CRISPR/Cas9-targeted enrichment and long-read sequencing of the Fuchs endothelial corneal dystrophy–associated TCF4 triplet repeat

Abstract

PurposeTo 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).MethodsWe 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.ResultsCRISPR-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.ConclusionCRISPR-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.

A novel framework for characterizing genomic haplotype diversity in the human immunoglobulin heavy chain locus

“…Furthermore, the sensitivity of SV detection is improved by resolving variants in a haplotype-specific manner 38,42 . When long-read sequencing has been combined with specific target enrichment methods, using either a CRISPR/Cas9 system 43,44 or DNA probes 45,46 , it has been shown to yield accurate and contiguous assemblies. Such targeted approaches have enabled higher resolution genotyping of the HLA loci 47 and KIR regions 48,49 .…”

Detecting genetic variation and base modifications together in the same single molecules of DNA and RNA at base pair resolution using a magnetic tweezer platform

“…Our MT platform is well suited to this dual analysis in the same single molecules of DNA. To analyze specific gene loci without DNA amplification, we developed the CRISPR/Cas9 enrichment method that achieved target specificities that are far greater than we have seen reported elsewhere in the published literature [10][11][12] . From human genomic DNA, we enriched fragments containing FMR1 and observed that the allele frequency of expanded repeats to normal was in the expected 1:1 ratio for a heterozygous sample.…”

“…Compared to full genome sequencing, these protocols dramatically reduce per-sample costs as multiple samples can be pooled and multiplexed; however, because these methods involve sample amplification, this benefit comes at the penalty of loss of epigenetic information and the introduction of bias into the results 9 . Recently amplification-free Cas9-based enrichment strategies have been attempted by several groups [10][11][12] but typically only achieve an enrichment of 20-to 60-fold (compared to 10,000-fold for PCR) 13 .…”

High-throughput multiplexed tandem repeat genotyping using targeted long-read sequencing

“…An amplification free targeted analysis with long-read sequencing is an ideal option for accurate genotyping TRs. Targeted cleavage with Cas9 enzyme followed by Nanopore sequencing [26] or PacBio sequencing [27] has been recently reported as alternative option for enrichment of regions of interest. This method does not have any amplifications and can be adapted for multiple targets in a single assay.…”