Results 1-25 of about 1,000
  1. Genome-wide association studies (GWAS) have linked thousands of genetic variants to various complex traits or diseases. However, most identified variants have weak individual effects, are correlated with nearby polymorphisms due to linkage disequilibrium (LD), and are located in non-coding cis-regulatory elements (CREs). These characteristics complicate the assessment of the direct impact of each variant on tissue specific gene expression and phenotype. To address this challenge, we have developed a novel algorithm that leverages polymer folding and 3D chromatin interactions to prioritize and identify putative causal variants and their target genes. From the millions of eQTL-Gene pairs identified by GTEx in human somatic tissues, we classify only ~10-20% as putative functional eQTL-Gene pairs supported by phenotypic associations confirmed through CRISPR deletion experiments. Our findings show that unlike most variants, functional eQTL-Gene pairs predominantly reside within the same topologically associating domain (TAD) and have strong associations with cell-type specific cis-regulatory elements (CREs), enriched for binding sites of tissue-specific transcription factors. Unlike most approaches that rely on linear distance or other chromatin features (histone code, accessibility), our algorithm emphasizes the importance of physical interactions and 3D chromatin folding in gene regulation, as the identified eQTL-Gene pairs are all among the small fraction of physical chromatin interactions sufficient for chromatin locus folding. Overall, our algorithm reduces false positive associations between DNA variants and genes identified by eQTL analysis and uncovers novel variant-gene pair associations. These findings suggest a mechanism where a small number of regulatory variants control tissue specific gene expression via their physical association with target genes confined within the same TAD. Our approach provides new insights into the molecular mechanisms driving GWAS phenotypes.
    Date: 2024-06-14
    Authors: Farooq H, Du L, Delafrouz P, Jiang W, Chronis C, Liang J.
    Ref: bioRxiv
  2. Transcriptional enhancers can regulate individual or multiple genes through long-range three-dimensional (3D) genome interactions, and these interactions are commonly altered in cancer. Yet, the functional relationship between changes in 3D interactions associated with regulatory regions and differential gene expression appears context-dependent. In this study, we used HiChiP to capture changes in 3D genome interactions between active regulatory regions of endometrial cancer cells in response to estrogen treatment and uncovered significant differential long-range interactions that are strongly enriched for estrogen receptor α (ER) bound sites (ERBS). The ERBS anchoring differential loops with either a gene's promoter or distal regions were correlated with larger transcriptional responses to estrogen compared to ERBS not involved in differential interactions. To functionally test this observation, CRISPR-based Enhancer-i was used to deactivate specific ERBS, which revealed a wide range of effects on the transcriptional response to estrogen. However, these effects are only subtly and not significantly stronger for ERBS in differential loops. In addition, we observed an enrichment of 3D interactions between the promoters of estrogen up-regulated genes and found that looped promoters can work together cooperatively. Overall, our work suggests that changes in 3D genome structure upon estrogen treatment identify some functionally important regulatory regions; however, these changes are not required for a transcriptional response to E2 in endometrial cancer cells.
    Date: 2024-06-14
    Authors: Abewe H, Richey A, Vahrenkamp JM, Ginley-Hidinger M, Rush CM, Kitchen N, Zhang X, Gertz J.
    Ref: bioRxiv
  3. Plasmid-mediated DNA transformation is a foundational molecular technique and the basis for most CRISPR-Cas9 gene editing systems. While plasmid transformations are well established for many agricultural Phytophthora pathogens, development of this technique in forest Phytophthoras is lacking. Given our long-term research objective to develop CRISPR-Cas9 gene editing in a forest pathogenic Phytophthora species, we sought to establish the functionality of polyethylene glycol (PEG)-mediated plasmid transformation in five species: P. cactorum, P. cinnamomi, P. cryptogea, P. ramorum, and P. syringae. We used the agricultural pathogen P. sojae, a species for which PEG-mediated transformations are well-established, as a transformation control. Using a protocol previously optimized for P. sojae, we tested transformations in the five forest Phytophthoras with three different plasmids: two developed for CRISPR-Cas9 gene editing and one developed for fluorescent protein tagging. Out of the five species tested, successful transformation, as indicated by stable growth of transformants on a high concentration of antibiotic selective growth medium and diagnostic PCR, was achieved only with P. cactorum and P. ramorum. However, while transformations in P. cactorum were consistent and stable, transformations in P. ramorum were highly variable and yielded transformants with very weak mycelial growth and abnormal morphology. Our results indicate that P. cactorum is the best candidate to move forward with CRISPR-Cas9 protocol development and provide insight for future optimization of plasmid transformations in forest Phytophthoras.
    Date: 2024-06-14
    Authors: Dort EN, Hamelin RC.
    Ref: bioRxiv
  4. We present an efficient approach for synthesizing cationic poly(ethylene imine) derivatives using the multicomponent split-Ugi reaction to rapidly create a library of complex functional ionizable lipopolymers. We synthesized a diverse library of 155 polymers, formulated them into polyplexes to establish structure-activity relationships crucial for endosomal escape and efficient transfection. After discovering a lead structure, lipopolymer-lipid hybrid nanoparticles are introduced to preferentially deliver to and elicit effective mRNA transfection in lung endothelium and immune cells, including T cells with low in vivo toxicity. The lipopolymer-lipid hybrid nanoparticles showed 300-fold improvement in systemic mRNA delivery to the lung compared to in vivo-JetPEI. Lipopolymer-lipid hybrid nanoparticles demonstrated efficient delivery of mRNA-based therapeutics for treatment of two different disease models. Lewis Lung cancer progression was significantly delayed after treatment with loaded IL-12 mRNA in U155@lipids after repeated i.v. administration. Systemic delivery of human CFTR (hCFTR) mRNA resulted in production of functional form of CFTR protein in the lungs. The functionality of hCFTR protein was confirmed by restoration of CFTR-mediated chloride secretion in conductive airway epithelia in CFTR knockout mice after nasal instillation of hCFTR mRNA loaded U155@lipids. We further showed that, U155@lipids nanoparticles can deliver complex CRISPR-Cas9 based RNA cargo to the lung, achieving 5.6±2.4 % gene editing in lung tissue. Moreover, we demonstrated successful PD-1 gene knockout of T cells in vivo. Our results highlight a versatile delivery platform for systemic delivering of mRNA of various sizes for gene therapy for a variety of therapeutics.
    Date: 2024-06-13
    Authors: Vlasova KY, Kerr A, Pennock ND, Jozic A, Sahel D, Gautam M, Murthy N, Roberts A, Ali MW, MacDonald KD, Walker J, Luxenhofer R, Sahay G.
    Ref: bioRxiv
  5. The CRISPR-Cas system is a bacterial adaptative immune system which protects against infection by phages: viruses that infect bacteria. To develop immunity, bacteria integrate spacers – fragments of the invading nucleic acids – into their CRISPR array to serve as the basis for sequence-targeted DNA cleavage. However, upon infection, phages quickly take over the metabolism of the bacteria, leaving little time for the bacteria to acquire new spacers, transcribe them and use them to cut the invading DNA. To develop CRISPR immunity, bacteria must be safely exposed to phage DNA. Phage infection releases eDNA which could be involved in the development of CRISPR immunity. Using S. thermophilus and phages 2972 and 858 as a model for CRISPR immunity, we show that eDNA is crucial to the development of optimal CRISPR immunity, as generation of phage-immune bacterial colonies decrease with eDNA digestion. Furthermore, it is phage eDNA specifically that impacts CRISPR immunity since its addition increases the generation of phage-immune colonies. We also show that the effect of eDNA is phage-specific, sequence specific and can even be traced to a region of the genome covering the early-expressed genes which differ between phages 2972 and 858. However, we also show that eDNA is not used as a source of genetic information for spacer acquisition. This suggests that the effect of eDNA involves a new mechanism of phage resistance. Moreover, the effect of eDNA is highly dependent on environmental conditions as variation in media suppliers are sufficient to interfere with this effect. These results link environmental conditions, specifically eDNA, to the CRISPR-Cas system, providing a better understanding of the context of the emergence of CRISPR immunity and could inform our understanding of the mechanisms through which bacteria detect the presence of phages before infection.
    Date: 2024-06-13
    Authors: Croteau FR, Tran J, Hynes AP.
    Ref: bioRxiv
  6. Climate change is leading to more frequent and severe extreme temperature events, negatively impacting agricultural productivity and threatening global food security. Plant reproduction, the process underpinning crop yield, is highly susceptible to heatwaves, affecting pollen development and ultimately affecting seed set and crop yields. Recent research has increasingly focused on understanding how pollen grains from various crops react to heat stress at the molecular and cellular levels. This surge in interest over the last decade has been underpinned by advances in genomic technologies, such as single-cell RNA sequencing, which holds significant potential for revealing the underlying regulatory reprogramming triggered by heat stress throughout the various stages of pollen development. This review focuses on how heat stress affects gene regulatory networks, including the heat stress response, the unfolded protein response, and autophagy, and discusses the impact of these changes on various stages of pollen development. It highlights the potential of pollen selection as a key strategy for improving heat tolerance in crops by leveraging the genetic variability among pollen grains. Additionally, genome-wide association studies and population screenings have shed light on the genetic underpinnings of traits in major crops that respond to high temperatures during male reproductive stages. Moreover, gene-editing tools like CRISPR/Cas systems could facilitate precise genetic modifications to boost pollen heat resilience. The information covered in this review is valuable for selecting traits and employing molecular genetic approaches to develop heat-tolerant genotypes.
    Date: 2024-06-13
    Authors: Lohani N, Singh MB, Bhalla P.
    Ref: Authorea Preprints
  7. Abstract Non-response in cancer immunotherapy poses a significant challenge, often attributed to poorly understood immune resistance mechanisms. Indoleamine 2,3-dioxygenase (IDO1), frequently expressed in advanced-stage cancers, contributes to this resistance by fostering an immunosuppressive environment through tryptophan degradation along the kynurenine pathway. While the association between IDO1 and CD8 T lymphocyte dysfunction is widely accepted, the precise mechanisms underlying T lymphocyte dysfunction via tryptophan catabolism remain debated. To elucidate these mechanisms, we used genome-wide CRISPR/Cas9 knockout screening in murine CD8 + T lymphocytes, searching for genes whose inactivation favored proliferation in low-tryptophan conditions. Strikingly, a majority of identified genes coded for proteins involved in mitochondrial function. By further exploring the impact of tryptophan deprivation on metabolism, we observed that CD8 + T cells, when activated under tryptophan shortage, fail to shift their metabolism towards glycolysis and instead rely on oxidative phosphorylation (OXPHOS) for energy production. Inactivating OXPHOS restored proliferation by forcing glycolysis despite tryptophan shortage. Since the activation-induced metabolic shift is essential to provide the macromolecules required for proliferation, our results provide a mechanistic rationale to explain the IDO1-induced proliferation arrest of CD8 + T lymphocytes.
    Date: 2024-06-13
    Authors: Zhu J, Bombart R, Corbet C, Gerin I, Bommer G, Gomard T, Klaessens S, Boudhan L, Eynde BVD.
    Ref: Research Square
  8. Abstract The advent of clustered regularly interspaced short palindromic repeat (CRISPR) genome editing initiated an era of accelerated understanding of gene function. A key approach to achieve this goal is functional validation, notably by knock-out (KO) generation of candidate genes in a genetic background harboring the character of interest. CRISPR genome editing enables precise genetic targeting, but the generated mutations are random. In this study, we propose a simple and straightforward method for precise KO (PKO) generation, using CRISPR-Cas9 for precise targeting coupled with a 32bp multi-stop cassette. This cassette is harboring a STOP codon in every reading-frame, such as its insertion guarantees the presence of a stop codon at the position of insertion. The CRISPR-Cas9 system and multi-stop cassette was bombarded on wheat embryos using biolistic. From the 391 embryos bombarded, 177 events of insertion were detected. Sanger sequencing and Next Generation Sequencing (NGS) showed that, in the majority of events, the cassette was truncated prior to its insertion, on average by 5bp. By design, this means that in the majority of detected events a multi-stop sequence was successfully inserted at the targeted position. Only one perfect insertion event was observed and the number of cells reached by the cassette is low, which was expected from this design, indicating that improvements can be made to the method. Our findings showed that PKO is achievable in wheat calli, which opens the way to improvements on wheat, and future developments of optimized KO on regenerated wheat plants.
    Date: 2024-06-13
    Authors: Kadner M, BENEDIT S, PAUL W, PICHON J, TASSY C, BARRET P.
    Ref: Research Square
  9. Abstract Background PIF1 helicase (5ʹ→3ʹ DNA helicase) is a member of helicase superfamily 1. It has unwinding activity and plays a crucial role in maintaining genome stability and coordinating DNA damage repair processes. Overexpression of PIF1 is common in several cancers; however, its role in ovarian cancer remains unclear. This study aimed to elucidate the regulatory role of PIF1 in ovarian cancer and explore its mechanism. Results Analysis of patient samples and public database datasets revealed a negative correlation between PIF1 overexpression and the overall survival rate of the patients. We found through molecular biology experiments and xenograft tumor models in nude mice that CRISPR/Cas9-mediated PIF1 partial knockdown in ovarian cancer cell lines significantly inhibited proliferation and clonogenicity, promoted senescence, and induced G2 cell cycle arrest. Moreover, PIF1 partial deficiency enhanced DNA damage in ovarian cancer cells, particularly sensitive to cisplatin. RAD51 serves as a central scaffold protein for homologous recombination repair and is crucial for timely and accurate DNA repair. We observed that PIF1 partial knockdown resulted in significant reduction of RAD51 in ovarian cancer cells. Notably, RAD51 overexpression in PIF1 partially deficient ovarian cancer cells rescued cell proliferation and DNA damage by increasing PIF1 expression. Immunofluorescence revealed the co-localization of EGFP-PIF1 and RAD51 in the cell nucleus, suggesting that the interaction between PIF1 and RAD51 may regulate the DNA damage response and cell survival in ovarian cancer cells. Conclusions Our study revealed that PIF1 is a druggable target for inducing DNA damage in ovarian cancer cells and provides insights into the potential synergistic mechanisms of action between PIF1 and RAD51 in ovarian cancer therapy.
    Date: 2024-06-13
    Authors: Zhou Q, Hua Y, Sun Q, Zou S, Guo J, Ni M, Zhang S, Liu S, Guo Y, Ao L, Che X, Zhang X, Heger M, Zheng X, Wu Z, Wang X, Cheng S, Pan W.
    Ref: Research Square
  10. One problem that has hampered the use of red fluorescent proteins in the fast-developing nematode C. elegans has been the substantial time delay in maturation of several generations of red fluorophores. The recently described mScarlet-I3 protein has properties that may overcome this limitation. We compare here the brightness and maturation time of CRISPR/Cas9 genome-engineered mScarlet, mScarlet3, mScarlet-I3 and GFP reporter knock-ins. Comparing the onset and brightness of expression of reporter alleles of C. elegans golg-4 , encoding a broadly expressed Golgi resident protein, we found that the onset of detection of mScarlet-I3 in the embryo is several hours earlier than older versions of mScarlet and comparable to GFP. These findings were further supported by comparing mScarlet-I3 and GFP reporter alleles for pks-1 , a gene expressed in the CAN neuron and cells of the alimentary system, as well as reporter alleles for the panneuronal, nuclear marker unc-75 . Hence, the relative properties of mScarlet-I3 and GFP do not depend on cellular or subcellular context. In all cases, mScarlet-I3 reporters also show improved signal-to-noise ratio compared to GFP.
    Date: 2024-06-13
    Authors: Cao WX, Merritt D, Pe K, Cesar M, Hobert O.
    Ref: bioRxiv
  11. Type IV CRISPR-Cas effector complexes are often encoded on plasmids and are proposed to prevent the replication of competing plasmids. The Type IV-A1 CRISPR-Cas system of Pseudomonas oleovorans additionally harbors a CRISPR RNA (crRNA) that tightly regulates the transcript levels of a chromosomal target and represents a natural CRISPR interference (CRISPRi) tool. This study investigates CRISPRi effects of this system using synthetic crRNAs against genome and plasmid sequences. Targeting of reporter genes revealed extended interference in P. oleovorans and Escherichia coli cells producing recombinant CRISPR ribonucleoprotein (crRNP) complexes. RNA-Seq analyses of Type IV-A1 CRISPRi-induced transcriptome alterations demonstrated highly effective long-range down-regulation of histidine operon expression, whereas CRISPRi effects of dCas9 remained limited to the vicinity of its binding site. Single-molecule microscopy uncovered the localization dynamics of crRNP complexes. The tracks of fluorescently labeled crRNPs co-localized with regions of increased plasmid replication, supporting efficient plasmid targeting. These results identify mechanistic principles that facilitate the application of Type IV-A1 CRISPRi for the regulation of gene expression and plasmid replication.
    Date: 2024-06-12
    Authors: Sanchez-Londono M, Rust S, Hernandez-Tamayo R, Gomes-Filho JV, Thanbichler M, Randau L.
    Ref: bioRxiv
  12. Over 50 hereditary degenerative disorders are caused by expansions of short tandem DNA repeats (STRs). (GAA) n repeat expansions are responsible for Friedreich's ataxia as well as late-onset cerebellar ataxias (LOCAs). Thus, the mechanisms of (GAA) n repeat expansions attract broad scientific attention. To investigate the role of DNA nicks in this process, we utilized a CRISPR-Cas9 nickase system to introduce targeted nicks adjacent to the (GAA) n repeat tract. We found that DNA nicks 5' of the (GAA) 100 run led to a dramatic increase in both the rate and scale of its expansion in dividing cells. Strikingly, they also promoted large-scale expansions of carrier- and large normal-size (GAA) n repeats, recreating, for the first time in a model system, the expansion events that occur in human pedigrees. DNA nicks 3' of the (GAA) 100 repeat led to a smaller but significant increase in the expansion rate as well. Our genetic analysis implies that in dividing cells, conversion of nicks into double-strand breaks (DSBs) during DNA replication followed by DSB or fork repair leads to repeat expansions. Finally, we showed that 5' strand nicks increase expansion frequency in non-dividing yeast cells, albeit to a lesser extent that in dividing cells.
    Date: 2024-06-12
    Authors: Li L, Scott WS, Khristich AN, Armenia JF, Mirkin SM.
    Ref: bioRxiv
  13. Rickettsia rickettsii is an obligate intracellular, tick-borne bacterium that causes Rocky Mountain spotted fever. The demanding nature of cultivating these bacteria within host cells and the labor involved in obtaining clonal isolates has severely limited progress regarding the development of compatible genetic tools to study this pathogen. Conditional expression of genes which might be toxic or have an otherwise undesirable effect is the next logical goal to expand upon the constitutive expression plasmids generated thus far. We describe the construction of an inducible promoter system based on the tet-On system, leveraging design elements from the anhydrotetracycline inducible promoter system used for Borrelia burgdorferi and one of the few characterized rickettsial promoters for the outer membrane gene, rompB (sca5). The functionality of this promoter is demonstrated via fluorescence of induced mScarlet production and was then used to construct a generalized inducible expression vector for R. rickettsii. The development of a functional inducible promoter was then applied to the construction of a CRISPR interference plasmid as a means to reduce or essentially silence the transcription of targeted genes. We demonstrate the viability of a simplified, single vector CRISPRi system to disrupt gene expression in R. rickettsii targeting the type IV secreted effector rarP2 and autotransporter peptidase rapL as examples.
    Date: 2024-06-12
    Authors: Nock A, Clark TR, Hackstadt T.
    Ref: bioRxiv
  14. Abstract The endoplasmic reticulum (ER) orchestrates major cellular processes, including protein synthesis, folding, assembly and degradation, to maintain cellular proteostasis. Central to these processes are highly stringent quality control machineries like the ER-associated protein degradation (ERAD). Key players in ERAD include HRD1 and SEL1L, which target misfolded proteins for ubiquitination and facilitate their retro-translocation to the cytosol. Bi-allelic loss-of-function of HRD1 and SEL1L is considered lethal, with hypomorphic variants linked to human diseases, including neurodevelopmental disorders. Despite their well-known roles, a comprehensive transcriptomic characterization of their bi-allelic loss has been lacking. In this study, we employed CRISPR/Cas9 to generate bi-allelic HRD1-KO and SEL1L-KO HEK293 cell models. Through differential gene expression analysis and co-expression network construction, we identified hub genes and novel regulatory networks. HRD1-KO cells displayed enrichment solely in ER-related processes, suggesting its specific role in ER protein quality control. Conversely, SEL1L-KO cells exhibited a broader impact, affecting mitochondrial function, ERAD-ribosomal quality control interactions, ER-Golgi transport, and Wnt signaling pathway. These results highlight the distinct roles of HRD1 and SEL1L in ERAD. By unraveling their whole transcriptome impact, our study sheds light on their potential involvement in diverse cellular processes, potentially enhancing our understanding of their cellular processes and disease mechanisms.
    Date: 2024-06-12
    Authors: Badawi S, Gariballa N, Kizhakkedath P, Ali BR.
    Ref: Research Square
  15. SETD8 is a methyltransferase that is overexpressed in several cancers, which monomethylates H4K20 as well as other non-histone targets such as PCNA or p53. We here report novel SETD8 inhibitors, which were discovered while trying to identify chemicals that prevent 53BP1 foci formation, an event mediated by H4K20 methylation. Consistent with previous reports, SETD8 inhibitors induce p53 expression, although they are equally toxic for p53-deficient cells. Thermal stability proteomics revealed that the compounds had a particular impact on nucleoli, which was confirmed by fluorescent and electron microscopy. Similarly, Setd8 deletion generated nucleolar stress and impaired ribosome biogenesis, confirming that this was an on-target effect of SETD8 inhibitors. Furthermore, a genome-wide CRISPR screen identified an enrichment of nucleolar factors among those modulating the toxicity of SETD8 inhibitors. Accordingly, the toxicity of SETD8 inhibitors correlated with MYC or mTOR activity, key regulators of ribosome biogenesis. Together, our study provides a new class of SETD8 inhibitors and a novel biomarker to identify tumors most likely to respond to this therapy.
    Date: 2024-06-12
    Authors: Murga M, Lopez-Pernas G, Soliva R, Fueyo-Marcos E, Amor C, Faustino I, Serna M, Serrano AG, Diaz L, Martinez S, Blanco-Aparicio C, Anton ME, Seashore-Ludlow B, Pastor J, Jafari R, Lafarga M, Llorca O, Orozco M, Fernandez-Capetillo O.
    Ref: bioRxiv
  16. Terminal deoxynucleotidyl transferase (TdT) is a unique DNA polymerase capable of template-independent extension of DNA with random nucleotides. TdT’s de novo DNA synthesis ability has found utility in DNA recording, DNA data storage, oligonucleotide synthesis, and nucleic acid labeling, but TdT’s intrinsic nucleotide biases limit its versatility in such applications. Here, we describe a multiplexed assay for profiling and engineering the bias and overall activity of TdT variants in high throughput. In our assay, a library of TdTs is encoded next to a CRISPR-Cas9 target site in HEK293T cells. Upon transfection of Cas9 and sgRNA, the target site is cut, allowing TdT to intercept the double strand break and add nucleotides. Each resulting insertion is sequenced alongside the identity of the TdT variant that generated it. Using this assay, 25,623 unique TdT variants, constructed by site-saturation mutagenesis at strategic positions, were profiled. This resulted in the isolation of several altered-bias TdTs that expanded the capabilities of our TdT-based DNA recording system, Cell History Recording by Ordered Insertion (CHYRON), by increasing the information density of recording through an unbiased TdT and achieving dual-channel recording of two distinct inducers (hypoxia and Wnt) through two differently biased TdTs. Select TdT variants were also tested in vitro , revealing concordance between each variant’s in vitro bias and the in vivo bias determined from the multiplexed high throughput assay. Overall, our work, and the multiplex assay it features, should support the continued development of TdT-based DNA recorders, in vitro applications of TdT, and further study of the biology of TdT.
    Date: 2024-06-11
    Authors: Carlson CK, Loveless TB, Milisavljevic M, Kelly PI, Mills JH, Tyo KEJ, Liu CC.
    Ref: bioRxiv
  17. Abstract Developmental anomalies of the hearing organ, the cochlea, are diagnosed in approximately one-fourth of individuals with congenital deafness. Most patients with cochlear malformations remain etiologically undiagnosed due to insufficient knowledge about underlying genes or the inability to make conclusive interpretations of identified genetic variants. We used exome sequencing for genetic evaluation of hearing loss associated with cochlear malformations in three probands from unrelated families. We subsequently generated monoclonal induced pluripotent stem cell (iPSC) lines, bearing patient-specific knockins and knockouts using CRISPR/Cas9 to assess pathogenicity of candidate variants. We detected FGF3 (p.Arg165Gly) and GREB1L (p.Cys186Arg), variants of uncertain significance in two recognized genes for deafness, and PBXIP1 (p.Trp574*) in a candidate gene. Upon differentiation of iPSCs towards inner ear organoids, we observed significant developmental aberrations in knockout lines compared to their isogenic controls. Patient-specific single nucleotide variants (SNVs) showed similar abnormalities as the knockout lines, functionally supporting their causality in the observed phenotype. Therefore, we present human inner ear organoids as a tool to rapidly validate the pathogenicity of DNA variants associated with cochlear malformations.
    Date: 2024-06-11
    Authors: Zafeer MF, Ramzan M, Duman D, Mutlu A, Seyhan S, Kalcioglu T, Fitoz S, DeRosa BA, Guo S, Dykxhoorn DM, Tekin M.
    Ref: Research Square
  18. Background: Motor learning is central to human existence, such as learning to speak or walk, sports moves, or rehabilitation after injury. Evidence suggests that all forms of motor learning share an evolutionarily conserved molecular plasticity pathway. Here, we present novel insights into the neural processes underlying operant self-learning, a form of motor learning in the fruit fly Drosophila. Methods We operantly trained wild type and transgenic Drosophila fruit flies, tethered at the torque meter, in a motor learning task that required them to initiate and maintain turning maneuvers around their vertical body axis (yaw torque). We combined this behavioral experiment with transgenic peptide expression, CRISPR/Cas9-mediated, spatio-temporally controlled gene knock-out and confocal microscopy. Results We find that expression of atypical protein kinase C (aPKC) in direct wing steering motoneurons co-expressing the transcription factor FoxP is necessary for this type of motor learning and that aPKC likely acts via non-canonical pathways. We also found that it takes more than a week for CRISPR/Cas9-mediated knockout of FoxP in adult animals to impair motor learning, suggesting that adult FoxP expression is required for operant self-learning. Conclusions Our experiments suggest that, for operant self-learning, a type of motor learning in Drosophila , co-expression of atypical protein kinase C (aPKC) and the transcription factor FoxP is necessary in direct wing steering motoneurons. Some of these neurons control the wing beat amplitude when generating optomotor responses, and we have discovered modulation of optomotor behavior after operant self-learning. We also discovered that aPKC likely acts via non-canonical pathways and that FoxP expression is also required in adult flies.
    Date: 2024-06-11
    Authors: Ehweiner A, Duch C, Brembs B.
    Ref: F1000Res
  19. Clustered regularly interspaced short palindromic repeats (CRISPR) based gene-editing has begun to transform the treatment landscape of genetic diseases. The history of the discovery of CRISPR/CRISPR-associated (Cas) proteins/single guide RNA (sgRNA)-based gene-editing since the first report of repetitive sequences of unknown significance in 1987 is fascinating, instructive, and inspiring for future advances. The recent approval of CRISPR-Cas9-based gene therapy to treat patients with severe sickle cell anemia and transfusion-dependent beta thalassemia has renewed hope for treating other hematologic diseases, including patients with germline predisposition to hematologic malignancies, who would benefit greatly from the development of CRISPR-based gene therapies. The purpose of this manuscript is three-fold: first, a chronological description of the history of CRISPR-Cas9-sgRNA-based gene editing; second, a brief description of the current state of clinical research in hematologic diseases, including selected applications in treating hematologic diseases with CRISPR-based gene therapy; and third, the current progress in gene therapies in inherited hematologic diseases and bone marrow failure syndromes, to hopefully stimulate efforts towards developing these therapies for patients with inherited bone marrow failure syndromes and other inherited conditions with germline predisposition to hematologic malignancies.
    Date: 2024-06-10
    Authors: Kansal R.
    Ref: Preprints.org
  20. Hypoimmune gene edited human pluripotent stem cells (hPSCs) are a promising platform for developing reparative cellular therapies that evade immune rejection. Existing first-generation hypoimmune strategies have used CRISPR/Cas9 editing to modulate genes associated with adaptive (e.g., T cell) immune responses, but have largely not addressed the innate immune cells (e.g., monocytes, neutrophils) that mediate inflammation and rejection processes occurring early after graft transplantation. We identified the adhesion molecule ICAM-1 as a novel hypoimmune target that plays multiple critical roles in both adaptive and innate immune responses post-transplantation. In a series of studies, we found that ICAM-1 blocking or knock-out (KO) in hPSC-derived cardiovascular therapies imparted significantly diminished binding of multiple immune cell types. ICAM-1 KO resulted in diminished T cell proliferation responses in vitro and in longer in vivo retention/protection of KO grafts following immune cell encounter in NeoThy humanized mice. The ICAM-1 KO edit was also introduced into existing first-generation hypoimmune hPSCs and prevented immune cell binding, thereby enhancing the overall hypoimmune capacity of the cells. This novel hypoimmune editing strategy has the potential to improve the long-term efficacy and safety profiles of regenerative therapies for cardiovascular pathologies and a number of other diseases. Highlights Antibody blocking of ICAM-1 on human pluripotent stem cell-derived cells inhibits immune cell adhesion CRISPR/Cas9 knock-out of ICAM-1 ablates surface and secreted ICAM-1 protein and inhibits immune adhesion ICAM-1 knock-out results in decreased T cell proliferative responses to human pluripotent stem cell-derived grafts in vitro , and resistance to immune-mediated graft loss in vivo Addition of ICAM-1 knock-out to first generation MHC knock-out human pluripotent stem cells confers protection against immune adhesion Graphical Abstract ICAM-1 Knock-out in Transendothelial Migration and at the Immune Synapse. Abbreviations: PSC-EC – pluripotent stem cell-derived endothelial cells; KO – knock-out; dSMAC – distal supramolecular activation complex; pSMAC – peripheral supramolecular activation complex; cSMAC – central supramolecular activation complex.
    Date: 2024-06-09
    Authors: Saha S, Haynes WJ, Del Rio NM, Young EE, Zhang J, Seo J, Huang L, Holm AM, Blashka W, Murphy L, Scholz MJ, Henrichs A, Suresh Babu J, Steill J, Stewart R, Kamp TJ, Brown ME.
    Ref: bioRxiv
  21. Huntington’s disease (HD), one of >50 inherited repeat expansion disorders (Depienne and Mandel, 2021), is a dominantly-inherited neurodegenerative disease caused by a CAG expansion in HTT (The Huntington’s Disease Collaborative Research Group, 1993). Inherited CAG repeat length is the primary determinant of age of onset, with human genetic studies underscoring that the property driving disease is the CAG length-dependent propensity of the repeat to further expand in brain (Swami et al ., 2009; GeM-HD, 2015; Hensman Moss et al ., 2017; Ciosi et al ., 2019; GeM-HD, 2019; Hong et al ., 2021). Routes to slowing somatic CAG expansion therefore hold great promise for disease-modifying therapies. Several DNA repair genes, notably in the mismatch repair (MMR) pathway, modify somatic expansion in HD mouse models (Wheeler and Dion, 2021). To identify novel modifiers of somatic expansion, we have used CRISPR-Cas9 editing in HD knock-in mice to enable in vivo screening of expansion-modifier candidates at scale. This has included testing of HD onset modifier genes emerging from human genome-wide association studies (GWAS), as well as interactions between modifier genes, thereby providing new insight into pathways underlying CAG expansion and potential therapeutic targets.
    Date: 2024-06-09
    Authors: Mouro Pinto R, Murtha R, Azevedo A, Douglas C, Kovalenko M, Ulloa J, Crescenti S, Burch Z, Oliver E, Vitalo A, Mota-Silva E, Riggs MJ, Correia K, Elezi E, Demelo B, Carroll JB, Gillis T, Gusella JF, MacDonald ME, Wheeler VC.
    Ref: bioRxiv
  22. ABSTRACT Xanthomonas spp. employ transcription activator-like effectors (TALEs) to promote pathogenicity by activating host susceptibility ( S ) genes. Cotton GhSWEET10 is an S gene targeted by a TALE in an early isolate of Xanthomonas citri pv. malvacearum ( Xcm ), but not by recent field Xcm isolates. To understand the pathogenicity shift in Xcm and its adaptation to cotton, we assembled the whole genome and the TALE repertoire of three recent Xcm Texas field isolates. A newly evolved TALE, Tal7b, activated different GhSWEET genes, GhSWEET14a and GhSWEET14b . Simultaneous activation of GhSWEET14a and GhSWEET14b resulted in pronounced water-soaked lesions. Transcriptome profiling coupled with TALE-binding element prediction identified a pectin lyase as an additional Tal7b target, quantitatively contributing to Xcm virulence alongside GhSWEET14a/b . CRISPR-Cas9-based gene editing supported the function of GhSWEETs as S genes in cotton bacterial blight and the promise of disrupting the TALE-binding site in these genes to control the disease. Collectively, our findings elucidate the rapid evolution of TALEs in Xanthomonas field isolates and highlight the virulence mechanism wherein TALEs induce multiple S genes simultaneously to promote pathogenicity.
    Date: 2024-06-09
    Authors: Mormile B, Bauer T, Wang L, Rivero R, Carpenter SC, Danmaigona-Clement C, Cox K, Zhang L, Ma X, Wheeler TA, Dever JK, He P, Bogdanove AJ, Shan L.
    Ref: bioRxiv
  23. Gene-editing technologies promise to create a new class of therapeutics that can achieve permanent correction with a single intervention. Besides eliminating mutant alleles in familial disease, gene-editing can also be used to favorably manipulate upstream pathophysiologic events and alter disease-course in wider patient populations, but few such feasible therapeutic avenues have been reported. Here we use CRISPR-Cas9 to edit the last exon of amyloid precursor protein ( App ), relevant for Alzheimer’s disease (AD). Our strategy effectively eliminates an endocytic (YENPTY) motif at APP C-terminus, while preserving the N-terminus and compensatory APP-homologues. This manipulation favorably alters events along the amyloid-pathway – inhibiting toxic APP-β-cleavage fragments (including Aβ) and upregulating neuroprotective APP-α-cleavage products. AAV-driven editing ameliorates neuropathologic, electrophysiologic, and behavioral deficits in an AD knockin mouse model. Effects persist for many months, and no abnormalities are seen in WT mice even after germline App -editing; underlining overall efficacy and safety. Pathologic alterations in the glial-transcriptome of App -KI mice, as seen by single nuclei RNA-sequencing (sNuc-Seq), are also normalized by App C-terminus editing. Our strategy takes advantage of innate transcriptional rules that render terminal exons insensitive to nonsense-decay, and the upstream manipulation is expected to be effective for all forms of AD. These studies offer a path for a one-time disease-modifying treatment for AD.
    Date: 2024-06-09
    Authors: Aulston BD, Gimse K, Bazick HO, Kramar EA, Pizzo DP, Parra-Rivas LA, Sun J, Branes-Guerrero K, Checka N, Bagheri N, Satyadev N, Carlson-Stevermer J, Saito T, Saido TC, Audhya A, Wood MA, Zylka MJ, Saha K, Roy S.
    Ref: bioRxiv
  24. CLN3 Batten disease (also referred to as Juvenile Neuronal Ceroid Lipofuscinosis; JNCL) is a lysosomal storage disorder that typically initiates with retinal degeneration but is followed by seizure onset, motor decline and premature death. Patient-derived iPSC-RPE cells show defective phagocytosis of photoreceptor outer segments (POSs). Because modifier genes are implicated in CLN3 disease, our goal here was to investigate a direct link between CLN3 mutation and POS phagocytosis defect by diseased RPE cells. Isogenic control and CLN3 mutant stem cell lines were generated by CRISPR-Cas9-mediated biallelic deletion of exons 7 and 8. A transgenic Yucatan miniswine model of CLN3 disease ( CLN3 Δ7–8/Δ7-8 pigs) was also used to study the impact of CLN3 Δ7-8/Δ7-8 mutation on POS phagocytosis. POS phagocytosis by cultured RPE cells was analyzed by Western blotting and immunohistochemistry. Electroretinogram, optical coherence tomography and histological analysis of CLN3 Δ7-8 and wild-type miniswine eyes were carried out at 6-, 36-, or 48-month age. CLN3 Δ7-8/Δ7-8 RPE ( CLN3 RPE) displayed reduced POS binding and consequently decreased uptake of POS compared to isogenic control RPE cells . Furthermore, wild-type miniswine RPE cells phagocytosed POS isolated from CLN3 Δ7-8/Δ7-8 ( CLN3 ) Yucatan miniswine less efficiently than POS isolated from wild-type miniswines. Consistent with decreased POS phagocytosis, lipofuscin/autofluorescence was decreased in CLN3 miniswine RPE at 36 months-of-age and was followed by almost complete loss of photoreceptors at 48 months of age. These results show that CLN3 Δ7-8/Δ7-8 mutation (that affects up to 85% patients) is independently sufficient for promoting both impaired POS phagocytosis and photoreceptor cell loss in CLN3 disease.
    Date: 2024-06-09
    Authors: Han J, Chear S, Talbot J, Swier V, Booth C, Reuben-Thomas C, Dalvi S, Weimer JM, Hewitt AW, Cook AL, Singh R.
    Ref: bioRxiv
  25. The common clownfish, Amphiprion ocellaris , is an iconic coral reef fish, ubiquitous in the marine aquarium hobby and useful for studying a variety of biological processes (e.g., mutual symbiosis, ultraviolet vision, and protandrous sex change). Recently, CRISPR/Cas9 methods were developed for knocking out specific genes for mechanistic studies. Here, we expand the genetic toolkit for A. ocellaris by creating the first transgenic line using the Tol2 transposon system. Fertilized eggs were co-injected with Tol2 transposase mRNA and a plasmid encoding an Elongation factor 1 α ( Ef1 α): Green fluorescent protein ( GFP) cassette at various concentrations, needle tip dimensions and timepoints post-fertilization. We compared various injection parameters and sterilization methods to maximize the survival of injected eggs. F0s (n=10) that were genotyped GFP+ were then raised to 6 months of age and crossed with wild-type (WT) females to confirm germline transmission. F1 offspring were also raised and crossed in the same manner. The highly efficient Tol2 transposon system resulted in a 37% rate of transgenesis for surviving eggs amounting to a 2.7% yield of all injected eggs surviving and being GFP+ (n= 160). Of these, 10 were raised to adulthood, 8 spawned, and 5/8 (62.5 %) produced GFP+ offspring. Further, 53.8% of offspring from an F1 paired with a WT female were GFP+ confirming the creation of a stable line. This is, to our knowledge, the first generation of a transgenic line in any coral reef fish. The ability to express transgenes of interest in the iconic anemonefish opens the door to a new era of exploration into their fascinating biology.
    Date: 2024-06-09
    Authors: Graham GJ, Ibanez EM, Mitchell LJ, Weis KE, Raetzman LT, Cortesi F, Rhodes JS.
    Ref: bioRxiv
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