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CRISPR/Cas9-targeted enrichment and long-read sequencing of the Fuchs endothelial corneal dystrophy–associated TCF4 triplet repeat
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CRISPR/Cas9-targeted enrichment and long-read sequencing of the Fuchs endothelial corneal dystrophy–associated TCF4 triplet repeat
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CRISPR/Cas9-targeted enrichment and long-read sequencing of the Fuchs endothelial corneal dystrophy–associated TCF4 triplet repeat
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Journal Article
CRISPR/Cas9-targeted enrichment and long-read sequencing of the Fuchs endothelial corneal dystrophy–associated TCF4 triplet repeat
2019
Overview
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).
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.
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.
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.
Publisher
Elsevier Inc,Nature Publishing Group US,Elsevier Limited
Subject
/ Aged
/ Alleles
/ amplification-free sequencing
/ Biomedical and Life Sciences
/ CRISPR
/ CRISPR-Cas Systems - genetics
/ Female
/ Fuchs endothelial corneal dystrophy
/ Fuchs' Endothelial Dystrophy - genetics
/ Fuchs' Endothelial Dystrophy - pathology
/ Genetic Predisposition to Disease
/ Genotype
/ Humans
/ Male
/ Transcription Factor 4 - genetics
/ Trinucleotide Repeat Expansion - genetics
