Results 1-25 of about 1,000
  1. The dogma “One gene, one protein” is clearly obsolete since cells use alternative splicing and generate multiple transcripts which are translated into protein isoforms, but also use alternative translation initiation sites and termination sites on a given transcripts. Alternative open reading frames for individual transcripts give proteins (the alternative Proteins: AltProts) originate from the 5'- and 3'- UTR mRNA regions, frameshifts of mRNA ORFs or from non-coding RNAs. To gain insight into the role of these newly identified alternative proteins in the regulation of cellular functions, it is crucial to assess their dynamic modulation within a framework of altered physiological modifications such as experimental spinal cord injury (SCI). Here, we carried out a longitudinal proteomic study on rat SCI from 12h to 10 days. Based on AltProt predictions, it was possible to identify a plethora of newly predicted protein hits. Among these proteins, some presented a special interest due to high homology with variable chain regions of immunoglobulins. We focus our interest on the one related to Kappa variable light chains which is similarly highly produced by B-cells in the Bence jones disease, but here expressed in astrocytes. This protein, name Heimdall is an Intrinsically disordered protein which is secreted under inflammatory conditions. Immunoprecipitation experiments showed that the Heimdall interactome contained proteins related to astrocyte fate keepers such as “NOTCH1, EPHA3, IPO13”. However, when Heimdall protein was neutralized utilizing a specific antibody or its gene knocked out by CRISPR-Cas9, sprouting elongations were observed in the corresponding astrocytes. Interestingly, depolarization assays and intracellular calcium measurements in Heimdall KO, established a depolarization effect on astrocyte membranes KO cells impacting the astrocyte phenotype sustained by the decrease of NOTCH2. Taken together, Heimdall is a novel neural key player involved in astrocytes gatekeeper phenotype.
    Date: 2023-01-24
    Authors: Capuz A, Osien S, Tristan C, karnoub M, Aboulouard S, Romero AR, Duhamel M, Cizkova D, Trerotola M, Devos D, Kobaissy F, Abeele FV, Bonnefond A, Fournier I, Rodet F, Salzet M.
    Ref: Research Square
  2. Gain-of-function mutations in NOTCH1 are among the most frequent genetic alterations in T cell acute lymphoblastic leukemia (T-ALL), making the Notch signaling pathway a promising therapeutic target for personalized medicine. Yet, a major limitation for long-term success of targeted therapy is relapse due to tumor heterogeneity or acquired resistance. Thus, we performed a genome-wide CRISPR-Cas9 screen to identify prospective resistance mechanisms to pharmacological NOTCH inhibitors and novel targeted combination therapies to efficiently combat T-ALL. Mutational loss of Phosphoinositide-3-Kinase regulatory subunit 1 (PIK3R1) causes resistance to Notch inhibition. PIK3R1 deficiency leads to increased PI3K/AKT signaling which regulates the cell cycle and spliceosome machinery, both at the transcriptional and post-translational level. Moreover, several therapeutic combinations have been identified, where simultaneous targeting of the cyclin-dependent kinases 4 and 6 (CDK4/6) and NOTCH proved to be the most efficacious in T-ALL xenotransplantation models.
    Date: 2023-01-24
    Authors: Radtke F, Cao L, Buendía GR, Fournier N, Liu Y, Armand F, Hamelin R, Pavlou M.
    Ref: Research Square
  3. CRISPR-Cas adaptive immune systems capture DNA fragments from invading mobile genetic elements and integrate them into the host genome to provide a template for RNA-guided immunity. CRISPR systems maintain genome integrity and avoid autoimmunity by distinguishing between self and non-self, a process for which the CRISPR-Cas1:Cas2 integrase is necessary but not sufficient. In some microbes, the Cas4 endonuclease assists CRISPR adaptation, but many CRISPR-Cas systems lack Cas4. We show here that an elegant alternative pathway employs an internal exonuclease to select and process DNA for integration using the protospacer adjacent motif (PAM). A natural Cas1:Cas2-exonuclease fusion (trimmer-integrase) catalyzes coordinated DNA capture, trimming and integration. Five cryo-EM structures of the CRISPR trimmer-integrase, visualized both before and during DNA integration, show how asymmetric processing generates size-defined, PAM-containing substrates. Before genome integration, the PAM sequence is released by Cas1 and cleaved by the exonuclease, marking inserted DNA as self and preventing aberrant CRISPR targeting of the host. Together, these data support a model in which CRISPR systems lacking Cas4 use fused or recruited exonucleases for faithful acquisition of new CRISPR immune sequences.
    Date: 2023-01-24
    Authors: Wang JY, Tuck OT, Skopintsev P, Soczek KM, Li G, Al-Shayeb B, Zhou J, Doudna JA.
    Ref: bioRxiv
  4. ABSTRACT Many positive-strand RNA viruses, including all known coronaviruses, employ programmed –1 ribosomal frameshifting (–1 PRF) to regulate the translation of polycistronic viral RNAs. However, only a few host factors have been shown to regulate –1 PRF. Through a reporter-based genome-wide CRISPR/Cas9 knockout screen, we identified several host factors that either suppressed or enhanced –1 PRF of SARS-CoV-2. One of these factors is eukaryotic translation initiation factor 2A (eIF2A), which specifically and directly enhanced –1 PRF in vitro and in cells. Consistent with the crucial role of efficient –1 PRF in transcriptase/replicase expression, loss of eIF2A reduced SARS-CoV-2 replication in cells. Transcriptome-wide analysis of eIF2A-interacting RNAs showed that eIF2A primarily interacted with 18S ribosomal RNA near the contacts between the SARS-CoV-2 frameshift-stimulatory element (FSE) and the ribosome. Thus, our results revealed an unexpected role for eIF2A in modulating the translation of specific RNAs independent of its previously described role during initiation.
    Date: 2023-01-24
    Authors: Wei L, Sun Y, Guo JU.
    Ref: bioRxiv
  5. ABSTRACT Mutations in the promoter of the telomerase reverse transcriptase ( TERT ) gene are the paradigm of a cross-cancer alteration in a non-coding region. TERT promoter mutations (TPMs) are biomarkers of poor prognosis in several tumors, including thyroid cancers. TPMs enhance TERT transcription, which is otherwise silenced in adult tissues, thus reactivating a bona fide oncoprotein. To study TERT deregulation and its downstream consequences, we generated a Tert mutant promoter mouse model via CRISPR/Cas9 engineering of the murine equivalent locus (Tert -123C>T ) and crossed it with thyroid-specific Braf V600E -mutant mice. We also employed an alternative model of Tert overexpression (K5-Tert). Whereas all Braf V600E animals developed well-differentiated papillary thyroid tumors, 29% and 36% of Braf V600E +Tert -123C>T and Braf V600E +K5-Tert mice progressed to poorly differentiated thyroid cancers at week 20, respectively. Braf+Tert tumors showed increased mitosis and necrosis in areas of solid growth, and older animals from these cohorts displayed anaplastic-like features, i.e., spindle cells and macrophage infiltration. Murine Tert promoter mutation increased Tert transcription in vitro and in vivo , but temporal and intra-tumoral heterogeneity was observed. RNA-sequencing of thyroid tumor cells showed that processes other than the canonical Tert-mediated telomere maintenance role operate in these specimens. Pathway analysis showed that MAPK and PI3K/AKT signaling, as well as processes not previously associated with this tumor etiology, involving cytokine and chemokine signaling, were overactivated. Braf+Tert animals remained responsive to MAPK pathway inhibitors. These models constitute useful pre-clinical tools to understand the cell-autonomous and microenvironment-related consequences of Tert-mediated progression in advanced thyroid cancers and other aggressive tumors carrying TPMs.
    Date: 2023-01-24
    Authors: Landa I, Thornton CE, Xu B, Haase J, Krishnamoorthy GP, Hao J, Knauf JA, Herbert ZT, Blasco MA, Ghossein R, Fagin JA.
    Ref: bioRxiv
  6. Parkinson's disease (PD) is one of the most common neurodegenerative diseases, but no disease-modifying therapies have been successful in clinical translation presenting a major unmet medical need. A promising target is alpha-synuclein or its aggregated form, which accumulates in the brain of PD patients as Lewy bodies. While it is not entirely clear which alpha-synuclein protein species is disease relevant, mere overexpression of alpha-synuclein in hereditary forms leads to neurodegeneration. To specifically address gene regulation of alpha-synuclein, we developed a CRISPR interference (CRISPRi) system based on the nuclease dead S. aureus Cas9 (SadCas9) fused with the transcriptional repressor domain Krueppel-associated box to controllably repress alpha-synuclein expression at the transcriptional level. We screened single guide (sg)RNAs across the SNCA promoter and identified several sgRNAs that mediate downregulation of alpha-synuclein at varying levels. CRISPRi downregulation of alpha-synuclein in iPSC-derived neuronal cultures from a patient with an SNCA genomic triplication showed functional recovery by reduction of oxidative stress and mitochondrial DNA damage. Our results are proof-of-concept in vitro for precision medicine by targeting the SNCA gene promoter. The SNCA CRISPRi approach presents a new model to understand safe levels of alpha-synuclein downregulation and a novel therapeutic strategy for PD and related alpha-synucleinopathies.
    Date: 2023-01-24
    Authors: Sastre D, Zafar F, Torres CAM, Piper D, Kirik D, Sanders LH, Qi S, Schuele B.
    Ref: bioRxiv
  7. Inborn Errors of Metabolism (IEM) and Immunity (IEI) are Mendelian diseases in which complex phenotypes and patient rarity can limit clinical annotations. Few genes are assigned to both IEM and IEI, but immunometabolic demands suggest functional overlap is underestimated. We applied CRISPR screens to test IEM genes for immunologic roles and IEI genes for metabolic effects and found considerable crossover. Analysis of IEM showed N-linked glycosylation and the de novo hexosamine synthesis enzyme, Gfpt1 , are critical for T cell expansion and function. Interestingly, Gfpt1 -deficient TH1 cells were more affected than TH17 cells, which had increased Nagk for salvage UDP-GlcNAc synthesis. Screening IEI genes showed the transcription factor Bcl11b promotes CD4+ T cell mitochondrial activity and Mcl1 expression necessary to prevent metabolic stress. These data illustrate a high degree of functional overlap of IEM and IEI genes and point to potential immunometabolic mechanisms for a previously unappreciated set of these disorders.
    Date: 2023-01-24
    Authors: Patterson AR, Needle GA, Sugiura A, Chi C, Steiner KK, Fisher EL, Robertson GL, Bodnya C, Markle JG, Gama V, Rathmell JC.
    Ref: bioRxiv
  8. Pooled CRISPR screens with single-cell RNA-seq readout (Perturb-seq) have emerged as a key technique in functional genomics, but are limited in scale by cost and combinatorial complexity. Here, we reimagine Perturb-seq’s design through the lens of algorithms applied to random, low-dimensional observations. We present compressed Perturb-seq, which measures multiple random perturbations per cell or multiple cells per droplet and computationally decompresses these measurements by leveraging the sparse structure of regulatory circuits. Applied to 598 genes in the immune response to bacterial lipopolysaccharide, compressed Perturb-seq achieves the same accuracy as conventional Perturb-seq at 4 to 20-fold reduced cost, with greater power to learn genetic interactions. We identify known and novel regulators of immune responses and uncover evolutionarily constrained genes with downstream targets enriched for immune disease heritability, including many missed by existing GWAS or trans-eQTL studies. Our framework enables new scales of interrogation for a foundational method in functional genomics.
    Date: 2023-01-23
    Authors: Yao D, Binan L, Bezney J, Simonton B, Freedman J, Frangieh CJ, Dey K, Geiger-Schuller K, Eraslan B, Gusev A, Regev A, Cleary B.
    Ref: bioRxiv
  9. Cytotrophoblasts fuse to form and renew syncytiotrophoblasts necessary to maintain placental health throughout gestation. During cytotrophoblast to syncytiotrophoblast differentiation, cells undergo regulated metabolic and transcriptional reprogramming. Mitochondria play a critical role in differentiation events in cellular systems, thus we hypothesized that mitochondrial metabolism played a central role in trophoblast differentiation. In this work, we employed static and stable isotope tracing untargeted metabolomics methods along with gene expression and histone acetylation studies in an established cell culture model of trophoblast differentiation. Trophoblast differentiation was associated with increased abundance of the TCA cycle intermediates citrate and α-ketoglutarate. Citrate was preferentially exported from mitochondria in the undifferentiated state but was retained to a larger extent within mitochondria upon differentiation. Correspondingly, differentiation was associated with decreased expression of the mitochondrial citrate transporter (CIC). CRISPR/Cas9 disruption of the mitochondrial citrate carrier showed that CIC is required for biochemical differentiation of trophoblasts. Loss of CIC resulted in broad alterations in gene expression and histone acetylation. These gene expression changes were partially rescued through acetate supplementation. Taken together, these results highlight a central role for mitochondrial citrate metabolism in orchestrating histone acetylation and gene expression during trophoblast differentiation.
    Date: 2023-01-22
    Authors: Mahr RM, Jena S, Nashif SK, Nelson AB, Rauckhorst AJ, Rome FI, Sheldon RD, Hughey CC, Puchalska P, Gearhart MD, Taylor EB, Crawford PA, Wernimont SA.
    Ref: bioRxiv
  10. The ability to map genetic interactions has been essential for determining gene function and defining biological pathways. Therefore, a system to readily perform genome-wide genetic modifier screens in human cells is a powerful platform for dissecting complex processes in mammalian cells, where redundancy and adaptation commonly mask the phenotype of a single genetic perturbation. Here, we report a CRISPR interference (CRISPRi) based platform, compatible with Fluorescence Activated Cell Sorting (FACS)-based reporter screens, that can be used to query epistatic relationships at scale. This is enabled by a flexible dual-sgRNA library design that allows for the simultaneous delivery and selection of a fixed sgRNA and a second randomized guide, comprised of a genome-wide library, with a single transduction. As a proof of principle, we apply our approach to study the pathways that mediate tail-anchored (TA) protein insertion at the endoplasmic reticulum (ER). We show that this dual-guide library approach can be successfully coupled with FACS-based reporter screening, to identify genetic epistasis and thereby place TA biogenesis factors in their respective parallel pathways. We demonstrate that this dual-guide approach is both more sensitive and specific than traditional growth screening approaches, and is ideally suited for dissecting the complex interplay between factors in human cells.
    Date: 2023-01-22
    Authors: Guna A, Page KR, Replogle JR, Esantsi TK, Wang ML, Weissman JS, Voorhees RM.
    Ref: bioRxiv
  11. Although mechanisms of telomere protection are well-defined in differentiated cells, it is poorly understood how stem cells sense and respond to telomere dysfunction. Recent efforts have characterized the DNA damage response (DDR) following progressive telomere erosion in human pluripotent cells, yet the broader impact of telomeric double-strand breaks (DSBs) in these cells is poorly characterized. Here, we report on DNA damage signaling, cell cycle, and transcriptome-level changes in human induced pluripotent stem cells (iPSCs) in response to telomere-internal DSBs. We engineered a novel human iPSC line with a targeted doxycycline-inducible TRF1-FokI fusion protein to acutely induce DSBs at telomeres. Using this model, we demonstrate that TRF1-FokI DSBs activate an ATR-dependent DDR in iPSCs, in contrast to an established ATM-dependent response to telomeric FokI breaks in differentiated cells. ATR activation leads to a potent cell cycle arrest in G2, which we show is p53-independent and can be rescued by treatment with an ATR inhibitor. Telomere lengths are remarkably well-maintained in the face of persistent TRF1-FokI induction. Using CRISPR-Cas9 to cripple the catalytic domain of telomerase, we show that telomerase is largely dispensable for survival and telomere length maintenance following telomeric breaks, which instead appear to be repaired by a mechanism bearing hallmarks of lengthening mediated by homologous recombination, so-called alternative lengthening of telomeres (ALT). Our findings suggest a previously unappreciated role for ALT in telomere maintenance in telomerase-positive iPSCs and reveal distinct iPSC-specific responses to targeted telomeric damage.
    Date: 2023-01-21
    Authors: Estep KN, Tobias JW, Fernandez RJ, Beveridge BM, Johnson FB.
    Ref: bioRxiv
  12. The stable incorporation of transgenes and recombinant DNA material into the host genome is a bottleneck in many bioengineering applications. Due to the low efficiency, identifying the transgenic animals is often a needle in the haystack. Thus, optimal conditions require efficient screening procedures, but also known and safe landing sites that do not interfere with host expression, low input material and strong expression from the new locus. Here, we leverage an existing library of ≈ 300 different loci coding for fluorescent markers that are distributed over all 6 chromosomes in Caenorhabditis elegans as safe harbors for versatile transgene integration sites using CRISPR/Cas9. We demonstrated that a single crRNA was sufficient for cleavage of the target region and integration of the transgene of interest, which can be easily followed by loss of the fluorescent marker. The same loci can also be used for extrachromosomal landing sites and as co-CRISPR markers without affecting body morphology or animal behavior. Thus, our method overcomes the uncertainty of transgene location during random mutagenesis, facilitates easy screening through fluorescence interference and can be used as co-CRISPR markers without further influence in phenotypes.
    Date: 2023-01-21
    Authors: Malaiwong N, Porta-de-la-Riva M, Krieg M.
    Ref: bioRxiv
  13. The seven subunit Arp2/3 complex drives the formation of branched actin networks that are essential for many cellular processes including cell migration. In humans, the ARPC5 subunit of the Arp2/3 complex is encoded by two paralogous genes ( ARPC5 and ARPC5L ), resulting in proteins with 67% identity. Through whole-exome sequencing, we identified a biallelic ARPC5 frameshift variant in a female child who presented with recurrent infections, multiple congenital anomalies, diarrhea, and thrombocytopenia, and suffered early demise from sepsis. Her consanguineous parents also had a previous child who died with similar clinical features. Using CRISPR/Cas9-mediated approaches, we demonstrate that loss of ARPC5 affects actin cytoskeleton organization and function, as well as chemokine-dependent cell migration in vitro . Homozygous Arpc5 -/- mice do not survive past embryonic day 9 due to severe developmental defects, including loss of the second pharyngeal arch which contributes to craniofacial and heart development. Our results indicate that ARPC5 is important for both prenatal development and postnatal immune signaling, in a non-redundant manner with ARPC5L. Moreover, our observations add the ARPC5 locus to the list of genes that should be considered when patients present with syndromic early-onset immunodeficiency, particularly if recessive inheritance is suspected.
    Date: 2023-01-21
    Authors: Sindram E, Caballero-Oteyza A, Kogata N, Huang S, Alizadeh Z, Gamez-Diaz L, Fazlollhi MR, Peng X, Grimbacher B, Way M, Proietti M.
    Ref: bioRxiv
  14. Viral vectors for gene therapy, such as recombinant Adeno-Associated Viruses (rAAV), are produced in Human Embryonic Kidney (HEK) 293 cells. However, the presence of the SV40 T-antigen-encoding CDS SV40GP6 and SV40GP7 in the HEK293T genome raises safety issues when these cells are used in manufacturing for clinical purposes. We developed a new T-antigen-negative HEK cell line from ExcellGene’s proprietary HEKExpress®, using the CRISPR-Cas9 strategy. We obtained a high number of clonally-derived cell populations and all of them were demonstrated T-antigen negative. Stability study and AAV production evaluation showed that the deletion of the T-antigen-encoding locus did not impact neither cell growth nor viability nor productivity. The resulting CMC-compliant cell line, named HEKzeroT®, is able to produce high AAV titers, from small to large scale.
    Date: 2023-01-20
    Authors: Pino P, Croissant C, Armitano J, Lazuech B, Švec D, Pugin C, Guesdon A, Bryan L, Castro A, Neuhaus L, Fonti G, Martinis J, Wurm M, Wurm FM.
    Ref: Authorea Preprints
  15. Background: Allergy health problems worldwide are mainly caused by allergens from domestic cats ( Felis catus ). Fel d 1 is a major allergen that causes severe allergic reactions in humans, including rhinitis, conjunctivitis, and life-threatening asthma.out cats. Fel d 1 level in CH2 knockout cats was assessed by enzyme-linked immunosorbent assay (ELISA). Cytoplasm injection clone technology (CICT) was used to clone the CH2 knockout cat. Results: : We report the first successful generation and cloning of CH2 knockout cats using the CRISPR-Cas9 system and CICT. CH2 knockout cats were confirmed using T7E1 and Sanger sequencing. Cloned CH2 knockout cat was verified by microsatellite analysis. Remarkably, ELISA proved that Fel d 1 level of CH2 knockout cats was extremely low compared with that of wild type domestic cats. Conclusion: CH2 knockout cats we generated showed an extremely low level of Fel d 1 and could be hypoallergenic cats. Our study indicates that creating hypoallergenic cats using the CRISPR-Cas9 system is a significant step forward in the consequence that these cats can safely approach allergic patients.
    Date: 2023-01-20
    Authors: Lee SR, Lee K, Song S, Joo M, Lee S, Kang J, Kang S, Idrees M, Kim J, Ko J, Kong I.
    Ref: Authorea Preprints
  16. Background: Epiretinal membranes in patients with proliferative vitreoretinopathy (PVR) consist of extracellular matrix and a number of cell types including retinal pigment epithelial (RPE) cells and fibroblasts, whose contraction causes retinal detachment. In RPE cells depletion of platelet-derived growth factor (PDGF) receptor (PDGFR)β suppresses vitreous-induced Akt activation, whereas in fibroblasts Akt activation through indirect activation of PDGFRα by growth factors outside the PDGF family (non-PDGFs) plays an essential role in experimental PVR. Whether non-PDGFs in the vitreous, however, were also able to activate PDGFRβ in RPE cells remained elusive. Methods We showed that expression of a truncated PDGFRβ lacking a PDGF-binding domain in the RPE cells whose PDGFRB gene had been silent using the CRISPR/Cas9 technology restored vitreous-induced Akt activation as well as cell proliferation, epithelial-mesenchymal transition, migration and contraction. Results We found that scavenging reactive oxygen species (ROS) with N-acetyl-cysteine and inhibiting Src family kinases (SFKs) with their specific inhibitor SU6656 blunted the vitreous-induced activation of the truncated PDGFRβ and Akt as well as the cellular events related to the PVR pathogenesis. Conclusions These discoveries suggest that in RPE cells PDGFRβ can be activated indirectly by non-PDGFs in the vitreous via an intracellular pathway of ROS/SFKs to facilitate the development of PVR, thereby providing novel opportunities for PVR therapeutics.
    Date: 2023-01-20
    Authors: Duan Y, Wu W, Cui J, Matsubara JA, Kazlauskas A, Li X, Lei H.
    Ref: Research Square
  17. The frontline therapy R-CHOP for patients with diffuse large B-cell lymphoma (DLBCL) has remained unchanged for two decades despite numerous phase III clinical trials investigating new alternatives. Multiple large studies have uncovered genetic subtypes of DLBCL enabling a targeted approach. To further pave the way for precision oncology, we perform genome-wide CRISPR screening to uncover the cellular response to one of the components of R-CHOP, vincristine, in the DLBCL cell line SU-DHL-5. We discover important pathways and subnetworks using gene-set enrichment analysis and protein-protein interaction networks and identify genes related to mitotic spindle organization that are essential during vincristine treatment. Inhibition of KIF18A, a mediator of chromosome alignment, using the small molecule inhibitor BTB-1 causes complete cell death in a synergistic manner when administered together with vincristine. We also identify the genes KIF18B and USP28 for which CRISPR/Cas9-directed knockout induces vincristine resistance across two DLBCL cell lines. Mechanistic studies show that lack of KIF18B or USP28 counteracts a vincristine-induced p53 response involving the mitotic surveillance pathway (USP28-53BP1-p53). Collectively, our CRISPR screening data uncover potential drug targets and mechanisms behind vincristine resistance, which may support the development of future drug regimens. Key points Inhibition of the mitotic surveillance pathway (USP28-53BP1-p53) and KIF18B induces resistance to vincristine Substantial synergistic effects observed when using the KIF18A-inhibitor BTB-1 with vincristine in eradicating GCB-subtype DLBCL cells
    Date: 2023-01-20
    Authors: Rovsing AB, Thomsen EA, Nielsen I, Skov TW, Luo Y, Dybkær K, Mikkelsen JG.
    Ref: bioRxiv
  18. ABSTRACT Focused Ultrasound Blood-Brain Barrier Opening (FUS-BBBO) can deliver adeno-associated viral vectors (AAVs) to treat genetic disorders of the brain. However, such disorders often affect large brain regions. Moreover, the applicability of FUS-BBBO in the treatment of brain-wide genetic disorders has not yet been evaluated. Herein, we evaluated the transduction efficiency and safety of opening up to 105 sites simultaneously. Increasing the number of targeted sites increased gene delivery efficiency at each site. We achieved transduction of up to 60% of brain cells with comparable efficiency in the majority of the brain regions. Furthermore, gene delivery with FUS-BBBO was safe even when all 105 sites were targeted simultaneously without negative effects on animal weight, neuronal loss, or astrocyte activation. To evaluate the application of multi-site FUS-BBBO for gene therapy, we used it for gene editing using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system, and found effective gene editing, but also a loss of neurons at the targeted sites. Overall, this study provides a brain-wide map of transduction efficiency and the first example of gene editing after site-specific noninvasive gene delivery to a large brain region.
    Date: 2023-01-20
    Authors: Nouraein S, Lee S, Saenz VA, Del Mundo HC, Yiu J, Szablowski JO.
    Ref: bioRxiv
  19. Gene editing in the mammalian brain has been challenging because of the restricted transport imposed by the blood-brain barrier (BBB). Current approaches rely on local injection to bypass the BBB. However, such administration is highly invasive and not amenable to treating certain delicate regions of the brain. We demonstrate a safe and effective gene editing technique by using focused ultrasound (FUS) to transiently open the BBB for the transport of intravenously delivered CRISPR/Cas9 machinery to the brain.
    Date: 2023-01-20
    Authors: Leong K, Lao Y, Ji R, Zhou J, Snow K, Kwon N, Saville E, He S, Chauhan S, Chi C, Datta M, Zhang H, Quek CH, Cai S, Li M, Gaitan Y, Bechtel L, Wu S, Lutz C, Tomer R, Murray S, Chavez A, Konofagou E.
    Ref: Research Square
  20. Despite their pivotal role in evaluating (patho)physiological cell states, traditional gene reporters can follow gene expression but leave marks on proteins or significantly modify mature mRNA. Multi-time point readouts of non-coding RNAs are, to date, not applicable without changing their nucleotide sequence, which may affect their natural function, half-life, and localization. We thus created INSPECT (Intron-encoded Scarless Programmable Extranuclear Cistronic Transcripts) as a minimally invasive transcriptional reporter nested within an intron of a gene of interest. Post-transcriptional excision of INSPECT results in a mature RNA with no sequence changes and an additional synthetic transcript that leaves the nucleus by hijacking the nuclear host export machinery for cytosolic translation into a reporter protein. Here, we first illustrate the cloning of such an INSPECT DNA donor construct and an auxiliary CRISPR/Cas9 plasmid to insert INSPECT into a gene of interest and, secondly, exemplary for NEAT1 _long, we showed the generation of such a cell line.
    Date: 2023-01-19
    Authors: TRUONG DJ, Armbrust N, Geilenkeuser J, Westmeyer GG.
    Ref: Research Square
  21. Antimicrobial peptides (AMPs) are at the interface of interactions between hosts and microbes and are therefore expected to be fast evolving in a coevolutionary arms race with pathogens. In contrast, previous work demonstrated that one AMP, Metchikowin (Mtk), has a single residue that segregates as either proline (P) or arginine (R) in populations of four different Drosophila species, some of which diverged more than 10 million years ago. The recurrent finding of this polymorphism regardless of geography or host species, coupled with evidence of balancing selection in Drosophila AMPs, suggest there is a distinct functional importance to each allele. The most likely hypotheses involve alleles having specificity to different pathogens or the more potent allele conferring a cost on the host. To assess their functional differences, we created D. melanogaster lines with the P allele, R allele, or Mtk null mutation using CRISPR/Cas9 genome editing. Here, we report results from experiments assessing the two hypotheses using these lines. In males, testing of systemic immune responses to a repertoire of bacteria and fungi demonstrated that the R allele performs as well or better than the P and null alleles with most infections. With some pathogens, however, females show results in contrast with males where Mtk alleles either do not contribute to survival or where the P allele outperforms the R allele. In addition, measurements of life history traits demonstrate that the R allele is more costly in the absence of infection for both sexes. These results provide strong in vivo evidence that differential fitness with or without infection and sex-based functional differences in alleles may be adaptive mechanisms of maintaining immune gene polymorphisms in contrast with expectations of rapid evolution. Therefore, a complex interplay of forces including pathogen species and host sex may lead to balancing selection for immune genotypes. Strikingly, this selection may act on even a single amino acid polymorphism in an AMP.
    Date: 2023-01-19
    Authors: Perlmutter JI, Chapman JR, Wilkinson MC, Nevarez-Saenz I, Unckless RL.
    Ref: bioRxiv
  22. CRISPR-Cas13 systems are unique among Class II CRISPR systems, as they exclusively target RNA. In vitro and in prokaryotic cells, Cas13 cleaves both target and non-target RNA indiscriminately upon activation by a specific target RNA. This property has been exploited for development of diagnostic nucleic acid detection tools. In eukaryotic cells, CRISPR-Cas13 initially seemed to exclusively cleave the target RNA and consequently, CRISPR-Cas13 has been adopted as a specific RNA knockdown tool. Recently, several groups have reported unexpected toxicity or collateral cleavage when using CRISPR-Cas13 in eukaryotic cells, which seems difficult to reconcile with the reported target specificity. To understand these seemingly contradicting findings, we explored the collateral cleavage activity of six Cas13 systems, and show that only the most active ortholog in vitro , LbuCas13a, exhibits strong collateral RNA cleavage activity in human cells. LbuCas13a displayed collateral cleavage in all tested cell lines, targeting both exogenous and endogenous transcripts and using different RNP delivery methods. Using Nanopore sequencing, we found that cytoplasmic RNAs are cleaved without bias by LbuCas13a. Furthermore, the cleavage sites are highly specific and often present in Uracil containing single stranded RNA loops of stem-loop structures. In response to collateral RNA cleavage, cells upregulate stress and innate immune response genes and depending on target transcript levels, RNA degradation resulted in apoptotic cell death. We demonstrate that LbuCas13a can serve as a cell selection tool, killing cells in a target RNA specific manner. As such, CRISPR-Cas13 is a promising new technology that might be useful in anti-tumor applications.
    Date: 2023-01-19
    Authors: Bot JF, Zhao Z, Kammeron D, Shang P, Geijsen N.
    Ref: bioRxiv
  23. Introduction Genome-wide association studies along with expression quantitative trait loci (eQTL) have identified hundreds of single nucleotide polymorphisms (SNPs) and their target genes in prostate cancer (PrCa). Although these genetic associations to PrCa have been widely reported, functional characterization of these risk loci remains challenging. Methods To screen for regulatory SNPs, we designed a library containing 9133 guide RNAs (gRNAs) to target 2,166 candidate SNP sites implicated in PrCa. We performed negative screening in dCas9-KRAB stable prostate cell lines and applied the RIGOR program to identify the essential SNPs for cell proliferation. We further characterized the regulatory role of a selected single nucleotide polymorphim (SNP, rs60464856) using luciferase reporter assay, ChIP-qPCR, and xCas9 base editing in prostate cells. Finally, we investigated the biological impact of the SNP-regulated gene RUVBL1 on cell proliferation and tumor growth via gene knockdown using in vitro and in vivo assays. Results From interference of 2,166 candidate SNPs via CRISPR interference screening, the RIGOR program identified 117 SNPs that could regulate genes to promote growth advantage in prostate cancer cell lines. Compared to unselected SNPs, the 117 candidates tended to reside near 5 kb flanking the transcription start sites (p = 0.01). To characterize the regulatory role of these SNPs, we selected one SNP (rs60464856) for detailed analysis. This SNP was covered by multiple gRNAs significantly depleted in the screening (FDR<0.05). Pooled SNP association analysis in the PRACTICAL cohort showed significantly higher PrCa risk for the G allele (pvalue=1.2E-16). eQTL analysis showed that the G allele is associated with an increased expression of RUVBL1 in multiple datasets. To further validate the CRISPR interference effect, we transfected a gRNA targeting the rs60464856 site in the dCas9 stable cell lines and observed significant inhibition of the RUVBL1 expression. We also applied the xCas9 adenine base editor to convert the rs60464856 A into G allele and observed an increased RUVBL1 expression in subclones carrying the rs60464856 G allele in prostate cell lines. To test if any protein showed allele-specific binding at rs60464856, we used SILAC-based DNA pull-down proteomics and observed that cohesin subunits (including SMC3) preferred the A allele. ChIP qPCR assays showed significant enrichment of CTCF and SMC3 signals at the rs60464856 site with preferential binding to the A allele. To evaluate the potential role of this locus in maintaining long-range chromatin structure, we analyzed a HiC dataset and found that the rs60464856 locus enriched consistent chromatin interactions in prostate cell lines. To determine the potential role of the rs60464856 target gene, we knocked down the RUVBL1 via shRNA and observed significant proliferation inhibition in prostate cell lines. We also tested PC3 cells with RUVBL1 knockdown in nude mice xenografts and observed reduced tumorigenesis. Gene set enrichment analysis showed that RUVBL1 expression was associated with the enrichment of cell cycle related pathways in both cell line and TCGA prostate cancer cohorts. Lastly, we showed that an increased RUVBL1 expression and its relevant pathway activation were associated with poor survival. Conclusion We applied the CRISPR interference screening at selected prostate cancer risk loci and identified over a hundred regulatory SNPs essential for prostate cell proliferation. Further analysis confirmed the important role of rs60464856 and its target gene RUVBL1 in prostate cell growth and tumorigenesis.
    Date: 2023-01-19
    Authors: Tian Y, Dong D, Wu L, Park JY, Wei G, Wang L, PRACTICAL/ELLIPSE consortium.
    Ref: bioRxiv
  24. In the mammalian central nervous system (CNS), astrocytes are indispensable for brain development, function, and health. However, non-invasive tools to study astrocyte biology and function in vivo have been limited to genetically modified mice. CRISPR/Cas9-based genome engineering enables rapid and precise gene manipulations in the CNS. Here, we developed a non-invasive astrocyte-specific method utilizing a single AAV vector, GEARBOCS (Gene Editing in AstRocytes Based On CRISPR/Cas9 System). We verified GEARBOCS’ specificity to mouse cortical astrocytes and demonstrated its utility for three types of gene manipulations: knockout (KO); tagging (TagIN); and reporter gene knock-in (Gene-TRAP) strategies. We deployed GEARBOCS to determine whether cortical astrocytes express Vamp2 protein. The presence of Vamp2-positive vesicles in cultured astrocytes is well-established, however, Vamp2 protein expression in astrocytes in vivo has proven difficult to ascertain due to its overwhelming abundance in neurons. Using GEARBOCS, we delineated the in vivo astrocytic Vamp2 expression and found that it is required for maintaining excitatory and inhibitory synapse numbers in the visual cortex. GEARBOCS strategy provides fast and efficient means to study astrocyte biology in vivo . Graphical Abstract (Main Points) • GEARBOCS is a single AAV-based CRISPR tool to target mouse astrocytes in vivo . • GEARBOCS can be used to knockout, tag, or gene-trap genes of interest in astrocytes in vivo . • Using GEARBOCS, we confirmed astrocytes express Vamp2 and found that astrocytic Vamp2 is required for maintenance of excitatory and inhibitory synapse numbers in vivo .
    Date: 2023-01-19
    Authors: Bindu DS, Tan CX, Savage JT, Eroglu C.
    Ref: bioRxiv
  25. ABSTRACT CRISPR-associated transposons (CASTs) direct DNA integration downstream of target sites using the RNA-guided DNA binding activity of nuclease-deficient CRISPR-Cas systems. Transposition relies on several key protein-protein and protein-DNA interactions, but little is known about the explicit sequence requirements governing efficient transposon DNA integration activity. Here, we exploit pooled library screening and high-throughput sequencing to reveal novel sequence determinants during transposition by the Type I-F Vibrio cholerae CAST system. On the donor DNA, large mutagenic libraries identified core binding sites recognized by the TnsB transposase, as well as an additional conserved region that encoded a consensus binding site for integration host factor (IHF). Remarkably, we found that VchCAST requires IHF for efficient transposition, thus revealing a novel cellular factor involved in CRISPR-associated transpososome assembly. On the target DNA, we uncovered preferred sequence motifs at the integration site that explained previously observed heterogeneity with single-base pair resolution. Finally, we exploited our library data to design modified transposon variants that enable in-frame protein tagging. Collectively, our results provide new clues about the assembly and architecture of the paired-end complex formed between TnsB and the transposon DNA, and inform the design of custom payload sequences for genome engineering applications of CAST systems.
    Date: 2023-01-19
    Authors: Walker MW, Klompe SE, Zhang DJ, Sternberg SH.
    Ref: bioRxiv
first · previous · 1 · 2 · 3 · 4 · 5 · 6 · 7 · 8 · 9 · 10 · next · last