway 15. Because HDR efficiency is relatively low, applications of HDR strategy for gene therapy, especially in human germline, have been limited 16. To address the concerns raised by germline editing, especially in humans, extensive experiments should be conducted, thus, increasing the demand for oocytes and embryos, of which the source is rare, and the use strictly regulated in some species.
Interspecies somatic cell nuclear transfer (iSCNT) is a cloning technique that utilizes recipient oocytes and donor cells derived from two different species. The iSCNT experiments primarily focused on applications of preservation/rescue of endangered species and establishment of embryonic stem cell lines for regenerative medicine research. Reprogramming of human somatic cells by iSCNT has been conducted in many labs without generating the ethical issues surrounding the use of human oocytes 17–19. It has been proved that iSCNT embryos between two species that are closely related can develop to term and lead to healthy cloned animals 20. The in vitro development of morulae or blastocysts has been achieved when nuclear donor cells and recipient oocytes had a very distant taxonomical relation, as in the case of interfamily bovine - pig, interorder cat and panda - rabbit, human - rabbit, - cattle, - sheep, and - goat, though remains ineffective and results are not always reproducible 21. In the present study, we hypothesized that 1) iSCNT could be an alternative approach to produce embryos for testing gene-editing at embryonic stage in certain species, where oocytes are rare or difficult to obtain, such as sheep in this study, and 2) CRISPR/Cas9-mediated HDR could be used for the correction of the G542X mutation in CF. First, we investigated the developmental potential of ovine-bovine iSCNT embryos. Based on the developmental results of reconstructed embryos, we optimized the concentrations of Cas9/gRNA for cytoplasmic injection and developed a strategy that allowed the correction of the CFTR/G542X mutation by CRISPR/Cas9 editing at embryonic stage.