Applications of CRISPR/Cas9 for Selective Sequencing and Clinical Diagnostics

In this chapter, we will discuss the applications of CRISPR/Cas9 in the context of clinical diagnostics. We will provide an overview of existing methods and their use cases in the diagnostic field. Special attention will be given to selective sequencing approaches using third-generation sequencing and PAM-site requirements. As target sequences in an AT-rich environment cannot easily be accessed by the commercially available SpCas9 due to rarity of NGG PAM-sites, new enzymes such as ScCas9 with PAM-site requirements of NNG will be highlighted. Original research on CRISPR/Cas9 systems to determine molecular glioma markers by enriching regions of interest will be discussed in the context of potential future applications in clinical diagnostics

Generation of an iPSC line of a patient with Angelman syndrome due to an imprinting defect

Angelman syndrome (AS) is a neurodevelopmental disorder with leading symptoms of happy demeanor, intellectual disability, ataxia and seizures. AS can be caused by genetic and epigenetic aberrations, resulting in the absence of functional UBE3A protein in the brain. UBE3A is an imprinted gene, which is, in neurons of the brain, expressed exclusively from maternal chromosome 15. The generated iPSC line was derived from skin fibroblasts of a patient with AS, who, due to an imprinting defect, lacked DNA methylation at the chromosome 15 imprinting center, which controls maternal-specific expression of UBE3A.Resource tableUnlabelled TableUnique stem cell line identifierZIPi015-KAlternative name(s) of stem cell lineAS_ID, ZIP15InstitutionZentrum für integrative Psychiatrie, University Hospital Kiel, Kiel, GermanyContact information of distributorFranz-Josef Müller, [email protected] Steenpass, [email protected] of cell lineiPSCOriginhumanAdditional origin infoAge: 12Sex: femaleEthnicity if known: caucasianCell Sourceskin fibroblastsClonalityclonalMethod of reprogrammingepisomal/transgene-freeGenetic Modificationepigenetic aberration – imprinting defectType of Modificationlack of DNA methylation establishment or maintenance in the germ line of the patient's motherAssociated diseaseAngelman syndrome (OMIM #105830)Gene/locusPrader-Willi/Angelman syndrome locus, chromosome 15q11q13Method of modificationNAName of transgene or resistanceNAInducible/constitutive systemNADate archived/stock date14.04.2017 (Essen)Cell line repository/bankNAEthical approvalEthikkommission der medizinischen Fakultät der Christian-Albrechts Universität zu Kiel, Approval number A145/11A145/1

Generation of two human isogenic iPSC lines from fetal dermal fibroblasts

Two isogenic hiPSC lines, ZIPi013-B and ZIPi013-E, were generated by reprogramming fetal dermal fibroblasts with episomal vectors. Previously, the same fetal fibroblasts were reprogrammed multiple times in a study comparing other reprogramming methods. As a consequence, the genomes have been sequenced multiple times. Both new cell lines offer the opportunity to study basic stem cell biology and model human disease. They can be applied as reference cell lines for creating isogenic clones bearing disease mutations on a well-characterized genomic background, as both cell lines have demonstrated excellent differentiation capacity in multiple labs.Resource tableUnlabelled TableUnique stem cell lines identifierZIPi013-BZIPi013-EAlternative names of stem cell linesZIP13K2 (ZIPi013-B)ZIP13K5 (ZIPi013-E)InstitutionZentrum für Integrative Psychiatrie gGmbH, Kiel, GermanyContact information of distributorPD Dr. Franz-Josef Müller, [email protected] of cell linesiPSCOriginhumanCell SourceFibroblastsClonalityClonalMethod of reprogrammingTransgene free, episomalMultiline rationaleIsogenic clonesGene modificationNOType of modificationN/AAssociated diseaseN/AGene/locusN/AMethod of modificationN/AName of transgene or resistanceN/AInducible/constitutive systemN/ADate archived/stock dateStock date ZIPi013-B 8th December 2017, stock date ZIPi013-E 12th December 2017Cell line repository/bankN/AEthical approvalhttps://www.sciencellonline.com/technical-support/ethical-statement.htmlEthikkommission der medizinischen Fakultät der Christian-Albrechts-Universität zu Kiel, approval number A145/1

Enhanced cortical neural stem cell identity through short SMAD and WNT inhibition in human cerebral organoids facilitates emergence of outer radial glial cells

Cerebral organoids exhibit broad regional heterogeneity accompanied by limited cortical cellular diversity despite the tremendous upsurge in derivation methods, suggesting inadequate patterning of early neural stem cells (NSCs). Here we show that a short and early Dual SMAD and WNT inhibition course is necessary and sufficient to establish robust and lasting cortical organoid NSC identity, efficiently suppressing non-cortical NSC fates, while other widely used methods are inconsistent in their cortical NSC-specification capacity. Accordingly, this method selectively enriches for outer radial glia NSCs, which cyto-architecturally demarcate well-defined outer sub-ventricular-like regions propagating from superiorly radially organized, apical cortical rosette NSCs. Finally, this method culminates in the emergence of molecularly distinct deep and upper cortical layer neurons, and reliably uncovers cortex-specific microcephaly defects. Thus, a short SMAD and WNT inhibition is critical for establishing a rich cortical cell repertoire that enables mirroring of fundamental molecular and cyto-architectural features of cortical development and meaningful disease modelling

An Organoid-Based Model of Cortical Development Identifies Non-Cell-Autonomous Defects in Wnt Signaling Contributing to Miller-Dieker Syndrome

Miller-Dieker syndrome (MDS) is caused by a heterozygous deletion of chromosome 17p13.3 involving the genes LIS1 and YWHAE (coding for 14.3.3ε) and leads to malformations during cortical development. Here, we used patient-specific forebrain-type organoids to investigate pathological changes associated with MDS. Patient-derived organoids are significantly reduced in size, a change accompanied by a switch from symmetric to asymmetric cell division of ventricular zone radial glia cells (vRGCs). Alterations in microtubule network organization in vRGCs and a disruption of cortical niche architecture, including altered expression of cell adhesion molecules, are also observed. These phenotypic changes lead to a non-cell-autonomous disturbance of the N-cadherin/β-catenin signaling axis. Reinstalling active β-catenin signaling rescues division modes and ameliorates growth defects. Our data define the role of LIS1 and 14.3.3ε in maintaining the cortical niche and highlight the utility of organoid-based systems for modeling complex cell-cell interactions in vitro

Generation of an induced pluripotent stem cell line, ZIPi021-A, from fibroblasts of a Prader-Willi syndrome patient with maternal uniparental disomy (mUPD)

Prader-Willi syndrome (PWS) is a neurodevelopmental disorder caused by loss of paternal expression of imprinted genes on chromosome 15q11-q13. We established a human induced pluripotent stem cell line (hiPSC), ZIPi021-A, from fibroblasts of a 4-year-old female PWS patient with the subtype of maternal uniparental disomy (mUPD). The generated hiPSC line was transgene-free, expressed pluripotency markers and showed the ability to differentiate into all three germ layers in vitro. The ZIPi021-A hiPSC line could be used as a cellular model for PWS in humans

Comparison of methylation estimates obtained via MinION nanopore sequencing and sanger bisulfite sequencing in the TRPA1 promoter region

Abstract Background Bisulfite sequencing has long been considered the gold standard for measuring DNA methylation at single CpG resolution. However, in recent years several new approaches like nanopore sequencing have been developed due to hints for a partial error-proneness of bisulfite sequencing. Since these errors were shown to be sequence-specific, we aimed to verify the methylation data of a particular region of the TRPA1 promoter from our previous studies obtained by bisulfite sequencing. Methods We compared methylation rates determined by direct bisulfite sequencing and nanopore sequencing following Cas9-mediated PCR-free enrichment. Results We could show that CpG methylation levels above 20% corroborate well with our previous data. Within the range between 0 and 20% methylation, however, Sanger sequencing data have to be interpreted cautiously, at least in the investigated region of interest (TRPA1 promotor region). Conclusion Based on the investigation of the TRPA1- region as an example, the present work can help in choosing the right method out of the two current main approaches for methylation analysis for different individual settings regarding many factors like cohort size, costs and prerequisites that should be fulfilled for each method. All in all, both methods have their raison d’être. Furthermore, the present paper contains and illustrates some important basic information and explanation of how guide RNAs should be located for an optimal outcome in Cas9 mediated PCR free target enrichment

Dnmt1 has de novo activity targeted to transposable elements

DNA methylation plays a critical role during development, particularly in repressing retrotransposons. The mammalian methylation landscape is dependent on the combined activities of the canonical maintenance enzyme Dnmt1 and the de novo Dnmts, 3a and 3b. Here, we demonstrate that Dnmt1 displays de novo methylation activity in vitro and in vivo with specific retrotransposon targeting. We used whole-genome bisulfite and long-read Nanopore sequencing in genetically engineered methylation-depleted mouse embryonic stem cells to provide an in-depth assessment and quantification of this activity. Utilizing additional knockout lines and molecular characterization, we show that the de novo methylation activity of Dnmt1 depends on Uhrf1, and its genomic recruitment overlaps with regions that enrich for Uhrf1, Trim28 and H3K9 trimethylation. Our data demonstrate that Dnmt1 can catalyze DNA methylation in both a de novo and maintenance context, especially at retrotransposons, where this mechanism may provide additional stability for long-term repression and epigenetic propagation throughout development.ISSN:1545-9993ISSN:1545-998