@article{discovery10067655,
            year = {2019},
           month = {February},
         journal = {Genetics in Medicine},
           title = {CRISPR/Cas9-targeted enrichment and long-read sequencing of the Fuchs endothelial corneal dystrophy-associated TCF4 triplet repeat},
            note = {{\copyright} The Author(s) 2019. This article is licensed under a Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/).},
        abstract = {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 (nā€‰=ā€‰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 ({$\ge$}50 repeats) phased CTG18.1 alleles analyzed. Furthermore, higher levels of repeat instability were associated with increased CTG18.1 allele length (mode length {$\ge$}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.},
          author = {Hafford-Tear, NJ and Tsai, Y-C and Sadan, AN and Sanchez-Pintado, B and Zarouchlioti, C and Maher, GJ and Liskova, P and Tuft, SJ and Hardcastle, AJ and Clark, TA and Davidson, AE},
             url = {https://doi.org/10.1038/s41436-019-0453-x},
            issn = {1530-0366},
        keywords = {Fuchs endothelial corneal dystrophy, amplification-free sequencing, no-amp targeted sequencing, somatic mosaicism, triplet repeat-mediated disease}
}