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