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CRISPR/Cas9-targeted enrichment and long-read sequencing of the Fuchs endothelial corneal dystrophy-associated TCF4 triplet repeat
NJ. Hafford-Tear, YC. Tsai, AN. Sadan, B. Sanchez-Pintado, C. Zarouchlioti, GJ. Maher, P. Liskova, SJ. Tuft, AJ. Hardcastle, TA. Clark, AE. Davidson,
Jazyk angličtina Země Spojené státy americké
Typ dokumentu časopisecké články, práce podpořená grantem
Grantová podpora
Department of Health - United Kingdom
NLK
ProQuest Central
od 2011-01-01 do 2021-12-31
Health & Medicine (ProQuest)
od 2011-01-01 do 2021-12-31
ROAD: Directory of Open Access Scholarly Resources
od 1998
- MeSH
- alely MeSH
- CRISPR-Cas systémy genetika MeSH
- dospělí MeSH
- expanze trinukleotidových repetic genetika MeSH
- Fuchsova endoteliální dystrofie genetika patologie MeSH
- genetická predispozice k nemoci * MeSH
- genotyp MeSH
- introny genetika MeSH
- lidé středního věku MeSH
- lidé MeSH
- sekvenční analýza DNA MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- transkripční faktor 4 genetika MeSH
- trinukleotidové repetice genetika MeSH
- zobrazení jednotlivé molekuly MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
PURPOSE: To demonstrate the utility of an amplification-free long-read sequencing method to characterize the Fuchs endothelial corneal dystrophy (FECD)-associated intronic TCF4 triplet repeat (CTG18.1). METHODS: We applied an amplification-free method, utilizing the CRISPR/Cas9 system, in combination with PacBio single-molecule real-time (SMRT) long-read sequencing, to study CTG18.1. FECD patient samples displaying a diverse range of CTG18.1 allele lengths and zygosity status (n = 11) were analyzed. A robust data analysis pipeline was developed to effectively filter, align, and interrogate CTG18.1-specific reads. All results were compared with conventional polymerase chain reaction (PCR)-based fragment analysis. RESULTS: CRISPR-guided SMRT sequencing of CTG18.1 provided accurate genotyping information for all samples and phasing was possible for 18/22 alleles sequenced. Repeat length instability was observed for all expanded (≥50 repeats) phased CTG18.1 alleles analyzed. Furthermore, higher levels of repeat instability were associated with increased CTG18.1 allele length (mode length ≥91 repeats) indicating that expanded alleles behave dynamically. CONCLUSION: CRISPR-guided SMRT sequencing of CTG18.1 has revealed novel insights into CTG18.1 length instability. Furthermore, this study provides a framework to improve the molecular diagnostic accuracy for CTG18.1-mediated FECD, which we anticipate will become increasingly important as gene-directed therapies are developed for this common age-related and sight threatening disease.
Pacific Biosciences Menlo Park CA USA
UCL Institute of Ophthalmology London UK
UCL Institute of Ophthalmology London UK Moorfields Eye Hospital London UK
Citace poskytuje Crossref.org
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