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 Previously, we generated CFTRF508del/F508del and CFTRG542X/G542X lambs (unpublished) using CRISPR/Cas9 and somatic cell nuclear transfer (SCNT) techniques.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.

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.

132 Introduction of F508del human mutation into the CFTR gene of sheep fetal fibroblasts using CRISPR/Cas9 ribonucleoprotein

2020 ◽  
Vol 32 (2) ◽  
pp. 192 ◽  
Author(s):  
I. Viotti Perisse ◽  
Z. Fan ◽  
A. Van Wettere ◽  
Z. Wang ◽  
A. Harris ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Gene Editing ◽  
Severe Disease ◽  
The United States ◽  
Cftr Gene ◽  
Common Mutation ◽  
Mutation Site ◽  
Fetal Fibroblasts ◽  
Cftr Protein ◽  
Exon 11

Cystic fibrosis (CF) is an autosomal recessive genetic disease that affects over 30 000 people in the United States and is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The CFTR protein is a cAMP-regulated C− channel responsible for regulation of anion transport, primarily in the epithelial cells. We have previously generated a sheep model of CF by genetically inactivating the CFTR gene (Fan et al. 2018 JCI Insight 3, e123529). The newborn CFTR −/− sheep develops severe disease consistent with CF pathology in humans. The CF model is extremely valuable for understanding the developmental aspects of CF disease, as sheep have been used extensively in the study of human fetal growth and development. Sheep, like humans, typically give birth to only one or two offspring in each pregnancy, which make them more suitable than many other species for testing prenatal gene-editing treatments. Thus, in this new study, we are working on the generation of F508del sheep CF model. The F508del mutation was chosen because it is the most common mutation in the human CFTR gene (~70%). This mutation is characterised by the deletion of the CTT nucleotides, which ultimately deletes the phenylalanine residue at position 508. The F508del mutation causes misfolding of the CFTR protein, which is further degraded by proteases. Even though several CFTR modulators are available, they are not effective in all patients. Additionally, they cannot reverse deleterious prenatal CF manifestations. Hence, this model will be valuable for evaluating both prenatal drug and gene therapies. Here, we used a CRISPR/Cas9 gene-editing approach to introduce the F508del mutation into the sheep genome. We designed an sgRNA targeting exon 11 of the sheep CFTR gene using the Benchling software (https://benchling.com/academic). The sgRNA was synthesised by Synthego and Cas9 purchased from ThermoFisher. Using the Lonza-4D-Nucleofector system, Cas9/sgRNA ribonucleoprotein complex was transfected into sheep fetal fibroblasts (SFFs), along with 100bp single-stranded oligodeoxynucleotide, flanking the F508del mutation, for the homology-directed repair. The transfected cells were subsequently cultured in Dulbecco's modified Eagle's medium, supplemented with 15% fetal bovine serum and 1% penicillin, and incubated at 38.5°C. Two days post-transfection, SFFs were seeded individually into five 96-well plates by limited dilution. After seven days, the individual colonies were expanded into 24-well plates and cultured for three more days. A total of 56 single-cell-derived SFF colonies were isolated. The presence of F508del mutation was confirmed by amplifying the PCR products of the exon 11 flanking the mutation site and subjecting each amplicon to Sanger sequencing. The sequencing results indicated that the indels (insertion/deletion) were introduced in 49 out of 56 (87.5%) of the colonies, and four (7.14%) of them were confirmed to have biallelic F508del mutations based on sequencing peaks. Therefore, we successfully introduced the F508del mutation in SFFs that will be used for the production of F508del CF sheep by somatic cell nuclear transfer.

Epigenetics in Cystic Fibrosis: Epigenetic Targeting of a Genetic Disease

2015 ◽  
Vol 16 (9) ◽  
pp. 976-987 ◽  
Author(s):  
Nualpun Sirinupong ◽  
Zhe Yang
Keyword(s):  
Cystic Fibrosis ◽  
Genetic Disease

Etiology of Human Genetic Disease on the Fly

2017 ◽  
Vol 33 (6) ◽  
pp. 391-398 ◽  
Author(s):  
Clement Y. Chow ◽  
Lawrence T. Reiter
Keyword(s):  
Genetic Disease ◽  
Human Genetic Disease

scholarly journals Surveillance of Colorectal Cancer (CRC) in Cystic Fibrosis (CF) Patients

2021 ◽  
Vol 3 (2) ◽  
pp. 84-95
Author(s):  
Fabio Ingravalle ◽  
Giovanni Casella ◽  
Adriana Ingravalle ◽  
Claudio Monti ◽  
Federica De Salvatore ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cystic Fibrosis ◽  
General Population ◽  
Genetic Disorder ◽  
The United States ◽  
Screening Colonoscopy ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Increased Risk ◽  
Speciality Training

Cystic Fibrosis (CF) is the commonest inherited genetic disorder in Caucasians due to a mutation in the gene CFTR (Cystic Fibrosis Transmembrane Conductance Regulator), and it should be considered as an Inherited Colorectal Cancer (CRC) Syndrome. In the United States, physicians of CF Foundation established the “Developing Innovative Gastroenterology Speciality Training Program” to increase the research on CF in gastrointestinal and hepatobiliary diseases. The risk to develop a CRC is 5–10 times higher in CF patients than in the general population and even greater in CF patients receiving immunosuppressive therapy due to organ transplantation (30-fold increased risk relative to the general population). Colonoscopy should be considered the best screening for CRC in CF patients. The screening colonoscopy should be started at the age of 40 in CF patients and, if negative, a new colonoscopy should be performed every 5 years and every 3 years if adenomas are detected. For transplanted CF patients, the screening colonoscopy could be started at the age of 35, in transplanted patients at the age of 30 and, if before, at the age of 30. CF transplanted patients, between the age of 35 and 55, must repeat colonoscopy every 3 years. Our review draws attention towards the clinically relevant development of CRC in CF patients, and it may pave the way for further screenings and studies.

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