Efficient CRISPR/Cas9-mediated editing of trinucleotide repeat expansion in myotonic dystrophy patient-derived iPS and myogenic cells

International audienceCRISPR/Cas9 is an attractive platform to potentially correct dominant genetic diseases by gene editing with unprecedented precision. In the current proof-of-principle study, we explored the use of CRISPR/Cas9 for gene-editing in myotonic dys-trophy type-1 (DM1), an autosomal-dominant muscle disorder, by excising the CTG-repeat expansion in the 3-untranslated-region (UTR) of the human myotonic dystrophy protein kinase (DMPK) gene in DM1 patient-specific induced pluripotent stem cells (DM1-iPSC), DM1-iPSC-derived myogenic cells and DM1 patient-specific myoblasts. To eliminate the pathogenic gain-of-function mutant DMPK transcript , we designed a dual guide RNA based strategy that excises the CTG-repeat expansion with high efficiency , as confirmed by Southern blot and single molecule real-time (SMRT) sequencing. Correction efficiencies up to 90% could be attained in DM1-iPSC as confirmed at the clonal level, following ribonucle-oprotein (RNP) transfection of CRISPR/Cas9 components without the need for selective enrichment. Expanded CTG repeat excision resulted in the disappearance of ribonuclear foci, a quintessential cellular phenotype of DM1, in the corrected DM1-iPSC, DM1-iPSC-derived myogenic cells and DM1 myoblasts. Consequently, the normal intracellular localization of the muscleblind-like splicing regulator 1 (MBNL1) was restored, resulting in the normalization of splicing pattern of SERCA1. This study validates the use of CRISPR/Cas9 for gene editing of repeat expansions

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

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

Determination of the Variability and Associated Epigenetic Signature of Tandem Repeats by Single Molecule Sequencing

Around 1.5 million short tandem repeats (STRs) are spread across the entire human genome. STRs are functionally important elements that are able to modulate the phenotype of an individual. They can modify cellular biology by influencing the genome, transcriptome and proteome of a cell. The most extreme examples of the functional impact of STRs are the more than 40 repeat expansion disorders like fragile X syndrome (FXS) and myotonic dystrophy 1 (DM1). Up to now, many aspects of STRs remain illusive. Due to an historical underestimation of their importance and the lack of adequate technologies they remain understudied. The rise of Single Molecule Real-Time (SMRT) sequencing from Pacific Biosciences changes this paradigm and arms researchers with better tools to investigate STRs. In this thesis we studied different aspects of STRs, thereby exploring different assets of SMRT sequencing. SMRT sequencing can span long, GC-rich repeats, whilst simultaneously revealing DNA modifications in the sequenced region. Unfortunately, the throughput of the technology is limited, making it economically unfeasible to sequence an entire genome if one is only interested in a single locus or a subset of the genome. Therefore, enrichment strategies like PCR are commonly being used. These strategies aver nevertheless very error-prone, especially when amplifying repeats. Furthermore, they remove all epigenetic marks. Thus, amplification impedes the complete genetic and epigenetic characterization of STRs. To tackle this, we developed a CRISPR-CAS9 approach to excise the FMR1 CGG repeat in combination with restriction enzymes to remove off-target genomic DNA. This generated a very accurate picture of the FMR1 CGG repeat variability of the BAC molecule and made it possible to identify DNA methylation. Indeed, besides avoiding amplification biases, this method permits native DNA capture and, hence, allows for direct detection of base modifications. On human DNA, enrichment factors over 100X were achieved while up to 5 reads covering the FMR1 CGG repeat could be retrieved from one SMRT cell. Albeit further improvements are necessary to allow wide spread implementation, this method has the potential to significantly further unravel the complex genotype-phenotype correlations in FXS. In addition, it could be used to screen for long, methylated CGG alleles in diagnostics where it could replace complicated and laborious Southern blots. FXS arises from the FMR1 CGG expansion of a premutation (55-200 repeats) to a full mutation allele (>200 repeats) in females. This type of expansion is the most frequent cause of inherited X-linked intellectual disability. The risk for a premutation to expand to a full mutation allele depends on the repeat length and AGG triplets interrupting this repeat. Therefore, it is necessary to map these AGG interruptions in order to study the stability of the FMR1 allele. Additionally, easy access to accurate size estimates and AGG information is also of great importance in genetic counseling since they allow for women carrying a premutation allele to estimate the risk for expansion. Unfortunately, the detection of AGG interruptions is hampered by technical difficulties. We demonstrated that single-molecule sequencing enables the determination of not only the repeat size, but also of the complete repeat sequence, including AGG interruptions in male and female alleles. This approach outperforms current strategies because it allows for an unambiguous separation of the normal allele from the expanded one. This permits the determination of the repeat structure for each allele in every male or female. Hence, we implemented SMRT sequencing as a diagnostic tool to identify AGG interruptions in females with a FMR1 premutation. By doing so, we improved the risk assessments for genetic counseling and positively impacted the management of the disorder. Except for diagnostic use, single-molecule sequencing will also facilitate large-scale studies assessing the influence of AGG interruptions on the stability of the CGG repeat. We performed already a proof-of-principle study to investigate the influence of AGG's on the stability of intermediate FMR1 CGG alleles (45-54 repeat units). SMRT sequencing was also explored to study the DMPK CTG repeat underlying DM1. Firstly, the variability of long CTG repeats was determined by small-pool PCR followed by long-read sequencing. This approach resulted in a higher accuracy, higher throughput and less hands-on time compared to Southern blots. Therefore, this methodology is now used to study the influence of the mismatch repair system on DM1 repeat instability. Undoubtedly, this approach will be implemented more broadly in the future. Besides, long-read sequencing was also used to assess the efficiency of CRISPR/CAS9 excision of the DMPK CTG repeat region. To our knowledge, this is the first study tackling the DMPK CTG repeat by single-molecule long-read sequencing. Ultimately, a targeted amplification-free enrichment method for the DMPK CTG repeat would remove the need for PCR completely and could further improve the analysis of this repeat. To conclude, SMRT sequencing is a powerful tool forging ahead STR research and diagnostics. In this thesis novel methodologies were developed to make maximal use of the advantages of SMRT sequencing (high accuracy, long reads & detection of base modifications). It will be interesting to see how novel methodologies employing long-read sequencing developed in this thesis and by other research groups will move the STR field ahead in the future.status: publishe

The future of prenatal cytogenetics: from copy number variations to non-invasive prenatal testing

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Single molecule real-time (SMRT) sequencing comes of age: applications and utilities for medical diagnostics

Short read massive parallel sequencing has emerged as a standard diagnostic tool in the medical setting. However, short read technologies have inherent limitations such as GC bias, difficulties mapping to repetitive elements, trouble discriminating paralogous sequences, and difficulties in phasing alleles. Long read single molecule sequencers resolve these obstacles. Moreover, they offer higher consensus accuracies and can detect epigenetic modifications from native DNA. The first commercially available long read single molecule platform was the RS system based on PacBio's single molecule real-time (SMRT) sequencing technology, which has since evolved into their RSII and Sequel systems. Here we capsulize how SMRT sequencing is revolutionizing constitutional, reproductive, cancer, microbial and viral genetic testing.status: publishe

Single molecule real-time (SMRT) sequencing comes of age : applications and utilities for medical diagnostics

Short read massive parallel sequencing has emerged as a standard diagnostic tool in the medical setting. However, short read technologies have inherent limitations such as GC bias, difficulties mapping to repetitive elements, trouble discriminating paralogous sequences, and difficulties in phasing alleles. Long read single molecule sequencers resolve these obstacles. Moreover, they offer higher consensus accuracies and can detect epigenetic modifications from native DNA. The first commercially available long read single molecule platform was the RS system based on PacBio's single molecule realtime (SMRT) sequencing technology, which has since evolved into their RSII and Sequel systems. Here we capsulize how SMRT sequencing is revolutionizing constitutional, reproductive, cancer, microbial and viral genetic testing

Detecting AGG Interruptions in Male and Female FMR1 Premutation Carriers by Single-Molecule Sequencing

The FMR1 gene contains an unstable CGG repeat in its 5' untranslated region. Premutation alleles range between 55 and 200 repeat units and confer a risk for developing fragile X-associated tremor/ataxia syndrome or fragile X-associated primary ovarian insufficiency. Furthermore, the premutation allele often expands to a full mutation during female germline transmission giving rise to the fragile X syndrome. The risk for a premutation to expand depends mainly on the number of CGG units and the presence of AGG interruptions in the CGG repeat. Unfortunately, the detection of AGG interruptions is hampered by technical difficulties. Here, we demonstrate that single-molecule sequencing enables the determination of not only the repeat size, but also the complete repeat sequence including AGG interruptions in male and female alleles with repeats ranging from 45 to 100 CGG units. We envision this method will facilitate research and diagnostic analysis of the FMR1 repeat expansion. This article is protected by copyright. All rights reserved.status: publishe

Detecting AGG Interruptions in Females With a FMR1 Premutation by Long-Read Single-Molecule Sequencing: A 1 Year Clinical Experience

The fragile X syndrome arises from the FMR1 CGG expansion of a premutation (55-200 repeats) to a full mutation allele (>200 repeats) and is the most frequent cause of inherited X-linked intellectual disability. The risk for a premutation to expand to a full mutation allele depends on the repeat length and AGG triplets interrupting this repeat. In genetic counseling it is important to have information on both these parameters to provide an accurate risk estimate to women carrying a premutation allele and weighing up having children. For example, in case of a small risk a woman might opt for a natural pregnancy followed up by prenatal diagnosis while she might choose for preimplantation genetic diagnosis (PGD) if the risk is high. Unfortunately, the detection of AGG interruptions was previously hampered by technical difficulties complicating their use in diagnostics. Therefore we recently developed, validated and implemented a new methodology which uses long-read single-molecule sequencing to identify AGG interruptions in females with a FMR1 premutation. Here we report on the assets of AGG interruption detection by sequencing and the impact of implementing the assay on genetic counseling.status: publishe

Detecting AGG Interruptions in Females With a FMR1 Premutation by Long-Read Single-Molecule Sequencing: A 1 Year Clinical Experience

The fragile X syndrome arises from the FMR1 CGG expansion of a premutation (55–200 repeats) to a full mutation allele (>200 repeats) and is the most frequent cause of inherited X-linked intellectual disability. The risk for a premutation to expand to a full mutation allele depends on the repeat length and AGG triplets interrupting this repeat. In genetic counseling it is important to have information on both these parameters to provide an accurate risk estimate to women carrying a premutation allele and weighing up having children. For example, in case of a small risk a woman might opt for a natural pregnancy followed up by prenatal diagnosis while she might choose for preimplantation genetic diagnosis (PGD) if the risk is high. Unfortunately, the detection of AGG interruptions was previously hampered by technical difficulties complicating their use in diagnostics. Therefore we recently developed, validated and implemented a new methodology which uses long-read single-molecule sequencing to identify AGG interruptions in females with a FMR1 premutation. Here we report on the assets of AGG interruption detection by sequencing and the impact of implementing the assay on genetic counseling

De liefde voor het vak: op zoek naar een pedagogiek van meesterschap

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