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Results for “Molecular Biology”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2017

The Mediator co-activator complex regulates Ty1 retromobility via alternative transcription start site selection

The Ty1 retrotransposons present in the genome of Saccharomyces cerevisiae belong to the large class of mobile genetic elements that replicate via an RNA intermediary and constitute a significant portion of most eukaryotic genomes. The retromobility of Ty1 is regulated by numerous host factors, including several subunits of the Mediator transcriptional co-activator complex. In spite of its known function in the nucleus, previous studies have implicated Mediator in the regulation of post-translational steps in Ty1 retromobility. To resolve this paradox, we systematically examined the effects of deleting nonessential Mediator subunits on the frequency of Ty1 retromobility and levels of retromobility intermediates. Our findings reveal that loss of distinct Mediator subunits alters Ty1 retromobility positively or negatively over a >10,000-fold range by regulating the ratio of an internal transcript, Ty1i, to the genomic Ty1 transcript. Ty1i RNA encodes a dominant negative inhibitor of Ty1 retromobility that blocks virus-like particle maturation and cDNA synthesis. These results resolve the conundrum of Mediator exerting sweeping control of Ty1 retromobility with only minor effects on the levels of Ty1 genomic RNA and the capsid protein, Gag. Since the majority of characterized intrinsic and extrinsic regulators of Ty1 retromobility alter a post-translational step(s), Mediator could play a central role in integrating signals that influence Ty1i expression to modulate retromobility.\n\nAuthor SummaryRetrotransposons are mobile genetic elements that copy their RNA genomes into DNA and insert the DNA copies into the host genome. These elements contribute to genome instability, control of host gene expression and adaptation to changing environments. Retrotransposons depend on numerous host factors for their own propagation and control. The retrovirus-like retrotransposon, Ty1, in the yeast Saccharomyces cerevisiae has been an invaluable model for retrotransposon research, and hundreds of host factors that regulate Ty1 retrotransposition have been identified. Non-essential subunits of the Mediator transcriptional co-activator complex have been identified as one set of host factors implicated in Ty1 regulation. Here, we report a systematic investigation of the effects of loss of these non-essential subunits of Mediator on Ty1 retrotransposition. Our findings reveal a heretofore unknown mechanism by which Mediator influences the balance between transcription from two promoters in Ty1 to modulate expression of an autoinhibitory transcript known as Ty1i RNA. Our results provide new insights into host control of retrotransposon activity via promoter choice and elucidate a novel mechanism by which the Mediator co-activator governs this choice.

molecular biology↗

bioRxiv2017

PTE, a novel module of mammalian Polycomb Response Elements

Polycomb Group proteins are essential epigenetic repressors. They form multiple protein complexes of which two kinds, PRC1 and PRC2, are indispensable for repression. Although much is known about their biochemical properties, how PRC1 and PRC2 are targeted to specific genes is poorly understood. Here we establish the Cyclin D2 (CCND2) oncogene as a simple model to address this question. We provide the evidence that coordinated recruitment of PRC1 and PRC2 complexes to CCND2 involves a combination of a specialized PRC1 targeting element (PTE) and an adjacent CpG-island, which together act as a human Polycomb Response Element. Chromatin immunoprecipitation analysis of CCND2 in different transcriptional states indicates that histone modifications produced by PRC1 and PRC2 are not sufficient to recruit either of the complexes. However, catalytic activity of PRC2 helps to anchor PRC1 at the PTE. Our analyses suggest that coordinated targeting of PRC1 and PRC2 complexes by juxtaposed AT-rich PTEs and CpG-islands may be a general feature of Polycomb repression in mammals.

molecular biology↗

bioRxiv2017

Transient Reduction of DNA Methylation at the Onset of Meiosis in Male Mice

The quality of germ cells depends on successful chromatin organization in meiotic prophase I (MPI). To better understand the epigenetic context of MPI we studied the dynamics of DNA methylation in wild-type male mice. We discovered an extended period of genome-wide transient reduction of DNA methylation (TRDM) during early MPI. Our data show that TRDM arises by passive demethylation in the premeiotic S phase highlighting the abundance of hemimethylated DNA in MPI. Importantly, TRDM unmasks a deficit in retrotransposon LINE-1 DNA methylation contributing to its expression in early MPI. We propose that TRDM facilitates meiosis and gamete quality control.

molecular biology↗

bioRxiv2017

Sleeping Beauty transposon integrates into non-TA dinucleotides via an alternative mechanism

Sleeping Beauty transposon (SB) is an important genetic tool for generating mutations in vertebrates. It is well known that SB exclusively integrates into TA dinucleotides. However, this \"TA law\" has never been strictly tested in large number of insertion sites after next generation sequencing was widely utilized. In this study, we analyzed 600 million pairs of Illumina sequence reads and identified 28 thousand SB insertions in non-TA sites. We recovered some non-TA sites using PCR and confirmed that at least parts of the insertions at non-TA sites are real integrations. The consensus sequence of these non-TA sites showed an asymmetric pattern distinct from the symmetric pattern of the canonical TA sites. The right side of the consensus sequence is exactly the same as the sequence of SB transposon ends, indicating interaction between the transposon DNA and the target DNA. Based on these results we suggested that SB has an alternative integration mechanism besides the canonical one to integrate its DNA into non-TA sites.\n\nHighlights[~] For the first time, we proved that Sleeping Beauty transposon can integrate into non-TA dinucleotides.\n\n [~]For the first time, we provided evidence that transposon DNA can directly interact with target DNA.\n\n [~]And for the first time, we found that a transposon can have two independent integration mechanisms.

molecular biology↗

bioRxiv2017

Thiol-linked alkylation for the metabolic sequencing of RNA

Gene expression profiling by high-throughput sequencing reveals qualitative and quantitative changes in RNA species at steady-state but obscures the intracellular dynamics of RNA transcription, processing and decay. We developed thiol(SH)-linked alkylation for the metabolic sequencing of RNA (SLAM-seq), an orthogonal chemistry-based epitranscriptomics-sequencing technology that uncovers 4-thiouridine (s4U)-incorporation in RNA species at single-nucleotide resolution. In combination with well-established metabolic RNA labeling protocols and coupled to standard, low-input, high-throughput RNA sequencing methods, SLAM-seq enables rapid access to RNA polymerase II-dependent gene expression dynamics in the context of total RNA. When applied to mouse embryonic stem cells, SLAM-seq provides global and transcript-specific insights into pluripotency-associated gene expression. We validated the method by showing that the RNA-polymerase II-dependent transcriptional output scales with Oct4/Sox2/Nanog-defined enhancer activity; and we provide quantitative and mechanistic evidence for transcript-specific RNA turnover mediated by post-transcriptional gene regulatory pathways initiated by microRNAs and N6-methyladenosine. SLAM-seq facilitates the dissection of fundamental mechanisms that control gene expression in an accessible, cost-effective, and scalable manner.\n\nOne Sentence SummaryChemical nucleotide-analog derivatization provides global insights into transcriptional and post-transcriptional gene regulation

molecular biology↗

bioRxiv2017

The RS Domain of Human CFIm68 Plays a Key Role in Selection Between Alternative Sites of Pre-mRNA Cleavage and Polyadenylation

Many eukaryotic protein-coding genes give rise to alternative mRNA isoforms with identical protein-coding capacities but which differ in the extents of their 3{acute} untranslated regions (3{acute}UTRs), due to the usage of alternative sites of pre-mRNA cleavage and polyadenylation. By governing the presence of regulatory 3{acute}UTR sequences, this type of alternative polyadenylation (APA) can significantly influence the stability, localisation and translation efficiency of mRNA. Though a variety of molecular mechanisms for APA have been proposed, previous studies have identified a pivotal role for the multi-subunit cleavage factor I (CFIm) in this process in mammals. Here we show that, in line with previous reports, depletion of the CFIm 68 kDa subunit (CFIm68) by CRISPR/Cas9-mediated gene disruption in HEK293 cells leads to a shift towards the use of promoter-proximal poly(A) sites. Using these cells as the basis for a complementation assay, we show that CFIm68 lacking its arginine/serine-rich (RS) domain retains the ability to form a nuclear complex with other CFIm subunits, but selectively lacks the capacity to restore polyadenylation at promoter-distal sites. In addition, nanoparticle-mediated analysis indicates that the RS domain is extensively phosphorylated in vivo. Overall, these results suggest that the CFIm68 RS domain makes a key regulatory contribution to APA.

molecular biology↗

bioRxiv2017

Design and specificity of long ssDNA donors for CRISPR-based knock-in

CRISPR/Cas technologies have transformed our ability to manipulate genomes for research and gene-based therapy. In particular, homology-directed repair after genomic cleavage allows for precise modification of genes using exogenous donor sequences as templates. While both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) forms of donors have been used as repair templates, a systematic comparison of the performance and specificity of repair using ssDNA versus dsDNA donors is still lacking. Here, we describe an optimized method for the synthesis of long ssDNA templates and demonstrate that ssDNA donors can drive efficient integration of gene-sized reporters in human cell lines. We next define a set of rules to maximize the efficiency of ssDNA-mediated knock-in by optimizing donor design. Finally, by comparing ssDNA donors with equivalent dsDNA sequences (PCR products or plasmids), we demonstrate that ssDNA templates have a unique advantage in terms of repair specificity while dsDNA donors can lead to a high rate of off-target integration. Our results provide a framework for designing high-fidelity CRISPR-based knock-in experiments, in both research and therapeutic settings. O_TEXTBOXUpdate: November 12th, 2019 Dear bioRxiv community, The conclusions of this pre-print (originally posted in August 2017) are outdated. While the experiments we present here are accurate, a recent and more systematic analysis revealed that the integration outcomes driven by different forms of HDR donors are more complex than our methods could originally identify. We initially analyzed donor integration only in FACS-selected cells, which under-estimates alleles where the mis-integration of payload leads to non-functional selection markers, and we quantified integration by ddPCR, which is an indirect read-out of sequence properties. These approaches could not capture the full details of donor integration events in our experiments. To address this, we have now developed a new framework based on long-read amplicon sequencing and an integrated computational pipeline to precisely analyze knock-in repair outcomes across a wide range of experimental parameters. Our new data uncover a complex repair landscape in which both single-stranded and double-stranded donors can lead to high rates of imprecise integration in some cell types. Please read our new bioRxiv pre-print entitled "Deep profiling reveals substantial heterogeneity of integration outcomes in CRISPR knock-in experiments" for further information. I hope that this example highlights one of the powers of pre-prints: the ability to update scientific discussions (and set records straight) as new results are obtained, often fueled by the availability of new technologies. Please do not hesitate to contact me directly for any questions or comments. - Manuel Leonetti (manuel.leonetti@czbiohub.org) C_TEXTBOX

molecular biology↗

bioRxiv2017

Identification of an Essential IL15-STAT1-IRX3 Prosurvival Pathway in T Lymphocytes with Therapeutic Implications

IRX3 homeobox transcription factor plays a role in neural development and in the pathogenesis of obesity and insulin resistance. T lymphocytes play an essential role in the pathogenesis of obesity and their expansion and differentiation is a tightly controlled process. Circulating cytokines trough activation of cell death mechanisms controls the number of CD4 and CD8 T cells, and failure to activate apoptosis is considered the main cause for their uncontrolled expansion. Here, the identification an essential anti-apoptotic pathway in T lymphocytes is presented, whereby IL-15 activation of Stat/Jak signaling cascade leads to induction of the IRX3 expression. IRX3, in turn, promotes T cells survival and self-renewal, at least partially, by controlling the expression of T-bet transcription factor eomesodermin (EOMES). The Stat/Jak signaling inhibition by Jak inhibitor 1 suppresses IRX3 transcription in T lymphocytes and tumor cell lines and increases apoptosis after growth factor deprivation. Our results demonstrate that IRX3 is essential in T lymphocytes differentiation and reveal that the IL-15-dependent IRX3 pro-survival pathway provides potential therapeutic target for regulation of T cell proliferation that has implications for immune cell malignancies, obesity and autoimmune diseases.

molecular biology↗

bioRxiv2017

Modeling of Epigenetic Modification-Induced Changes in CRX-dependent Genes cis-Regulatory Elements

PurposeDNA methylation is a well characterized epigenetic repressor of mRNA transcription in many plant and vertebrate systems. However, the mechanism of this repression is not fully understood. The process of synthesizing a strand of RNA from DNA, or transcription, is controlled by proteins that regulate RNA polymerase activity by binding to specific gene regulatory sequences. Cone-rod homeobox (CRX) is a well-characterized mammalian transcription factor that controls photoreceptor cell specific gene expression. While much is known about the functions and DNA binding specificity of CRX, less is known about how DNA methylation modulates CRX binding affinity to genomic cis-regulatory elements.\n\nMethodsWe used bisulfite pyrosequencing of human ocular tissues to measure DNA methylation levels of the regulatory regions of RHO, PDE6B, PAX6, and LINE. To describe the molecular mechanism of repression, we used molecular modeling to illustrate the effect of DNA methylation on human RHO regulatory sequences.\n\nResultsIn this study, we demonstrate an inverse correlation between DNA methylation in regulatory regions adjacent to the human RHO and PDE6B genes and their subsequent transcription in human ocular tissues. Docking of CRX to our DNA models shows that CRX interacts with the grooves of these sequences, suggesting changes in groove structure could regulate binding. Molecular dynamics simulations of the RHO promoter and enhancer regions show changes in the flexibility and groove width upon epigenetic modification. Models also demonstrate that changes to the local dynamics of CRX binding sites within RHO regulatory sequences which may account for the repression of CRX dependent transcription.\n\nConclusionCollectively, these data demonstrate epigenetic regulation of CRX binding sites in human retinal tissue and provide insight into the mechanism of this mode of epigenetic regulation to be tested in future experiments.

molecular biology↗

bioRxiv2017

Role for Ribosome-Associated Complex and Stress-Seventy subfamily B (RAC-Ssb) in integral membrane protein translation

Targeting of most integral membrane proteins to the endoplasmic reticulum is controlled by the signal recognition particle (SRP), which recognizes a hydrophobic signal sequence near the protein N-terminus. Proper folding of these proteins is monitored by the unfolded protein response, and involves protein degradation pathways to ensure quality control. Here, we identify a new pathway for quality control of major facilitator superfamily transporters that occurs before the first transmembrane helix-the signal sequence recognized by SRP-is made by the ribosome. Increased rates of translation elongation of the N-terminal sequence of these integral membrane proteins can divert the nascent protein chains to the ribosome-associated complex (RAC) and Stress-Seventy Subfamily B (Ssb) chaperones. We also show that quality control of integral membrane proteins by RAC-Ssb couples translation rate to the unfolded protein response, which has implications for understanding mechanisms underlying human disease and protein production in biotechnology.

molecular biology↗

bioRxiv2017

Active ribosome profiling with RiboLace

Ribosome profiling, or Ribo-Seq, is based around large-scale sequencing of RNA fragments protected from nuclease digestion by ribosomes. Thanks to its unique ability to provide positional information concerning ribosomes flowing along transcripts, this method can be used to shed light on mechanistic aspects of translation. However, current Ribo-Seq approaches lack the ability to distinguish between fragments protected by ribosomes in active translation or by inactive ribosomes. To overcome these significant limitation, we developed RiboLace: a novel method based on an original puromycin-containing molecule capable of isolating active ribosomes by means of an antibody-free and tag-free pull-down approach. RiboLace is fast, works reliably with low amounts of input material, and can be easily and rapidly applied both in vitro and in vivo, thereby generating a global snapshot of active ribosome footprints at single nucleotide resolution.

molecular biology↗

bioRxiv2017

Efficient in situ barcode sequencing using padlock probe-based BaristaSeq

Cellular DNA/RNA tags (barcodes) allow for multiplexed cell lineage tracing and neuronal projection mapping with cellular resolution. Conventional approaches to reading out cellular barcodes trade off spatial resolution with throughput. Bulk sequencing achieves high throughput but sacrifices spatial resolution, whereas manual cell picking has low throughput. In situ sequencing could potentially achieve both high spatial resolution and high throughput, but current in situ sequencing techniques are inefficient at reading out cellular barcodes. Here we describe BaristaSeq, an optimization of a targeted, padlock probe-based technique for in situ barcode sequencing compatible with Illumina sequencing chemistry. BaristaSeq results in a five-fold increase in amplification efficiency, with a sequencing accuracy of at least 97%. BaristaSeq could be used for barcode-assisted lineage tracing, and to map long-range neuronal projections.\n\nKey PointsO_LIIn situ sequencing by gap-filling padlock probes is limited by the strand displacement of DNA polymerases\nC_LIO_LIIllumina sequencing chemistry offers superior signal-to-noise ratio in situ compared to sequencing by ligation\nC_LIO_LIBaristaSeq as an accurate method for barcode sequencing in situ with improved gap-filling efficiency\nC_LI

molecular biology↗

bioRxiv2017

Separable functions of the PHD finger protein Spp1 in the Set1 and the meiotic DSB forming complexes cooperate for meiotic DSB formation.

Histone H3K4 methylation is a feature of meiotic recombination hotspots shared by many organisms including plants and mammals. Meiotic recombination is initiated by programmed double-strand break (DSB) formation that in budding yeast takes place in gene promoters and is promoted by histone H3K4 di/trimethylation. This histone modification is recognized by Spp1, a PHD-finger containing protein that belongs to the conserved histone H3K4 methyltransferase Set1 complex. During meiosis, Spp1 binds H3K4me3 and interacts with a DSB protein, Mer2, to promote DSB formation close to gene promoters. How Set1 complex- and Mer2- related functions of Spp1 are connected is not clear. Here, combining genome-wide localization analyses, biochemical approaches and the use of separation of function mutants, we show that Spp1 is present within two distinct complexes in meiotic cells, the Set1 and the Mer2 complexes. Disrupting the Spp1-Set1 interaction mildly decreases H3K4me3 levels and does not affect meiotic recombination initiation. Conversely, the Spp1-Mer2 interaction is required for normal meiotic recombination initiation, but dispensable for Set1 complex-mediated histone H3K4 methylation. Finally, we evidence that Spp1 preserves normal H3K4me3 levels independently of the Set1 complex. We propose a model where the three populations of Spp1 work sequentially to promote recombination initiation: first by depositing histone H3K4 methylation (Set1 complex), next by \"reading\" and protecting histone H3K4 methylation, and finally by making the link with the chromosome axis (Mer2-Spp1 complex). This work deciphers the precise roles of Spp1 in meiotic recombination and opens perspectives to study its functions in other organisms where H3K4me3 is also present at recombination hotspots.\n\nAuthor summaryMeiotic recombination is a conserved pathway of sexual reproduction that is required to faithfully segregate homologous chromosomes and produce viable gametes. Recombination events between homologous chromosomes are triggered by the programmed formation of DNA breaks, which occur preferentially at places called hotspots. In many organisms, these hotspots are located close to a particular chromatin modification, the methylation of lysine 4 of histone H3 (H3K4me3). It was previously shown in the budding yeast model that one protein, Spp1, plays an important function in this process. We further explored the functional link between Spp1 and its interacting partners, and show that Spp1 shows genetically separable functions, by depositing the H3K4me3 mark on the chromatin, \"reading\" and protecting it, and linking it to the recombination proteins. We provide evidence that Spp1 is in three independent complexes to perform these functions. This work opens perspectives for understanding the process in other eukaryotes such as mammals, where most of the proteins involved are conserved.

molecular biology↗

bioRxiv2017

Cryo-EM shows how dynactin recruits two dyneins for faster movement

Dynein and its cofactor dynactin form a highly processive microtubule motor in the presence of an activating adaptor, such as BICD2. Different adaptors link dynein/dynactin to distinct cargos. Here we use electron microscopy (EM) and single molecule studies to show that adaptors can recruit a second dynein to dynactin. Whereas BICD2 is biased toward recruiting a single dynein, the adaptors BICDR1 and HOOK3 predominantly recruit two. We find that the shift toward a double dynein complex increases both force and speed. A 3.5 [A] cryo-EM reconstruction of a dynein tail/dynactin/BICDR1 complex reveals how dynactin can act as a scaffold to coordinate two dyneins side by side. Our work provides a structural basis for how diverse adaptors recruit different numbers of dyneins and regulate the motile properties of the dynein/dynactin transport machine.

molecular biology↗

bioRxiv2017

Infant Transmitted/Founder HIV-1 Viruses from Peripartum Transmission are Neutralization Resistant to Paired Maternal Plasma

Despite extensive genetic diversity of HIV-1 in chronic infection, infant HIV-1 infection involves selective transmission of a single or few maternal virus variants. These transmitted/founder (T/F) variants are of particular interest, as a maternal or infant HIV vaccine should raise envelope (Env)-specific IgG responses capable of blocking this group of viruses. However, the maternal or infant factors that contribute to selection of infant T/F viruses are not well understood. In this study, we isolated HIV-1 env genes by single genome amplification from 16 mother-infant transmitting pairs from the U.S. pre-antiretroviral era Women Infant Transmission Study (WITS). Infant T/F and representative maternal non-transmitted Env variants from plasma were identified and used to generate pseudoviruses for paired maternal plasma neutralization sensitivity analysis. Eighteen out of 21 (85%) infant T/F Env pseudoviruses were neutralization resistant to paired maternal plasma. Yet, all infant T/F viruses were neutralization sensitive to a panel of HIV-1 broadly neutralizing antibodies and variably sensitive to heterologous plasma neutralizing antibodies. Moreover, infant T/F pseudoviruses were overall more neutralization resistant compared to maternal non-transmitted plasma variants (p=0.012). Altogether, our findings suggest that autologous neutralization of circulating viruses by maternal plasma antibodies select for neutralization-resistant viruses that initiate peripartum transmission, raising the spector that enhancement of this response at the end of pregnancy could further reduce infant HIV infection risk.\n\nAuthor SummaryMother to child transmission (MTCT) of HIV-1 can occur during pregnancy (in utero), at the time of delivery (peripartum) or by breastfeeding (postpartum). With the availability of anti-retroviral therapy (ART), rate of MTCT of HIV-1 have been significantly lowered. However, significant implementation challenges remains in resource-poor areas, making it difficult to eliminate pediatric HIV. An improved understanding of the viral population (escape variants from autologous neutralizing antibodies) that lead to infection of infants at time of transmission will help in designing immune interventions to reduce vertical HIV-1 transmission. Here, we selected 16 HIV-1-infected mother-infant pairs from WITS cohort (from pre anti-retroviral era), where infants became infected peripartum. HIV-1 env gene sequences were obtained by the single genome amplification method. The sensitivity of these infant Env pseudoviruses against paired maternal plasma and a panel of broadly neutralizing monoclonal antibodies (bNAbs) was analyzed. We demonstrated that the infant T/F viruses were more resistant against maternal plasma than non-transmitted maternal variants, but sensitive to most (bNAbs). Signature sequence analysis of infant T/F and non-transmitted maternal variants revealed the potential importance of V3 and MPER region for resistance against to paired maternal plasma. These findings provide insights for the design of maternal immunization strategies to enhance neutralizing antibodies that target V3 region of autologous virus populations, which could work synergistically with maternal ARVs to further reduce the rate of peripartum HIV-1 transmission.

molecular biology↗

bioRxiv2017

Downstream promoter interactions of TFIID TAFs facilitate transcription reinitiation

TFIID binds promoter DNA to recruit RNA polymerase II and other basal factors for transcription. Although the TATA-Binding Protein (TBP) subunit of TFIID is necessary and sufficient for in vitro transcription, the TBP-Associated Factor (TAF) subunits recognize downstream promoter elements, act as co-activators, and interact with nucleosomes. Here we show that transcription induces stable TAF binding to downstream promoter DNA, independent of upstream contacts, TBP, or other basal transcription factors. This transcription-dependent TAF complex promotes subsequent activator-independent transcription, and promoter response to TAF mutations in vivo correlates with the level of downstream, rather than overall, Taf1 crosslinking. We propose a new model in which TAFs function as reinitiation factors, accounting for the differential responses of promoters to various transcription factor mutations.

molecular biology↗

bioRxiv2017

Reversal of molecular pathology by RNA-targeting Cas9 in a myotonic dystrophy mouse model

The dominantly inherited, multi-systemic disease myotonic dystrophy type I (DM1) is caused by triplet repeat CTG expansions in the DMPK gene and is the most common form of adult-onset muscular dystrophy. Elimination of the toxic, repetitive CUG RNA constitutes a therapeutic for this disease. We report an RNA-targeting Cas9 (RCas9) system that supports efficient reversal of DM1 phenotypes via delivery to adult poly(CUG) DM1 mouse muscle using adeno-associated virus (AAV). We observe elimination of CUG RNA, restoration of CUG foci-associated Mbnl1 protein to wild-type subcellular localization, correction of DM1-type alternative splicing patterns in candidate genes including the voltage-gated chloride channel 1 (Clcn1) responsible for characteristic myotonia, recovery of Clcn1 staining, and reduction in centralized myonuclei. Our results establish RCas9 as a potential long-term in vivo therapeutic for DM1.\n\nOne Sentence SummaryA repurposed CRISPR system termed RNA-targeting Cas9 reverses the molecular pathology associated with the most common type of adult onset muscular dystrophy in adult mouse muscle.

molecular biology↗

bioRxiv2017

Impeding transcription of expanded microsatellite repeats by deactivated Cas9

Transcription of expanded microsatellite repeats is associated with multiple human diseases, including myotonic dystrophy, Fuchs endothelial corneal dystrophy, and C9orf72-ALS/FTD. Eliminating or reducing production of RNA and proteins arising from these expanded loci holds therapeutic benefit. Here, we tested the hypothesis that a deactivated form of the Cas9 enzyme impedes transcription across expanded microsatellites. We observed a repeat length-, PAM-, and strand-dependent reduction in the abundance of repeat-containing RNAs upon targeting dCas9 directly to repeat sequences. Aberrant splicing patterns were rescued in DM1 cells, and production of RAN peptides characteristic of DM1, DM2, and C9orf72-ALS/FTD cells was drastically decreased. Systemic delivery of dCas9/gRNA by adeno-associated virus led to reductions in pathological RNA foci, rescue of chloride channel 1 protein expression, and decreased myotonia. These observations suggest that transcription of microsatellite repeat-containing RNAs is more sensitive to perturbation than transcription of other RNAs, indicating potentially viable strategies for therapeutic intervention.

molecular biology↗