92 Correction of the CFTR G542X mutation using CRISPR/Cas9 genome editing in ovine-bovine interspecies embryos

2021 ◽  
Vol 33 (2) ◽  
pp. 153
Author(s):  
Z. Fan ◽  
Y. Liu ◽  
I. V. Perisse ◽  
K. L. White ◽  
I. A. Polejaeva
Keyword(s):  
Cftr Gene ◽  
Transmembrane Conductance Regulator ◽  
Human Genetic Disease ◽  
Cell Stage ◽  
Homology Directed Repair ◽  
Guide Rnas ◽  
Significant Difference ◽  
Cftr Mutations ◽  
Developmental Capacity ◽  
Cf Transmembrane Conductance Regulator

Cystic fibrosis (CF) is a human genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. We have recently generated 3 CF sheep models: a CFTR−/− model (Fan et al. 2018 LCI Insight 3:e123529; https://doi.org/10.1172/jci.insight.123529) and 2 additional models where we introduced human G542X and F508del mutations into the sheep genome (unpublished). Correction of CFTR mutations in zygotes with gene-editing techniques could be a permanent solution to cure this disease. To assess the efficiency of mutation correction invitro by CRISPR/Cas9, we utilised embryos generated by ovine-bovine interspecies SCNT (iSCNT) due to limited access to sheep oocytes. First, we evaluated the developmental capacity of reconstructed iSCNT embryos, in which nucleus donors were derived from ovine fibroblasts and recipient cytoplasm from enucleated bovine oocytes. These iSCNT embryos were able to develop to 16- to 32-cell stage (3/30, 10.0%), which allowed the genotyping of each embryo using PCR-restriction fragment length polymorphism assays and Sanger sequencing. Then, specific single-guide RNAs (sgRNAs) and 101-bp single-stranded oligodeoxynucleotides (ssODNs) were designed and synthesised to correct the G542X mutation in the sheep CFTR gene. We optimized the concentrations of Cas9:sgRNA ribonucleoproteins (RNPs) for 1-cell stage embryonic injection. Mutation analysis of embryos was conducted at 3 days post injection. Genotyping results showed that we achieved high efficiencies (95.7–100%) of mutations (indels) at targeting loci after injection of different concentrations of Cas9:sgRNA RNPs (0.02 µg:0.6 pmol/µL to 1.4 µg:40 pmol/µL). Furthermore, when an RNP (1.4 µg:40 pmol/µL) was co-injected with a ssODN (80 pmol/µL), both targeting the G542X mutation, the mutation was successfully corrected in the genome of iSCNT embryos generated using G542X fibroblasts as nucleus donors at an efficiency of 5.7% (3/53) via homology-directed repair mechanism. During the invitro culture of iSCNT embryos, we did not observe significant difference (P>0.05, unpaired t-test) in cleavage rates between embryos with or without injection (85.5% vs. 89.0%). Off-target analysis of those mutated and G542X-corrected embryos is in progress. Our strategy overcomes the limitation of oocyte source and provides an opportunity to mimic the editing of any other gene in embryos of different species.

scholarly journals Correction of a CFTR/G542X Mutation Using CRISPR/Cas9 Editing in Ovine-bovine Interspecies Embryos

2021 ◽  
Author(s):  
Zhiqiang Fan ◽  
Ying Liu ◽  
Iuri Perisse ◽  
Ann Harris ◽  
Kenneth White ◽  
...  
Keyword(s):  
Transmembrane Conductance Regulator ◽  
Gene Correction ◽  
Human Genetic Disease ◽  
Cell Stage ◽  
Homology Directed Repair ◽  
Stage Embryo ◽  
Developmental Capacity ◽  
Embryo Stage ◽  
Cf Transmembrane Conductance Regulator

Abstract Cystic fibrosis (CF) is a human genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. Correction of CFTR mutations at embryo stage could be a permanent solution to cure this disease. To assess the efficiency of CFTR/G542X mutation correction in vitro by CRISPR/Cas9, we utilized embryos generated by ovine-bovine interspecies somatic cell nuclear transfer (iSCNT) due to a limited access to sheep oocytes. First, we evaluated the developmental capacity of reconstructed iSCNT embryos. These embryos were able to develop to 16-cell stage, allowing for individual embryo genotyping. Then, we optimized the concentrations of Cas9:gRNA ribonucleoprotein (RNP) for 1-cell stage embryo injection. Genotyping results showed that we achieved high efficiencies (88.9–100%) of indel mutations at the target locus after injection of different concentrations of RNP. When an RNP (0.09 µg/µl:2.3 µM) was co-injected with a ssODN (18 µM), the G542X mutation was corrected via the homology-directed repair in 11.1% (1/9) of iSCNT embryos. Taken together, we developed an effective strategy to correct the CFTR/G542X mutation in ovine-bovine iSCNT embryos by CRISRP/Cas9. Our strategy overcomes the limitation of oocyte source and provides the opportunity of mimicking the editing of any other genes in one-cell embryos of different species.

95 Invitro correction of F508del and G542X mutations in sheep fibroblasts of cystic fibrosis models

2021 ◽  
Vol 33 (2) ◽  
pp. 155
Author(s):  
K. Bunch ◽  
I. V. Perisse ◽  
Z. Fan ◽  
K. White ◽  
I. Polejaeva
Keyword(s):  
Cystic Fibrosis ◽  
Genetic Disease ◽  
The United States ◽  
Human Genetic Disease ◽  
Nonsense Mediated Decay ◽  
Homology Directed Repair ◽  
Mutant Transcript ◽  
Pcr Products ◽  
Exon 11 ◽  
Cf Transmembrane Conductance Regulator

Cystic fibrosis (CF) is a human genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. Among the ∼2000 known CF mutations, the F508del mutation is found in 84% and G542X in 4.6% of the CF patients in the United States. The F508del mutation occurs in exon 11 and is characterised by deletion of the “CTT” nucleotides, resulting in deletion on the phenylalanine residue at the position 508 of CFTR. This causes misfolding of the CFTR protein, which is further degraded by proteases. The G542X mutation is a nonsense mutation found in exon 12 and associated with nonsense-mediated decay of the mutant transcript causing the absence of protein production. Previously, we generated CFTRF508del/F508del and CFTRG542X/G542X lambs (unpublished) using CRISPR/Cas9 and somatic cell nuclear transfer (SCNT) techniques. We hypothesised that gene editing may be an effective tool to correct these mutations and permanently cure this genetic disease. Thus, in this study, we evaluated the efficiency of CRISPR/Cas9-meditated gene knock-in to correct the F508del and G542X mutations in sheep fibroblasts invitro. We designed single guide (sg)RNAs using the Benchling software (https://benchling.com/academic) and approximately 100bp of single-stranded oligodeoxynucleotides (ssODNs) targeting the mutation sites at exon 11 and 12 to introduce either “CTT” or change the “T” to “G” nucleotide in genome of F508del or G542X CF sheep cells, respectively. Each of Cas9/sgRNA ribonucleoproteins was transfected into sheep fibroblast cells along with ssODNs using the Lonza-4D-NucleofectorTM (Lonza) system for homology-directed repair. The transfected cells were subsequently cultured in Dulbecco’s modified Eagle medium, supplemented with 15% fetal bovine serum and 1% penicillin, and incubated at 38.5°C. DNA was extracted 48h post-transfection to validate mutation efficiency. PCR products of the exons 11 and 12 were ligated into T-vector, and bacterial colonies were selected based on blue/white screening. In total, we isolated 32 single cell bacterial colonies for each mutant. Sequencing results indicate that “CTT” was introduced in 4/26 (15.3%) plasmid colonies, and “T to G” replaced in 13/31 (41.9%) colonies. Therefore, our results indicate that the F508del and G542X mutations can be effectively corrected in CF sheep fibroblasts invitro using a CRISPR/Cas9 approach.

scholarly journals Restoration of R117H CFTR folding and function in human airway cells through combination treatment with VX-809 and VX-770

2016 ◽  
Vol 311 (3) ◽  
pp. L550-L559 ◽  
Author(s):  
Martina Gentzsch ◽  
Hong Y. Ren ◽  
Scott A. Houck ◽  
Nancy L. Quinney ◽  
Deborah M. Cholon ◽  
...  
Keyword(s):  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Ion Conductance ◽  
Bronchial Epithelial ◽  
Rare Mutation ◽  
Limited Success ◽  
Folding Defect ◽  
Cftr Mutations ◽  
Cf Transmembrane Conductance Regulator ◽  
And Function

Cystic fibrosis (CF) is a lethal recessive genetic disease caused primarily by the F508del mutation in the CF transmembrane conductance regulator (CFTR). The potentiator VX-770 was the first CFTR modulator approved by the FDA for treatment of CF patients with the gating mutation G551D. Orkambi is a drug containing VX-770 and corrector VX809 and is approved for treatment of CF patients homozygous for F508del, which has folding and gating defects. At least 30% of CF patients are heterozygous for the F508del mutation with the other allele encoding for one of many different rare CFTR mutations. Treatment of heterozygous F508del patients with VX-809 and VX-770 has had limited success, so it is important to identify heterozygous patients that respond to CFTR modulator therapy. R117H is a more prevalent rare mutation found in over 2,000 CF patients. In this study we investigated the effectiveness of VX-809/VX-770 therapy on restoring CFTR function in human bronchial epithelial (HBE) cells from R117H/F508del CF patients. We found that VX-809 stimulated more CFTR activity in R117H/F508del HBEs than in F508del/F508del HBEs. R117H expressed exclusively in immortalized HBEs exhibited a folding defect, was retained in the ER, and degraded prematurely. VX-809 corrected the R117H folding defect and restored channel function. Because R117 is involved in ion conductance, VX-770 acted additively with VX-809 to restore CFTR function in chronically treated R117H/F508del cells. Although treatment of R117H patients with VX-770 has been approved, our studies indicate that Orkambi may be more beneficial for rescue of CFTR function in these patients.

scholarly journals Three Novel CFTR Polymorphic Repeats Improve Segregation Analysis for Cystic Fibrosis

2009 ◽  
Vol 55 (7) ◽  
pp. 1372-1379 ◽  
Author(s):  
Ausilia Elce ◽  
Angelo Boccia ◽  
Giuseppe Cardillo ◽  
Sonia Giordano ◽  
Rossella Tomaiuolo ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Prenatal Diagnosis ◽  
Large Scale ◽  
Segregation Analysis ◽  
Preimplantation Diagnosis ◽  
Cftr Gene ◽  
Transmembrane Conductance Regulator ◽  
Large Scale Analysis ◽  
Allelic Distribution ◽  
Cftr Mutations

Abstract Background: Molecular diagnosis for cystic fibrosis (CF) is based on the direct identification of mutations in the CFTR gene [cystic fibrosis transmembrane conductance regulator (ATP-binding cassette sub-family C, member 7)] (detection rate about 90% with scanning procedures) and on segregation analysis of intragenic polymorphisms for carrier and prenatal diagnosis in about 20% of CF families in which 1 or both causal mutations are unknown. Methods: We identified 3 novel intragenic polymorphic repeats (IVS3polyA, IVS4polyA, and IVS10CA repeats) in the CFTR gene and developed and validated a procedure based on the PCR followed by capillary electrophoresis for large-scale analysis of these polymorphisms and the 4 previously identified microsatellites (IVS1CA, IVS8CA, IVS17bTA, and IVS17bCA repeats) in a single run. We validated the procedure for both single- and 2-cell samples (for a possible use in preimplantation diagnosis), and on a large number of CF patients bearing different genotypes and non-CF controls. Results: The allelic distribution and heterozygosity results suggest that the 3 novel polymorphisms strongly contribute to carrier and prenatal diagnosis of CF in families in which 1 or both causal mutations have not been identified. At least 1 of the 4 previously identified microsatellites was informative in 78 of 100 unrelated CF families; at least 1 of all 7 polymorphisms was informative in 98 of the families. Finally, the analysis of haplotypes for the 7 polymorphisms revealed that most CF mutations are associated with different haplotypes, suggesting multiple slippage events but a single origin for most CFTR mutations. Conclusions: The analysis of the 7 polymorphisms is a rapid and efficient tool for routine carrier, prenatal, and preimplantation diagnosis of CF.

scholarly journals CFTR mutations altering CFTR fragmentation

2012 ◽  
Vol 449 (1) ◽  
pp. 295-305 ◽  
Author(s):  
Kendra Tosoni ◽  
Michelle Stobbart ◽  
Diane M. Cassidy ◽  
Andrea Venerando ◽  
Mario A. Pagano ◽  
...  
Keyword(s):  
Bronchial Epithelial Cell ◽  
Epithelial Cell Line ◽  
Transmembrane Conductance Regulator ◽  
Wild Type ◽  
Ladder Pattern ◽  
Cftr Mutations ◽  
Cf Transmembrane Conductance Regulator ◽  
In Vitro Interaction ◽  
Kinase Ck2

Most CF (cystic fibrosis) results from deletion of a phenylalanine (F508) in the CFTR {CF transmembrane-conductance regulator; ABCC7 [ABC (ATP-binding cassette) sub-family C member 7]} which causes ER (endoplasmic reticulum) degradation of the mutant. Using stably CFTR-expressing BHK (baby-hamster kidney) cell lines we demonstrated that wild-type CTFR and the F508delCFTR mutant are cleaved into differently sized N- and C-terminal-bearing fragments, with each hemi-CFTR carrying its nearest NBD (nucleotide-binding domain), reflecting differential cleavage through the central CFTR R-domain. Similar NBD1-bearing fragments are present in the natively expressing HBE (human bronchial epithelial) cell line. We also observe multiple smaller fragments of different sizes in BHK cells, particularly after F508del mutation (ladder pattern). Trapping wild-type CFTR in the ER did not generate a F508del fragmentation fingerprint. Fragments change their size/pattern again post-mutation at sites involved in CFTR's in vitro interaction with the pleiotropic protein kinase CK2 (S511A in NBD1). The F508del and S511A mutations generate different fragmentation fingerprints that are each unlike the wild-type; yet, both mutants generate new N-terminal-bearing CFTR fragments that are not observed with other CK2-related mutations (S511D, S422A/D and T1471A/D). We conclude that the F508delCFTR mutant is not degraded completely and there exists a relationship between CFTR's fragmentation fingerprint and the CFTR sequence through putative CK2-interactive sites that lie near F508.

scholarly journals Multiplexed Genetic Analysis Using an Expanded Genetic Alphabet

2004 ◽  
Vol 50 (11) ◽  
pp. 2019-2027 ◽  
Author(s):  
Scott C Johnson ◽  
David J Marshall ◽  
Gerda Harms ◽  
Christie M Miller ◽  
Christopher B Sherrill ◽  
...  
Keyword(s):  
Newborn Screening ◽  
Cftr Gene ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Genetic Tests ◽  
Wide Range ◽  
Additional Base ◽  
A Prospective Study ◽  
Cystic Fibrosis Transmembrane ◽  
Made In

Abstract Background: All states require some kind of testing for newborns, but the policies are far from standardized. In some states, newborn screening may include genetic tests for a wide range of targets, but the costs and complexities of the newer genetic tests inhibit expansion of newborn screening. We describe the development and technical evaluation of a multiplex platform that may foster increased newborn genetic screening. Methods: MultiCode® PLx involves three major steps: PCR, target-specific extension, and liquid chip decoding. Each step is performed in the same reaction vessel, and the test is completed in ∼3 h. For site-specific labeling and room-temperature decoding, we use an additional base pair constructed from isoguanosine and isocytidine. We used the method to test for mutations within the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The developed test was performed manually and by automated liquid handling. Initially, 225 samples with a range of genotypes were tested retrospectively with the method. A prospective study used samples from >400 newborns. Results: In the retrospective study, 99.1% of samples were correctly genotyped with no incorrect calls made. In the perspective study, 95% of the samples were correctly genotyped for all targets, and there were no incorrect calls. Conclusions: The unique genetic multiplexing platform was successfully able to test for 31 targets within the CFTR gene and provides accurate genotype assignments in a clinical setting.

scholarly journals A Peptide-Nucleic Acid Targeting miR-335-5p Enhances Expression of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene with the Possible Involvement of the CFTR Scaffolding Protein NHERF1

Biomedicines ◽  
2021 ◽  
Vol 9 (2) ◽  
pp. 117
Author(s):  
Anna Tamanini ◽  
Enrica Fabbri ◽  
Tiziana Jakova ◽  
Jessica Gasparello ◽  
Alex Manicardi ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Nucleic Acid ◽  
Peptide Nucleic Acid ◽  
Stop Codon ◽  
Scaffolding Protein ◽  
Cftr Gene ◽  
Scaffolding Proteins ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Cystic Fibrosis Transmembrane

(1) Background: Up-regulation of the Cystic Fibrosis Transmembrane Conductance Regulator gene (CFTR) might be of great relevance for the development of therapeutic protocols for cystic fibrosis (CF). MicroRNAs are deeply involved in the regulation of CFTR and scaffolding proteins (such as NHERF1, NHERF2 and Ezrin). (2) Methods: Content of miRNAs and mRNAs was analyzed by RT-qPCR, while the CFTR and NHERF1 production was analyzed by Western blotting. (3) Results: The results here described show that the CFTR scaffolding protein NHERF1 can be up-regulated in bronchial epithelial Calu-3 cells by a peptide-nucleic acid (PNA) targeting miR-335-5p, predicted to bind to the 3′-UTR sequence of the NHERF1 mRNA. Treatment of Calu-3 cells with this PNA (R8-PNA-a335) causes also up-regulation of CFTR. (4) Conclusions: We propose miR-335-5p targeting as a strategy to increase CFTR. While the efficiency of PNA-based targeting of miR-335-5p should be verified as a therapeutic strategy in CF caused by stop-codon mutation of the CFTR gene, this approach might give appreciable results in CF cells carrying other mutations impairing the processing or stability of CFTR protein, supporting its application in personalized therapy for precision medicine.

Liquid secretion inhibitors reduce mucociliary transport in glandular airways

2002 ◽  
Vol 283 (2) ◽  
pp. L329-L335 ◽  
Author(s):  
Stephen T. Ballard ◽  
Laura Trout ◽  
Anil Mehta ◽  
Sarah K. Inglis
Keyword(s):  
Beat Frequency ◽  
Anion Channel ◽  
Ciliary Beat Frequency ◽  
Mucociliary Transport ◽  
Transmembrane Conductance Regulator ◽  
Inhibitory Effects ◽  
Transmembrane Conductance ◽  
Regulator Protein ◽  
Liquid Absorption ◽  
Cf Transmembrane Conductance Regulator

Because of its possible importance in cystic fibrosis (CF) pulmonary pathogenesis, the effect of anion and liquid secretion inhibitors on airway mucociliary transport was examined. When excised porcine tracheas were treated with ACh to induce gland liquid secretion, the rate of mucociliary transport was increased nearly threefold from 2.5 ± 0.5 to 6.8 ± 0.8 mm/min. Pretreatment with both bumetanide and dimethylamiloride (DMA), to respectively inhibit Cl− and HCO[Formula: see text]secretion, significantly reduced mucociliary transport in the presence of ACh by 92%. Pretreatment with the anion channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid similarly reduced mucociliary transport in ACh-treated airways by 97%. These agents did not, however, reduce ciliary beat frequency. Luminal application of benzamil to block liquid absorption significantly attenuated the inhibitory effects of bumetanide and DMA on mucociliary transport. We conclude that anion and liquid secretion is essential for normal mucociliary transport in glandular airways. Because the CF transmembrane conductance regulator protein likely mediates Cl−, HCO[Formula: see text], and liquid secretion in normal glands, we speculate that impairment of gland liquid secretion significantly contributes to defective mucociliary transport in CF.

Intrafollicular testosterone concentration and sex ratio in individually cultured bovine embryos

2010 ◽  
Vol 22 (3) ◽  
pp. 533 ◽  
Author(s):  
Manuel García-Herreros ◽  
Pablo Bermejo-Álvarez ◽  
Dimitrios Rizos ◽  
Alfonso Gutiérrez-Adán ◽  
Alan G. Fahey ◽  
...  
Keyword(s):  
Follicular Fluid ◽  
Coefficient Of Determination ◽  
Bovine Embryos ◽  
Linear Quadratic ◽  
Cell Stage ◽  
Testosterone Concentration ◽  
Cumulus Oocyte Complex ◽  
Significant Difference ◽  
Embryo Sex ◽  
Follicular Size

Recent studies have suggested a relationship between bovine follicular fluid testosterone concentration and the likelihood of the oocyte being fertilised by an X- or Y-bearing spermatozoon; however, this theory has been challenged. To further test this hypothesis, follicles were dissected from the ovaries of slaughtered heifers, measured and carefully ruptured. The cumulus–oocyte complex (COC) was removed and the follicular fluid collected and testosterone concentration determined by radioimmunoassay. COCs were matured, fertilised and cultured in an individually identifiable manner; all cleaved embryos (2- to 4-cell stage, n = 164) had their sex determined by PCR. Testosterone concentrations were positively skewed. There was no significant difference between follicular fluid testosterone concentrations in male and female embryos (mean ± s.e.m. 51.5 ± 5.59 and 49.5 ± 7.42 ng mL–1, respectively). Linear, quadratic and cubic logistical regression showed that follicular testosterone concentration could not reliably predict the sex of the embryo with odds ratios of 1.001, 1.013 and 1.066, repectively, and coefficient of determination (R2) values of 0.0003, 0.0126 and 0.0567, respectively. Follicular size and testosterone concentration were not related (R2 = 0.087). Finally, follicular size had no influence on embryo sex determination (P = 0.70). In conclusion, under the conditions of the present study, the likelihood of an oocyte being fertilised by an X- or Y-bearing spermatozoon was not affected by the size of the follicle from which it was derived, nor by the testosterone concentration in the follicular fluid.

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