eprintid: 10067655 rev_number: 30 eprint_status: archive userid: 608 dir: disk0/10/06/76/55 datestamp: 2019-02-12 12:38:41 lastmod: 2021-09-19 22:23:32 status_changed: 2019-02-12 12:38:41 type: article metadata_visibility: show creators_name: Hafford-Tear, NJ creators_name: Tsai, Y-C creators_name: Sadan, AN creators_name: Sanchez-Pintado, B creators_name: Zarouchlioti, C creators_name: Maher, GJ creators_name: Liskova, P creators_name: Tuft, SJ creators_name: Hardcastle, AJ creators_name: Clark, TA creators_name: Davidson, AE title: CRISPR/Cas9-targeted enrichment and long-read sequencing of the Fuchs endothelial corneal dystrophy–associated TCF4 triplet repeat ispublished: inpress subjects: MOOR divisions: UCL divisions: B02 divisions: C07 divisions: D08 divisions: C08 divisions: D09 keywords: Fuchs endothelial corneal dystrophy, amplification-free sequencing, no-amp targeted sequencing, somatic mosaicism, triplet repeat-mediated disease note: © 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 (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. date: 2019-02-08 date_type: published official_url: https://doi.org/10.1038/s41436-019-0453-x oa_status: green full_text_type: pub language: eng primo: open primo_central: open_green article_type_text: Journal Article verified: verified_manual elements_id: 1627562 doi: 10.1038/s41436-019-0453-x pii: 10.1038/s41436-019-0453-x language_elements: English lyricists_name: Davidson, Alice lyricists_name: Hafford Tear, Nathaniel lyricists_name: Hardcastle, Alison lyricists_name: Liskova, Petra lyricists_name: Tuft, Stephen lyricists_name: Zarouchlioti, Christina lyricists_id: AEDAV12 lyricists_id: NHAFF13 lyricists_id: AJHAR52 lyricists_id: PLISK79 lyricists_id: SJTUF57 lyricists_id: CZARO28 actors_name: Flynn, Bernadette actors_id: BFFLY94 actors_role: owner full_text_status: public publication: Genetics in Medicine event_location: United States issn: 1530-0366 citation: Hafford-Tear, NJ; Tsai, Y-C; Sadan, AN; Sanchez-Pintado, B; Zarouchlioti, C; Maher, GJ; Liskova, P; ... Davidson, AE; + view all <#> Hafford-Tear, NJ; Tsai, Y-C; Sadan, AN; Sanchez-Pintado, B; Zarouchlioti, C; Maher, GJ; Liskova, P; Tuft, SJ; Hardcastle, AJ; Clark, TA; Davidson, AE; - view fewer <#> (2019) CRISPR/Cas9-targeted enrichment and long-read sequencing of the Fuchs endothelial corneal dystrophy–associated TCF4 triplet repeat. Genetics in Medicine 10.1038/s41436-019-0453-x <https://doi.org/10.1038/s41436-019-0453-x>. (In press). Green open access document_url: https://discovery.ucl.ac.uk/id/eprint/10067655/1/Davidson_CRISPR%20or%20Cas9-targeted%20enrichment%20and%20long-read%20sequencing%20of%20the%20Fuchs%20endothelial%20corneal%20dystrophy-associated%20TCF4%20triplet%20repeat_Proof.pdf