Dataset Information

Efficient Gene Editing at Major CFTR Mutation Loci.

ABSTRACT: Cystic fibrosis (CF) is a lethal autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Nuclease-mediated precise gene editing (PGE) represents a promising therapy for CF, for which an efficient strategy that is free of viral vector, drug selection, and reporter enrichment (VDR free) is desirable. Here we compared different transfection methods (lipofectamine versus electroporation) and formats (plasmid DNA versus ribonucleoprotein) in delivering the CRISPR/Cas9 elements along with single-stranded oligodeoxynucleotides (ssODNs) to clinically relevant cells targeting major CFTR mutation loci. We demonstrate that, among different combinations, electroporation of CRISPR/Cas9 and guide RNA (gRNA) ribonucleoprotein (Cas9 RNP) is the most effective one. By using this VDR-free method, 4.8% to 27.2% efficiencies were achieved in creating dF508, G542X, and G551D mutations in a wild-type induced pluripotent stem cell (iPSC) line. When it is applied to a patient-derived iPSC line carrying the dF508 mutation, a greater than 20% precise correction rate was achieved. As expected, genetic correction leads to the restoration of CFTR function in iPSC-derived proximal lung organoids, as well as in a patient-derived adenocarcinoma cell line CFPAC-1. The present work demonstrates the feasibility of gene editing-based therapeutics toward monogenic diseases such as CF.

SUBMITTER: Ruan J

PROVIDER: S-EPMC6409404 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Efficient Gene Editing at Major CFTR Mutation Loci.

Ruan Jinxue J Hirai Hiroyuki H Yang Dongshan D Ma Linyuan L Hou Xia X Jiang Hong H Wei Hongguang H Rajagopalan Carthic C Mou Hongmei H Wang Guoshun G Zhang Jifeng J Li Kui K Chen Yuqing E YE Sun Fei F Xu Jie J

Molecular therapy. Nucleic acids 20190216

Cystic fibrosis (CF) is a lethal autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Nuclease-mediated precise gene editing (PGE) represents a promising therapy for CF, for which an efficient strategy that is free of viral vector, drug selection, and reporter enrichment (VDR free) is desirable. Here we compared different transfection methods (lipofectamine versus electroporation) and formats (plasmid DNA versus ribonucleoprotein ...[more]

PMID: 30852378

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Project description:BackgroundStickleback fish are widely used to study the genetic and ecological basis of phenotypic evolution. Although several major loci have now been identified that contribute to evolutionary differences between wild populations, further study of the phenotypes associated with particular genes and mutations has been limited by the difficulty of generating targeted mutations at precise locations in the stickleback genome.Approach and aimsWe compared different methods of expressing single-guide RNAs (sgRNAs) and Cas9 activity in fertilized stickleback eggs. We used an easily scored pigmentation gene (SLC24A5) to screen for molecular lesions, phenotypic effects, and possible germline transmission of newly induced alleles. We then used the optimized CRISPR methods to target two major evolutionary loci in sticklebacks, KITLG and EDA. We hypothesized that coding region mutations in the KITLG gene would alter body pigmentation and possibly sex determination, and that mutations in the EDA gene would disrupt the formation of most armor plates, fin rays, spines, teeth, and gill rakers.ResultsTargeted deletions were successfully induced at each target locus by co-injecting one-cell stage stickleback embryos with either Cas9 mRNA or Cas9 protein, together with sgRNAs designed to protein-coding exons. Founder animals were typically mosaic for multiple mutations, which they transmitted through the germline at overall rates of 21 to 100%. We found that the copy of KITLG on the X chromosome (KITLGX) has diverged from the KITLG on the Y chromosome (KITLGY). Predicted loss-of-function mutations in the KITLGX gene dramatically altered pigmentation in both external skin and internal organ, but the same was not true for KITLGY mutations. Predicted loss-of-function mutations in either the KITLGX or KITLGY genes did not lead to sex reversal or prevent fertility. Homozygous loss-of-function mutations in the EDA gene led to complete loss of armor plates, severe reduction or loss of most soft rays in the dorsal, anal, and caudal fins, and severe reductions in tooth and gill raker number. In contrast, long dorsal and pelvic spines remained intact in EDA mutant animals, suggesting that common co-segregation of plate loss and spine reduction in wild populations is unlikely to be due to pleiotropic effects of EDA mutations.ConclusionCRISPR-Cas9 approaches can be used to induce germline mutations in key evolutionary loci in sticklebacks. Targeted coding region mutations confirm an important role for KITLG and EDA in skin pigmentation and armor plate reduction, respectively. They also provide new information about the functions of these genes in other body structures.

Dataset Information

Efficient Gene Editing at Major CFTR Mutation Loci.

Publications

Efficient Gene Editing at Major CFTR Mutation Loci.

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