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1. Key HPI axis receptors facilitate light adaptive behavior in larval zebrafish

Author

Han B. Lee, Soaleha Shams, Viet Ha Dang Thi, Grace E. Boyum, Rodsy Modhurima, Emma M. Hall, Izzabella K. Green, Elizabeth M. Cervantes, Fernando E. Miguez, and Karl J. Clark

Subjects
GR (glucocorticoid receptor), Stress response, Zebrafish, Light assays, Light adaptation, GAM (generalized additive models), Medicine, Science
Abstract

Abstract The vertebrate stress response (SR) is mediated by the hypothalamic–pituitary–adrenal (HPA) axis and contributes to generating context appropriate physiological and behavioral changes. Although the HPA axis plays vital roles both in stressful and basal conditions, research has focused on the response under stress. To understand broader roles of the HPA axis in a changing environment, we characterized an adaptive behavior of larval zebrafish during ambient illumination changes. Genetic abrogation of glucocorticoid receptor (nr3c1) decreased basal locomotor activity in light and darkness. Some key HPI axis receptors (mc2r [ACTH receptor], nr3c1), but not nr3c2 (mineralocorticoid receptor), were required to adapt to light more efficiently but became dispensable when longer illumination was provided. Such light adaptation was more efficient in dimmer light. Our findings show that the HPI axis contributes to the SR, facilitating the phasic response and maintaining an adapted basal state, and that certain adaptations occur without HPI axis activity.

Published
2024
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2. Genetic therapy in a mitochondrial disease model suggests a critical role for liver dysfunction in mortality

Author

Ankit Sabharwal, Mark D Wishman, Roberto Lopez Cervera, MaKayla R Serres, Jennifer L Anderson, Shannon R Holmberg, Bibekananda Kar, Anthony J Treichel, Noriko Ichino, Weibin Liu, Jingchun Yang, Yonghe Ding, Yun Deng, Jean M Lacey, William J Laxen, Perry R Loken, Devin Oglesbee, Steven A Farber, Karl J Clark, Xiaolei Xu, and Stephen C Ekker

Subjects
Leigh Syndrome French Canadian Type, gene-breaking transposon, LRPPRC, mitochondria, zebrafish, Medicine, Science, Biology (General), QH301-705.5
Abstract

The clinical and largely unpredictable heterogeneity of phenotypes in patients with mitochondrial disorders demonstrates the ongoing challenges in the understanding of this semi-autonomous organelle in biology and disease. Previously, we used the gene-breaking transposon to create 1200 transgenic zebrafish strains tagging protein-coding genes (Ichino et al., 2020), including the lrpprc locus. Here, we present and characterize a new genetic revertible animal model that recapitulates components of Leigh Syndrome French Canadian Type (LSFC), a mitochondrial disorder that includes diagnostic liver dysfunction. LSFC is caused by allelic variations in the LRPPRC gene, involved in mitochondrial mRNA polyadenylation and translation. lrpprc zebrafish homozygous mutants displayed biochemical and mitochondrial phenotypes similar to clinical manifestations observed in patients, including dysfunction in lipid homeostasis. We were able to rescue these phenotypes in the disease model using a liver-specific genetic model therapy, functionally demonstrating a previously under-recognized critical role for the liver in the pathophysiology of this disease.

Published
2022
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3. A phenotype-based forward genetic screen identifies Dnajb6 as a sick sinus syndrome gene

Author

Yonghe Ding, Di Lang, Jianhua Yan, Haisong Bu, Hongsong Li, Kunli Jiao, Jingchun Yang, Haibo Ni, Stefano Morotti, Tai Le, Karl J Clark, Jenna Port, Stephen C Ekker, Hung Cao, Yuji Zhang, Jun Wang, Eleonora Grandi, Zhiqiang Li, Yongyong Shi, Yigang Li, Alexey V Glukhov, and Xiaolei Xu

Subjects
sick sinus syndrome, sinus arrest, Dnajb6, electrocardiogram, genetic diseases, zebrafish, Medicine, Science, Biology (General), QH301-705.5
Abstract

Previously we showed the generation of a protein trap library made with the gene-break transposon (GBT) in zebrafish (Danio rerio) that could be used to facilitate novel functional genome annotation towards understanding molecular underpinnings of human diseases (Ichino et al, 2020). Here, we report a significant application of this library for discovering essential genes for heart rhythm disorders such as sick sinus syndrome (SSS). SSS is a group of heart rhythm disorders caused by malfunction of the sinus node, the heart’s primary pacemaker. Partially owing to its aging-associated phenotypic manifestation and low expressivity, molecular mechanisms of SSS remain difficult to decipher. From 609 GBT lines screened, we generated a collection of 35 zebrafish insertional cardiac (ZIC) mutants in which each mutant traps a gene with cardiac expression. We further employed electrocardiographic measurements to screen these 35 ZIC lines and identified three GBT mutants with SSS-like phenotypes. More detailed functional studies on one of the arrhythmogenic mutants, GBT411, in both zebrafish and mouse models unveiled Dnajb6 as a novel SSS causative gene with a unique expression pattern within the subpopulation of sinus node pacemaker cells that partially overlaps with the expression of hyperpolarization activated cyclic nucleotide gated channel 4 (HCN4), supporting heterogeneity of the cardiac pacemaker cells.

Published
2022
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4. An optimized FusX assembly-based technique to introduce mitochondrial TC-to-TT variations in human cell lines

Author

Bibekananda Kar, Ankit Sabharwal, Santiago Restrepo-Castillo, Brandon W. Simone, Karl J. Clark, and Stephen C. Ekker

Subjects
Cell Biology, Genetics, Sequencing, Molecular Biology, CRISPR, Science (General), Q1-390
Abstract

Summary: The FusX TALE Based Editor (FusXTBE) is a programmable base editing platform that can introduce specific TC-to-TT variations in the mitochondrial DNA (mtDNA). Here, we provide a protocol describing the synthesis and testing of the FusXTBE plasmids in cultured human cell lines. This tool is designed to be easily modified to work in diverse applications where editing of mitochondrial DNA is desired.For complete details on the use and execution of this protocol, please refer to Sabharwal et al. (2021) and Ma et al. (2016).

Published
2022
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5. Cre/lox regulated conditional rescue and inactivation with zebrafish UFlip alleles generated by CRISPR-Cas9 targeted integration

Author

Fang Liu, Sekhar Kambakam, Maira P Almeida, Zhitao Ming, Jordan M Welker, Wesley A Wierson, Laura E Schultz-Rogers, Stephen C Ekker, Karl J Clark, Jeffrey J Essner, and Maura McGrail

Subjects
zebrafish, Cre/lox, conditional rescue and inactivation, CRISPR-Cas9, precision targeted integration, GeneWeld, Medicine, Science, Biology (General), QH301-705.5
Abstract

The ability to regulate gene activity spatially and temporally is essential to investigate cell-type-specific gene function during development and in postembryonic processes and disease models. The Cre/lox system has been widely used for performing cell and tissue-specific conditional analysis of gene function in zebrafish. However, simple and efficient methods for isolation of stable, Cre/lox regulated zebrafish alleles are lacking. Here, we applied our GeneWeld CRISPR-Cas9 targeted integration strategy to generate floxed alleles that provide robust conditional inactivation and rescue. A universal targeting vector, UFlip, with sites for cloning short homology arms flanking a floxed 2A-mRFP gene trap, was integrated into an intron in rbbp4 and rb1. rbbp4off and rb1off integration alleles resulted in strong mRFP expression,>99% reduction of endogenous gene expression, and recapitulated known indel loss-of-function phenotypes. Introduction of Cre led to stable inversion of the floxed cassette, loss of mRFP expression, and phenotypic rescue. rbbp4on and rb1on integration alleles did not cause phenotypes in combination with a loss-of-function mutation. Addition of Cre led to conditional inactivation by stable inversion of the cassette, gene trapping and mRFP expression, and the expected mutant phenotype. Neural progenitor Cre drivers were used for conditional inactivation and phenotypic rescue to showcase how this approach can be used in specific cell populations. Together these results validate a simplified approach for efficient isolation of Cre/lox-responsive conditional alleles in zebrafish. Our strategy provides a new toolkit for generating genetic mosaics and represents a significant advance in zebrafish genetics.

Published
2022
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6. Mitochondrial Base Editing: Recent Advances towards Therapeutic Opportunities

Author

Bibekananda Kar, Santiago R. Castillo, Ankit Sabharwal, Karl J. Clark, and Stephen C. Ekker

Subjects
mitochondria, mitochondrial DNA, heteroplasmy, base editing, DdCBE, TALED, Biology (General), QH301-705.5, Chemistry, QD1-999
Abstract

Mitochondria are critical organelles that form networks within our cells, generate energy dynamically, contribute to diverse cell and organ function, and produce a variety of critical signaling molecules, such as cortisol. This intracellular microbiome can differ between cells, tissues, and organs. Mitochondria can change with disease, age, and in response to the environment. Single nucleotide variants in the circular genomes of human mitochondrial DNA are associated with many different life-threatening diseases. Mitochondrial DNA base editing tools have established novel disease models and represent a new possibility toward personalized gene therapies for the treatment of mtDNA-based disorders.

Published
2023
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7. Endogenous zebrafish proneural Cre drivers generated by CRISPR/Cas9 short homology directed targeted integration

Author

Maira P. Almeida, Jordan M. Welker, Sahiba Siddiqui, Jon Luiken, Stephen C. Ekker, Karl J. Clark, Jeffrey J. Essner, and Maura McGrail

Subjects
Medicine, Science
Abstract

Abstract We previously reported efficient precision targeted integration of reporter DNA in zebrafish and human cells using CRISPR/Cas9 and short regions of homology. Here, we apply this strategy to isolate zebrafish Cre recombinase drivers whose spatial and temporal restricted expression mimics endogenous genes. A 2A-Cre recombinase transgene with 48 bp homology arms was targeted into proneural genes ascl1b, olig2 and neurod1. We observed high rates of germline transmission ranging from 10 to 100% (2/20 olig2; 1/5 neurod1; 3/3 ascl1b). The transgenic lines Tg(ascl1b-2A-Cre) is75 , Tg(olig2-2A-Cre) is76 , and Tg(neurod1-2A-Cre) is77 expressed functional Cre recombinase in the expected proneural cell populations. Somatic targeting of 2A-CreERT2 into neurod1 resulted in tamoxifen responsive recombination in the nervous system. The results demonstrate Cre recombinase expression is driven by the native promoter and regulatory elements of the targeted genes. This approach provides a straightforward, efficient, and cost-effective method to generate cell type specific zebrafish Cre and CreERT2 drivers, overcoming challenges associated with promoter-BAC and transposon mediated transgenics.

Published
2021
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8. Haploinsufficiency as a disease mechanism in GNB1‐associated neurodevelopmental disorder

Author

Laura Schultz‐Rogers, Ikuo Masuho, Filippo Pinto e Vairo, Christopher T. Schmitz, Tanya L. Schwab, Karl J. Clark, Lauren Gunderson, Pavel N. Pichurin, Klaas Wierenga, Kirill A. Martemyanov, and Eric W. Klee

Subjects
Genetics, QH426-470
Abstract

Abstract Background GNB1 encodes a subunit of a heterotrimeric G‐protein complex that transduces intracellular signaling cascades. Disruptions to the gene have previously been shown to be embryonic lethal in knockout mice and to cause complex neurodevelopmental disorders in humans. To date, the majority of variants associated with disease in humans have been missense variants in exons 5‐7. Methods Genetic sequencing was performed on two patients presenting with complex neurological phenotypes including intellectual disability, hypotonia, and in one patient seizures. Reported variants were assessed using RNA sequencing and functional BRET/BiFC assays. Results A splice variant reported in patient 1 was confirmed to cause usage of a cryptic splice site leading to a truncated protein product. Patient 2 was reported to have a truncating variant. BRET and BiFC assays of both patient variants confirmed both were deficient in inducing GPCR‐induced G protein activation due to lack of dimer formation with the Gγ subunit. Conclusion Here, we report two patients with functionally confirmed loss of function variants in GNB1 and neurodevelopmental phenotypes including intellectual disability, hypotonia, and seizures in one patient. These results suggest haploinsufficiency of GNB1 is a mechanism for neurodevelopmental disorders in humans.

Published
2020
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9. Building the vertebrate codex using the gene breaking protein trap library

Author

Noriko Ichino, MaKayla R Serres, Rhianna M Urban, Mark D Urban, Anthony J Treichel, Kyle J Schaefbauer, Lauren E Tallant, Gaurav K Varshney, Kimberly J Skuster, Melissa S McNulty, Camden L Daby, Ying Wang, Hsin-kai Liao, Suzan El-Rass, Yonghe Ding, Weibin Liu, Jennifer L Anderson, Mark D Wishman, Ankit Sabharwal, Lisa A Schimmenti, Sridhar Sivasubbu, Darius Balciunas, Matthias Hammerschmidt, Steven Arthur Farber, Xiao-Yan Wen, Xiaolei Xu, Maura McGrail, Jeffrey J Essner, Shawn M Burgess, Karl J Clark, and Stephen C Ekker

Subjects
protein trap, gene-break transposon, disease model, human genetic disorders, light sheet microscopy, gene reversion, Medicine, Science, Biology (General), QH301-705.5
Abstract

One key bottleneck in understanding the human genome is the relative under-characterization of 90% of protein coding regions. We report a collection of 1200 transgenic zebrafish strains made with the gene-break transposon (GBT) protein trap to simultaneously report and reversibly knockdown the tagged genes. Protein trap-associated mRFP expression shows previously undocumented expression of 35% and 90% of cloned genes at 2 and 4 days post-fertilization, respectively. Further, investigated alleles regularly show 99% gene-specific mRNA knockdown. Homozygous GBT animals in ryr1b, fras1, tnnt2a, edar and hmcn1 phenocopied established mutants. 204 cloned lines trapped diverse proteins, including 64 orthologs of human disease-associated genes with 40 as potential new disease models. Severely reduced skeletal muscle Ca2+ transients in GBT ryr1b homozygous animals validated the ability to explore molecular mechanisms of genetic diseases. This GBT system facilitates novel functional genome annotation towards understanding cellular and molecular underpinnings of vertebrate biology and human disease.

Published
2020
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11. Efficient targeted integration directed by short homology in zebrafish and mammalian cells

Author

Wesley A Wierson, Jordan M Welker, Maira P Almeida, Carla M Mann, Dennis A Webster, Melanie E Torrie, Trevor J Weiss, Sekhar Kambakam, Macy K Vollbrecht, Merrina Lan, Kenna C McKeighan, Jacklyn Levey, Zhitao Ming, Alec Wehmeier, Christopher S Mikelson, Jeffrey A Haltom, Kristen M Kwan, Chi-Bin Chien, Darius Balciunas, Stephen C Ekker, Karl J Clark, Beau R Webber, Branden S Moriarity, Stacy L Solin, Daniel F Carlson, Drena L Dobbs, Maura McGrail, and Jeffrey Essner

Subjects
CRISPR/Cas9, knock-in, targeted integration, human, pig fibroblasts, end joining, Medicine, Science, Biology (General), QH301-705.5
Abstract

Efficient precision genome engineering requires high frequency and specificity of integration at the genomic target site. Here, we describe a set of resources to streamline reporter gene knock-ins in zebrafish and demonstrate the broader utility of the method in mammalian cells. Our approach uses short homology of 24–48 bp to drive targeted integration of DNA reporter cassettes by homology-mediated end joining (HMEJ) at high frequency at a double strand break in the targeted gene. Our vector series, pGTag (plasmids for Gene Tagging), contains reporters flanked by a universal CRISPR sgRNA sequence which enables in vivo liberation of the homology arms. We observed high rates of germline transmission (22–100%) for targeted knock-ins at eight zebrafish loci and efficient integration at safe harbor loci in porcine and human cells. Our system provides a straightforward and cost-effective approach for high efficiency gene targeting applications in CRISPR and TALEN compatible systems.

Published
2020
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12. Disruption of pdgfra alters endocardial and myocardial fusion during zebrafish cardiac assembly

Author

Suzan El-Rass, Shahram Eisa-Beygi, Edbert Khong, Koroboshka Brand-Arzamendi, Antonio Mauro, Haibo Zhang, Karl J. Clark, Stephen C. Ekker, and Xiao-Yan Wen

Subjects
Pdgfra, Gene trapping, Heart development, Cardiac fusion, Zebrafish, Science, Biology (General), QH301-705.5
Abstract

Cardiac development in vertebrates is a finely tuned process regulated by a set of conserved signaling pathways. Perturbations of these processes are often associated with congenital cardiac malformations. Platelet-derived growth factor receptor α (PDGFRα) is a highly conserved tyrosine kinase receptor, which is essential for development and organogenesis. Disruption of Pdgfrα function in murine models is embryonic lethal due to severe cardiovascular defects, suggesting a role in cardiac development, thus necessitating the use of alternative models to explore its precise function. In this study, we generated a zebrafish pdgfra mutant line by gene trapping, in which the Pdgfra protein is truncated and fused with mRFP (Pdgfra-mRFP). Our results demonstrate that pdgfra mutants have defects in cardiac morphology as a result of abnormal fusion of myocardial precursors. Expression analysis of the developing heart at later stages suggested that Pdgfra-mRFP is expressed in the endocardium. Further examination of the endocardium in pdgfra mutants revealed defective endocardial migration to the midline, where cardiac fusion eventually occurs. Together, our data suggests that pdgfra is required for proper medial migration of both endocardial and myocardial precursors, an essential step required for cardiac assembly and development.

Published
2017
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13. Glucocorticoids Target Ependymal Glia and Inhibit Repair of the Injured Spinal Cord

Author

Craig M. Nelson, Vanda A. Lennon, Han Lee, Randall G. Krug, Aichurok Kamalova, Nicolas N. Madigan, Karl J. Clark, Anthony J. Windebank, and John R. Henley

Subjects
neural regeneration, spinal cord injury, glucocorticoid signaling, Nr3c1, ependymal glia, Biology (General), QH301-705.5
Abstract

Following injury, the mammalian spinal cord forms a glial scar and fails to regenerate. In contrast, vertebrate fish spinal cord tissue regenerates significantly to restore function. Cord transection in zebrafish (Danio rerio) initially causes paralysis and neural cell death. Subsequently, ependymal glia proliferate, bipolar glia extend across the lesion, and new neurons are born; axons from spared and nascent neurons extend along trans-lesional glial bridges to restore functional connectivity. Here we report that glucocorticoids, used in the clinical management of spinal cord injury, directly inhibit neural repair by targeting ependymal glia independently of hematogenous cells and microglia. After transecting injury, the glucocorticoid receptor in ependymal glia is regulated differentially in zebrafish (becoming inactive) vs. the rat (becoming active). Glucocorticoid blockade of neural regeneration via a direct effect on ependymal glia has important therapeutic implications for the putative benefit of corticosteroids in early management of spinal cord injury.

Published
2019
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14. Bi-allelic variants in HMGCR cause an autosomal-recessive progressive limb-girdle muscular dystrophy

Author

Joel A. Morales-Rosado, Tanya L. Schwab, Sarah K. Macklin-Mantia, A. Reghan Foley, Filippo Pinto e Vairo, Davut Pehlivan, Sandra Donkervoort, Jill A. Rosenfeld, Grace E. Boyum, Ying Hu, Anh T.Q. Cong, Timothy E. Lotze, Carrie A. Mohila, Dimah Saade, Diana Bharucha-Goebel, Katherine R. Chao, Christopher Grunseich, Christine C. Bruels, Hannah R. Littel, Elicia A. Estrella, Lynn Pais, Peter B. Kang, Michael T. Zimmermann, James R. Lupski, Brendan Lee, Matthew J. Schellenberg, Karl J. Clark, Klaas J. Wierenga, Carsten G. Bönnemann, and Eric W. Klee

Subjects
Genetics, Genetics (clinical)
Published
2023

16. Robust activation of microhomology-mediated end joining for precision gene editing applications.

Author

Hirotaka Ata, Thomas L Ekstrom, Gabriel Martínez-Gálvez, Carla M Mann, Alexey V Dvornikov, Kyle J Schaefbauer, Alvin C Ma, Drena Dobbs, Karl J Clark, and Stephen C Ekker

Subjects
Genetics, QH426-470
Abstract

One key problem in precision genome editing is the unpredictable plurality of sequence outcomes at the site of targeted DNA double stranded breaks (DSBs). This is due to the typical activation of the versatile Non-homologous End Joining (NHEJ) pathway. Such unpredictability limits the utility of somatic gene editing for applications including gene therapy and functional genomics. For germline editing work, the accurate reproduction of the identical alleles using NHEJ is a labor intensive process. In this study, we propose Microhomology-mediated End Joining (MMEJ) as a viable solution for improving somatic sequence homogeneity in vivo, capable of generating a single predictable allele at high rates (56% ~ 86% of the entire mutant allele pool). Using a combined dataset from zebrafish (Danio rerio) in vivo and human HeLa cell in vitro, we identified specific contextual sequence determinants surrounding genomic DSBs for robust MMEJ pathway activation. We then applied our observation to prospectively design MMEJ-inducing sgRNAs against a variety of proof-of-principle genes and demonstrated high levels of mutant allele homogeneity. MMEJ-based DNA repair at these target loci successfully generated F0 mutant zebrafish embryos and larvae that faithfully recapitulated previously reported, recessive, loss-of-function phenotypes. We also tested the generalizability of our approach in cultured human cells. Finally, we provide a novel algorithm, MENTHU (http://genesculpt.org/menthu/), for improved and facile prediction of candidate MMEJ loci. We believe that this MMEJ-centric approach will have a broader impact on genome engineering and its applications. For example, whereas somatic mosaicism hinders efficient recreation of knockout mutant allele at base pair resolution via the standard NHEJ-based approach, we demonstrate that F0 founders transmitted the identical MMEJ allele of interest at high rates. Most importantly, the ability to directly dictate the reading frame of an endogenous target will have important implications for gene therapy applications in human genetic diseases.

Published
2018
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17. The endocannabinoid gene faah2a modulates stress-associated behavior in zebrafish.

Author

Randall G Krug, Han B Lee, Louis Y El Khoury, Ashley N Sigafoos, Morgan O Petersen, and Karl J Clark

Subjects
Medicine, Science
Abstract

The ability to orchestrate appropriate physiological and behavioral responses to stress is important for survival, and is often dysfunctional in neuropsychiatric disorders that account for leading causes of global disability burden. Numerous studies have shown that the endocannabinoid neurotransmitter system is able to regulate stress responses and could serve as a therapeutic target for the management of these disorders. We used quantitative reverse transcriptase-polymerase chain reactions to show that genes encoding enzymes that synthesize (abhd4, gde1, napepld), enzymes that degrade (faah, faah2a, faah2b), and receptors that bind (cnr1, cnr2, gpr55-like) endocannabinoids are expressed in zebrafish (Danio rerio). These genes are conserved in many other vertebrates, including humans, but fatty acid amide hydrolase 2 has been lost in mice and rats. We engineered transcription activator-like effector nucleases to create zebrafish with mutations in cnr1 and faah2a to test the role of these genes in modulating stress-associated behavior. We showed that disruption of cnr1 potentiated locomotor responses to hyperosmotic stress. The increased response to stress was consistent with rodent literature and served to validate the use of zebrafish in this field. Moreover, we showed for the first time that disruption of faah2a attenuated the locomotor responses to hyperosmotic stress. This later finding suggests that FAAH2 may be an important mediator of stress responses in non-rodent vertebrates. Accordingly, FAAH and FAAH2 modulators could provide distinct therapeutic options for stress-aggravated disorders.

Published
2018
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18. Designed architectural proteins that tune DNA looping in bacteria

Author

Nicole A. Becker, Justin P. Peters, Tanya L. Schwab, Louis J. Maher, David H. Tse, Karl J. Clark, Robert T. Young, and Wilma K. Olson

Subjects
DNA, Bacterial, Effector, AcademicSubjects/SCI00010, Escherichia coli Proteins, Gene regulation, Chromatin and Epigenetics, Plasma protein binding, Gene Expression Regulation, Bacterial, Biology, Lac repressor, DNA-Binding Proteins, chemistry.chemical_compound, High-mobility group, chemistry, Lac Operon, Transcription (biology), Helix, Genetics, Biophysics, Escherichia coli, Nucleic Acid Conformation, Promoter Regions, Genetic, Gene, DNA, Protein Binding
Abstract

Architectural proteins alter the shape of DNA. Some distort the double helix by introducing sharp kinks. This can serve to relieve strain in tightly-bent DNA structures. Here, we design and test artificial architectural proteins based on a sequence-specific Transcription Activator-like Effector (TALE) protein, either alone or fused to a eukaryotic high mobility group B (HMGB) DNA-bending domain. We hypothesized that TALE protein binding would stiffen DNA to bending and twisting, acting as an architectural protein that antagonizes the formation of small DNA loops. In contrast, fusion to an HMGB domain was hypothesized to generate a targeted DNA-bending architectural protein that facilitates DNA looping. We provide evidence from Escherichia coli Lac repressor gene regulatory loops supporting these hypotheses in living bacteria. Both data fitting to a thermodynamic DNA looping model and sophisticated molecular modeling support the interpretation of these results. We find that TALE protein binding inhibits looping by stiffening DNA to bending and twisting, while the Nhp6A domain enhances looping by bending DNA without introducing twisting flexibility. Our work illustrates artificial approaches to sculpt DNA geometry with functional consequences. Similar approaches may be applicable to tune the stability of small DNA loops in eukaryotes., Graphical Abstract Graphical AbstractThe stability of Lac repressor loops in E. coli is tuned by designed artificial sequence-specific architectural DNA binding proteins that stiffen or kink looped DNA, impacting gene expression in opposite ways.

Published
2021

21. Cre/lox regulated conditional rescue and inactivation with zebrafish UFlip alleles generated by CRISPR-Cas9 targeted integration

Author

Zhitao Ming, Maira P Almeida, Sekhar Kambakam, Fang Liu, Jordan M Welker, Wesley A Wierson, Laura E Schultz-Rogers, Stephen C Ekker, Karl J Clark, Jeffrey J Essner, and Maura McGrail

Subjects
General Immunology and Microbiology, General Neuroscience, General Medicine, General Biochemistry, Genetics and Molecular Biology
Abstract

The ability to regulate gene activity spatially and temporally is essential to investigate cell-type-specific gene function during development and in postembryonic processes and disease models. The Cre/lox system has been widely used for performing cell and tissue-specific conditional analysis of gene function in zebrafish. However, simple and efficient methods for isolation of stable, Cre/lox regulated zebrafish alleles are lacking. Here, we applied our GeneWeld CRISPR-Cas9 targeted integration strategy to generate floxed alleles that provide robust conditional inactivation and rescue. A universal targeting vector, UFlip, with sites for cloning short homology arms flanking a floxed 2A-mRFP gene trap, was integrated into an intron in rbbp4 and rb1. rbbp4off and rb1off integration alleles resulted in strong mRFP expression,>99% reduction of endogenous gene expression, and recapitulated known indel loss-of-function phenotypes. Introduction of Cre led to stable inversion of the floxed cassette, loss of mRFP expression, and phenotypic rescue. rbbp4on and rb1on integration alleles did not cause phenotypes in combination with a loss-of-function mutation. Addition of Cre led to conditional inactivation by stable inversion of the cassette, gene trapping and mRFP expression, and the expected mutant phenotype. Neural progenitor Cre drivers were used for conditional inactivation and phenotypic rescue to showcase how this approach can be used in specific cell populations. Together these results validate a simplified approach for efficient isolation of Cre/lox-responsive conditional alleles in zebrafish. Our strategy provides a new toolkit for generating genetic mosaics and represents a significant advance in zebrafish genetics.

Published
2022

22. The NIH Somatic Cell Genome Editing program

Author

Ross C. Wilson, Kevin D. Wells, W. Mark Saltzman, Philip J. Santangelo, Guohua Yi, Aravind Asokan, Shengdar Q. Tsai, Nenad Bursac, R. Holland Cheng, Shaoqin Gong, Gang Bao, Jennifer A. Doudna, Venkata S. Sabbisetti, Jarryd M. Campbell, Ryuji Morizane, Charles A. Gersbach, Mary E. Dickinson, Jon D. Hennebold, Kit S. Lam, Zheng-Yi Chen, John T. Hinson, Melinda R. Dwinell, Daniel G. Anderson, William R. Lagor, Qiaobing Xu, Melissa C. Skala, Jennifer A. Lewis, David J. Segal, Samantha Maragh, Guoping Feng, Stephen C. Ekker, Benjamin E. Deverman, Jonathan K. Watts, Alice F. Tarantal, Moriel H. Vandsburger, George A. Truskey, Ionita Ghiran, Marina E. Emborg, Jeff W.M. Bulte, Scot A. Wolfe, James E. Dahlman, Niren Murthy, Paul B. McCray, Erik J. Sontheimer, John C. Tilton, David T. Curiel, Benjamin S. Freedman, Guangping Gao, Mary Shimoyama, Kam W. Leong, Jiangbing Zhou, P. J. Brooks, Samira Kiani, Krystof S. Bankiewicz, Karl J. Clark, Jillian F. Banfield, Jon E. Levine, Krishanu Saha, Todd C. McDevitt, David R. Liu, Randall S. Prather, Daniel F. Carlson, Peter M. Glazer, Elliot L. Chaikof, Jason D. Heaney, Subhojit Roy, John A. Ronald, Stephen A. Murray, Cathleen M. Lutz, Anastasia Khvorova, Wen Xue, Sushmita Roy, Oleg Mirochnitchenko, Danith H. Ly, and David M. Gamm

Subjects
Gene Editing, Multidisciplinary, Genome, Human, Computer science, Somatic cell, Cells, Targeted Gene Repair, Genetic Therapy, Computational biology, Genome, United States, Targeted gene repair, Human health, National Institutes of Health (U.S.), Genome editing, Research community, Perspective, Genetics research, Animals, Humans, In patient, Human genome, Goals
Abstract

The move from reading to writing the human genome offers new opportunities to improve human health. The United States National Institutes of Health (NIH) Somatic Cell Genome Editing (SCGE) Consortium aims to accelerate the development of safer and more-effective methods to edit the genomes of disease-relevant somatic cells in patients, even in tissues that are difficult to reach. Here we discuss the consortium’s plans to develop and benchmark approaches to induce and measure genome modifications, and to define downstream functional consequences of genome editing within human cells. Central to this effort is a rigorous and innovative approach that requires validation of the technology through third-party testing in small and large animals. New genome editors, delivery technologies and methods for tracking edited cells in vivo, as well as newly developed animal models and human biological systems, will be assembled—along with validated datasets—into an SCGE Toolkit, which will be disseminated widely to the biomedical research community. We visualize this toolkit—and the knowledge generated by its applications—as a means to accelerate the clinical development of new therapies for a wide range of conditions., This Perspective discusses how the Somatic Cell Genome Editing Consortium aims to accelerate the implementation of safe and effective genome-editing therapies in the clinic.

Published
2021

23. The canonical HPA axis contributes to locomotion during photoadaptation but is not required

Author

Han B. Lee, Viet Ha Dang Thi, Grace E. Boyum, Rodsy Modhurima, Emma M. Hall, Izzabella K. Green, Elizabeth Cervantes, Soaleha Shams, and Karl J. Clark

Abstract

The hypothalamic-pituitary-adrenal (HPA) axis and its effector molecules—glucocorticoids—modulate diverse aspects of physiology in vertebrates ranging from metabolism to immune function, behavior, and circadian cycle. While the glucocorticoid receptor (nr3c1) is known to be involved in light adaptation (photoadaptation) of the retinal cells, the role of nr3c1 and other HPA axis signaling molecules in the behavioral phenotypes observed during photoadaptation have not been delineated. Therefore, we investigated locomotor adaptation to various light/dark durations using larval zebrafish that carry a mutated allele in key HPA axis receptors. First, we established baseline locomotion and compared WT and nr3c1 mutant larvae in constantly lit and dark conditions (for 12-hrs). WT larval zebrafish showed highest locomotor activity in the middle of the day and low activity during the early and later parts of the day, which was generally higher in the light. The baseline locomotion was depressed in nr3c1 mutants throughout the day in both environments with a more significant change noted in the light. Next, groups of larvae mutant in nr3c1, nr3c2 (mineralocorticoid receptor), or mc2r (melanocortin receptor type 2; adrenocorticotropic hormone (ACTH) receptor) along with their wildtype (WT) siblings were acclimated in the dark and underwent four cycles of dark-light illumination changes with different durations of illumination: 7.5, 6, 4, or 2 min. The nr3c1 and mc2r mutant fish showed significantly decreased locomotion during the dark phase when the illumination was provided for 4 or 2 min. However, with 4 min or longer illumination, if locomotor deficits were observed in mutant larvae, they demonstrated a “catch-up” phenotype with increasing locomotion during the later dark phases of the assay and ultimately reaching the swimming distances indistinguishable from their WT siblings in the assays with 7.5-min illumination. Finally, we looked at effects of light intensity, beginning with our short light exposure. A lower intensity of light failed to elicit any response even from WT fish after 1-min illumination. However, this dim light still evoked a robust response from nr3c1 mutants and their WT siblings after 7.5-min illumination. The nr3c2 mutant larvae consistently showed locomotor response similar to their WT siblings regardless of the length of the light phase. Thus, activation of the canonical HPA axis (i.e. nr3c1, mc2r) was necessary to induce a rapid locomotor adaptation following after light to dark transition with shorter light exposure times (~≤4 min), but it was not needed for locomotor responses observed after longer exposure to light (~> 4 min). Moreover, the locomotor response after transitioning to dark from a short (1 min) exposure was dependent on the intensity of light, whereas the longer exposure was not. Together, these findings suggest that locomotion after longer exposure to light is not dependent on the HPA axis, suggesting that either parallel independent pathway(s) are responsible for the locomotor response in these cases or that the HPA axis facilitates a primary pathway to increase sensitivity to light exposure.

Published
2022

24. Genome engineering with TALE and CRISPR systems in neuroscience

Author

Han B Lee, Brynn N Sundberg, Ashley N Sigafoos, and Karl J Clark

Subjects
Neuroscience, CRISPR, Cas9, TALE, genome engineering, TALEN, Genetics, QH426-470
Abstract

Recent advancement in genome engineering technology is changing the landscape of biological research and providing neuroscientists with an opportunity to develop new methodologies to ask critical research questions. This advancement is highlighted by the increased use of programmable DNA-binding agents (PDBAs) such as transcription activator-like effector (TALE) and RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas) systems. These PDBAs fused or co-expressed with various effector domains allow precise modification of genomic sequences and gene expression levels. These technologies mirror and extend beyond classic gene targeting methods contributing to the development of novel tools for basic and clinical neuroscience. In this Review, we discuss the recent development in genome engineering and potential applications of this technology in the field of neuroscience.

Published
2016
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25. Widening of the genetic and clinical spectrum of Lamb-Shaffer syndrome, a neurodevelopmental disorder due to SOX5 haploinsufficiency

Author

Ashley N. Sigafoos, Salima El Chehadeh, Marcia C. Willing, Ela Akay, Florian Cherik, Anne-Marie E. Goyette, Vinodh Narayanan, Diane Masser-Frye, Catherine Karimov, Rhonda E. Schnur, Rebekah Bressi, Rhys H. Thomas, Gary D. Clark, Tina Barbaro-Dieber, Jill A. Rosenfeld, Carlos A. Bacino, Maria J. Guillen Sacoto, Laura Russell, Kristin Lindstrom, Caroline Schluth-Bolard, Xia Wang, Yvonne Hilhorst-Hofstee, Marcelo Vargas, Zehua Zhu, Ash Zawerton, Boris Keren, Mariëtte J.V. Hoffer, Isabelle Marey, Alice Poisson, Daphné Lehalle, Maries Joseph, Gaetan Lesca, Simon Zwolinski, Laurence Perrin, Rhoda Akilapa, Emilia K. Bijlsma, Christel Depienne, Amélie Piton, Claire G. Salter, Lucie Dupuis, Daryl A. Scott, Jolien S. Klein Wassink-Ruiter, Benjamin Cogné, Mathilde Nizon, Richard Chang, Kirsty McWalter, Myriam Srour, Perrine Charles, Anne-Claude Tabet, Natalie Canham, Sylvie Odent, Caroline Nava, Karl J. Clark, Elizabeth J. Bhoj, Jonathan Levy, Keri Ramsey, Yves Alembik, Lucia Ortega, Sophie Dupuis-Girod, Shoji Ichikawa, Christine Francannet, Marta Bertoli, Christèle Dubourg, Eric W. Klee, Ange-Line Bruel, Sebastien Moutton, Emily Fassi, Anthony Vandersteen, Abdul Haseeb, Antonina Wojcik, Patrick R. Blackburn, Lynne M. Bird, Patrick Rump, Véronique Lefebvre, Alma Kuechler, Sophie Nambot, Keren Machol, Cyril Mignot, Andreas Hartmann, Rossana Sanchez Russo, Erica H. Gerkes, Sylvie Jaillard, Roberto Mendoza-Londono, Trevor Cole, Pauline Monin, Cleveland Clinic, Institut du Cerveau et de la Moëlle Epinière = Brain and Spine Institute (ICM), Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Pitié-Salpêtrière [AP-HP], Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Service de Génétique Cytogénétique et Embryologie [CHU Pitié-Salpêtrière], CHU Pitié-Salpêtrière [AP-HP], Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Génétique humaine et fonctions cognitives - Human Genetics and Cognitive Functions (GHFC (UMR_3571 / U-Pasteur_1)), Institut Pasteur [Paris]-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Département de génétique [Robert Debré], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-AP-HP Hôpital universitaire Robert-Debré [Paris], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Centre de recherche en neurosciences de Lyon (CRNL), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Université Jean Monnet [Saint-Étienne] (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Hospices Civils de Lyon (HCL), Centre d'Etude et de Recherche Multimodal Et Pluridisciplinaire en imagerie du vivant (CERMEP - imagerie du vivant), Université de Lyon-Université de Lyon-CHU Grenoble-Hospices Civils de Lyon (HCL)-CHU Saint-Etienne-Université Jean Monnet [Saint-Étienne] (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA), Lipides - Nutrition - Cancer [Dijon - U1231] (LNC), Université de Bourgogne (UB)-Institut National de la Santé et de la Recherche Médicale (INSERM)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement, Centre de génétique - Centre de référence des maladies rares, anomalies du développement et syndromes malformatifs (CHU de Dijon), Centre Hospitalier Universitaire de Dijon - Hôpital François Mitterrand (CHU Dijon), Institut de Génétique et Développement de Rennes (IGDR), Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-Centre National de la Recherche Scientifique (CNRS)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), CHU Pontchaillou [Rennes], Institut de recherche en santé, environnement et travail (Irset), Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique )-Institut National de la Santé et de la Recherche Médicale (INSERM)-École des Hautes Études en Santé Publique [EHESP] (EHESP)-Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-Université d'Angers (UA), Center for Human and Clinical Genetics, Leiden University Medical Center (LUMC), Service de génétique médicale - Unité de génétique clinique [Nantes], Université de Nantes (UN)-Centre hospitalier universitaire de Nantes (CHU Nantes), unité de recherche de l'institut du thorax UMR1087 UMR6291 (ITX), Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Department of Health Sciences Research [Mayo Clinic] (HSR), Mayo Clinic, This research was funded in part by the Agence Nationale de la Recherche and European High-Functioning Autism Network (ANR EUHFAUTISM), the Assistance Publique–Hôpitaux de Paris (AP-HP), the Institut National de la Santé et de la Recherche Médicale (INSERM), the BioPsy labex (to Christel Depienne and C.N.) and the Association Française du Syndrome Gilles de la Tourette (AFSGT) to Christel Depienne. It was also funded by the Cleveland Clinic Lerner Research Institute (LRI Chair’s Innovative Research Award to V.L.), and by Harper’s Quest and the LAMSHF Syndrome Research Fund (donations to V.L.) and the Center for Individualized Medicine, Mayo Clinic. This study makes use of data generated by the DECIPHER community and the Deciphering Developmental Disorders (DDD) Study, which is funded by the Wellcome Trust. The DDD study presents independent research commissioned by the Health Innovation Challenge Fund (grant number HICF-1009-003), a parallel funding partnership between Wellcome and the Department of Health, and the Wellcome Sanger Institute (grant number WT098051). The views expressed in this publication are those of the author(s) and not necessarily those of Wellcome or the Department of Health. The study has UK Research Ethics Committee approval (10/H0305/83, granted by the Cambridge South REC, and GEN/284/12 granted by the Republic of Ireland REC). The research team acknowledges the support of the National Institute for Health Research, through the Comprehensive Clinical Research Network., We thank the patients and their families for their participation in this study, and the C4RCD Research Group (Newell Belnap, Amanda Courtright, Ana Claasen, David Craig, Matt Huentelman, Madison LaFleur, Sampathkumar Rangasamy, Ryan Richholt, Isabelle Schrauwen, Ashley L. Siniard, and Szabolics Szelinger) for providing clinical information on patient P18., Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Pitié-Salpêtrière [APHP]-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Service de Génétique et Cytogénétique [CHU Pitié-Salpêtrière], Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-CHU Pitié-Salpêtrière [APHP], Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-AP-HP Hôpital universitaire Robert-Debré [Paris], Centre d'Exploration et de Recherche Médicales par Émission de Positons (CERMEP), Université Joseph Fourier - Grenoble 1 (UJF)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-CHU Grenoble-Hospices Civils de Lyon (HCL)-CHU Saint-Etienne-Université Jean Monnet [Saint-Étienne] (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université de Bourgogne (UB)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Institut National de la Santé et de la Recherche Médicale (INSERM), Université d'Angers (UA)-Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-École des Hautes Études en Santé Publique [EHESP] (EHESP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), and Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)

Subjects
Male, Medizin, Haploinsufficiency, L-SOX5, VARIANTS, 0302 clinical medicine, Neurodevelopmental disorder, Intellectual disability, Missense mutation, 2.1 Biological and endogenous factors, Aetiology, Child, Genetics (clinical), Genetics, Pediatric, Genetics & Heredity, 0303 health sciences, Pedigree, FAMILY, DNA-Binding Proteins, developmental delay, TRANSCRIPTION FACTORS, Phenotype, intellectual disability, Child, Preschool, missense variants, Female, SOXD Transcription Factors, Adult, EXPRESSION, Adolescent, Intellectual and Developmental Disabilities (IDD), Clinical Sciences, Mutation, Missense, autism, Cell fate determination, Biology, LONG FORM, SEQUENCE, Article, 03 medical and health sciences, Young Adult, Rare Diseases, Clinical Research, CARTILAGE, Intellectual Disability, medicine, Animals, Humans, Language Development Disorders, Genetic Predisposition to Disease, Preschool, Transcription factor, Gene, 030304 developmental biology, [SDV.GEN]Life Sciences [q-bio]/Genetics, MUTATIONS, Human Genome, Infant, medicine.disease, Brain Disorders, Neurodevelopmental Disorders, Deciphering Developmental Disorder Study, Mutation, Autism, epilepsy, Missense, 030217 neurology & neurosurgery, GENERATION
Abstract

International audience; PURPOSE: Lamb-Shaffer syndrome (LAMSHF) is a neurodevelopmental disorder described in just over two dozen patients with heterozygous genetic alterations involving SOX5, a gene encoding a transcription factor regulating cell fate and differentiation in neurogenesis and other discrete developmental processes. The genetic alterations described so far are mainly microdeletions. The present study was aimed at increasing our understanding of LAMSHF, its clinical and genetic spectrum, and the pathophysiological mechanisms involved.METHODS: Clinical and genetic data were collected through GeneMatcher and clinical or genetic networks for 41 novel patients harboring various types of SOX5 alterations. Functional consequences of selected substitutions were investigated.RESULTS: Microdeletions and truncating variants occurred throughout SOX5. In contrast, most missense variants clustered in the pivotal SOX-specific high-mobility-group domain. The latter variants prevented SOX5 from binding DNA and promoting transactivation in vitro, whereas missense variants located outside the high-mobility-group domain did not. Clinical manifestations and severity varied among patients. No clear genotype-phenotype correlations were found, except that missense variants outside the high-mobility-group domain were generally better tolerated.CONCLUSIONS:This study extends the clinical and genetic spectrum associated with LAMSHF and consolidates evidence that SOX5 haploinsufficiency leads to variable degrees of intellectual disability, language delay, and other clinical features.

Published
2020

26. A phenotype-based forward genetic screen identifiesDnajb6as a sick sinus syndrome gene

Author

Yonghe Ding, Di Lang, Jianhua Yan, Haisong Bu, Hongsong Li, Kunli Jiao, Jingchun Yang, Tai Le, Karl J. Clark, Stephen C. Ekker, Hung Cao, Yuji Zhang, Yigang Li, Alexey V. Glukhov, and Xiaolei Xu

Abstract

Sick sinus syndrome (SSS) is a group of heart rhythm disorders caused by malfunction of the sinus node, the heart’s primary pacemaker. Partially owing to its aging-associated phenotypic manifestation and low expressivity, molecular mechanisms of SSS remain difficult to decipher. Here, we aim to develop a phenotype-based forward genetic approach in the zebrafish (Danio rerio) animal model for discovering essential genes which dysfunction could result in SSS-like phenotypes. Previously we showed the generation of protein trap library by using a revertible gene-breaking transposon (GBT)-based insertional mutagenesis system. Here, we reported the generation of a collection of 35 zebrafish insertional cardiac lines derived from this protein trap library, which was screened using electrocardiographic measurements. As a result, three mutants with SSS-like phenotypes were identified. We then focused on one of these 3 GBT mutants calledGBT411in whichdnajb6bgene was disrupted, and conducted expressional, genetic, transcriptome, and electrophysiological studies using both zebrafish and mouse models. These studies confirmed the identity ofDnajb6as a novel SSS causative gene with a unique expression pattern within the specialized population of sinus node pacemaker cardiomyocytes that lack the expression of HCN4 channels. Together, this study demonstrates the feasibility of a genetic screening approach in an adult vertebrate animal model for discovering new genetic factors for a heart rhythm disorder such as SSS.

Published
2022

27. Impact of integrated translational research on clinical exome sequencing

Author

Gavin R. Oliver, Jennifer L. Kemppainen, Ashley N. Sigafoos, Konstantinos N. Lazaridis, Megan M. Hager, Teresa M. Kruisselbrink, Jessica Jackson, Jessica M. Tarnowski, Laura Rust, Nicole J. Boczek, Cherisse A. Marcou, Nicole L. Bertsch, Marissa S. Ellingson, Pavel N. Pichurin, Brendan C. Lanpher, Sarah K. Macklin-Mantia, Dusica Babovic-Vuksanovic, Gianrico Farrugia, Eva Morava-Kozicz, Aditi Gupta, Lauren Gunderson, Paldeep S. Atwal, Jolene M. Summer Bolster, Michael T. Zimmermann, Marine I. Murphree, A. Keith Stewart, Carrie A. Lahner, Tanya L. Schwab, Zhiyv Niu, Tammy M. McAllister, Matthew J. Ferber, Lindsay A. Mulvihill, Ralitza H. Gavrilova, Kristen J. Rasmussen, Laura Schultz-Rogers, Sarah A. Kroc, Carri A. Prochnow, Scott A. Beck, Joel A. Morales-Rosado, Garrett Jenkinson, Eric W. Klee, Filippo Vairo, Karl J. Clark, Stacy L. Aoudia, Katherine Agre, Rebecca J. Lowy, David R. Deyle, Alejandro Ferrer, Erica L. Macke, Lisa A. Schimmenti, Sarah S. Barnett, Laura J. Fisher, Margot A. Cousin, Rory J. Olson, Radhika Dhamija, Linda Hasadsri, Patrick R. Blackburn, Raul Urrutia, Charu Kaiwar, and Klaas J. Wierenga

Subjects
0301 basic medicine, Pediatrics, medicine.medical_specialty, Multivariate analysis, business.industry, Translational research, Genomics, Disease, 030105 genetics & heredity, Omics, Undiagnosed Diseases, Translational Research, Biomedical, 03 medical and health sciences, 030104 developmental biology, Phenotype, Exome Sequencing, Medicine, Humans, Exome, Personalized medicine, Genetic Testing, business, Exome sequencing, Genetics (clinical)
Abstract

Purpose Exome sequencing often identifies pathogenic genetic variants in patients with undiagnosed diseases. Nevertheless, frequent findings of variants of uncertain significance necessitate additional efforts to establish causality before reaching a conclusive diagnosis. To provide comprehensive genomic testing to patients with undiagnosed disease, we established an Individualized Medicine Clinic, which offered clinical exome testing and included a Translational Omics Program (TOP) that provided variant curation, research activities, or research exome sequencing. Methods From 2012 to 2018, 1101 unselected patients with undiagnosed diseases received exome testing. Outcomes were reviewed to assess impact of the TOP and patient characteristics on diagnostic rates through descriptive and multivariate analyses. Results The overall diagnostic yield was 24.9% (274 of 1101 patients), with 174 (15.8% of 1101) diagnosed on the basis of clinical exome sequencing alone. Four hundred twenty-three patients with nondiagnostic or without access to clinical exome sequencing were evaluated by the TOP, with 100 (9% of 1101) patients receiving a diagnosis, accounting for 36.5% of the diagnostic yield. The identification of a genetic diagnosis was influenced by the age at time of testing and the disease phenotype of the patient. Conclusion Integration of translational research activities into clinical practice of a tertiary medical center can significantly increase the diagnostic yield of patients with undiagnosed disease.

Published
2023

28. Expanding the CRISPR Toolbox with ErCas12a in Zebrafish and Human Cells

Author

Brandon W. Simone, Stephen C. Ekker, Karl J. Clark, Drena Dobbs, Michael A. Barry, Michael J. Emch, Jordan M. Welker, William A.C. Gendron, Iddo Friedberg, Zachary WareJoncas, Bibekananda Kar, Jeffrey J. Essner, Wesley A. Wierson, Carla M. Mann, and Maura McGrail

Subjects
CRISPR-Associated Proteins, Computational biology, Biology, Genome engineering, Nucleic acid secondary structure, 03 medical and health sciences, 0302 clinical medicine, Genetics, CRISPR, Animals, Humans, Clustered Regularly Interspaced Short Palindromic Repeats, Guide RNA, Research Articles, Zebrafish, 030304 developmental biology, Gene Editing, 0303 health sciences, Genome, Base Sequence, Effector, Nucleic acid sequence, RNA, DNA, Protospacer adjacent motif, Gene Targeting, CRISPR-Cas Systems, Genetic Engineering, 030217 neurology & neurosurgery, Biotechnology, RNA, Guide, Kinetoplastida
Abstract

CRISPR and CRISPR-Cas effector proteins enable the targeting of DNA double-strand breaks to defined loci based on a variable length RNA guide specific to each effector. The guide RNAs are generally similar in size and form, consisting of a ∼20 nucleotide sequence complementary to the DNA target and an RNA secondary structure recognized by the effector. However, the effector proteins vary in protospacer adjacent motif requirements, nuclease activities, and DNA binding kinetics. Recently, ErCas12a, a new member of the Cas12a family, was identified in Eubacterium rectale. Here, we report the first characterization of ErCas12a activity in zebrafish and expand on previously reported activity in human cells. Using a fluorescent reporter system, we show that CRISPR-ErCas12a elicits strand annealing mediated DNA repair more efficiently than CRISPR-Cas9. Further, using our previously reported gene targeting method that utilizes short homology, GeneWeld, we demonstrate the use of CRISPR-ErCas12a to integrate reporter alleles into the genomes of both zebrafish and human cells. Together, this work provides methods for deploying an additional CRISPR-Cas system, thus increasing the flexibility researchers have in applying genome engineering technologies.

Published
2019

29. Chimeric RNA: DNA TracrRNA Improves Homology-Directed Repair

Author

Brandon W, Simone, Han B, Lee, Camden L, Daby, Hirotaka, Ata, Santiago, Restrepo-Castillo, Gabriel, Martínez-Gálvez, Bibekananda, Kar, William A C, Gendron, Karl J, Clark, and Stephen C, Ekker

Subjects
Gene Editing, Humans, RNA, DNA Breaks, Double-Stranded, DNA, CRISPR-Cas Systems
Abstract

Nearly 90% of human pathogenic mutations are caused by small genetic variations, and methods to correct these errors efficiently are critically important. One way to make small DNA changes is providing a single-stranded oligo deoxynucleotide (ssODN) containing an alteration coupled with a targeted double-strand break (DSB) at the target locus in the genome. Coupling an ssODN donor with a CRISPR-Cas9-mediated DSB is one of the most streamlined approaches to introduce small changes. However, in many systems, this approach is inefficient and introduces imprecise repair at the genetic junctions. We herein report a technology that uses spatiotemporal localization of an ssODN with CRISPR-Cas9 to improve gene alteration. We show that by fusing an ssODN template to the trans-activating RNA (tracrRNA), we recover precise genetic alterations, with increased integration and precision in

Published
2021

30. The FusX TALE Base Editor (FusXTBE) for Rapid Mitochondrial DNA Programming of Human Cells

Author

Ankit, Sabharwal, Bibekananda, Kar, Santiago, Restrepo-Castillo, Shannon R, Holmberg, Neal D, Mathew, Benjamin Luke, Kendall, Ryan P, Cotter, Zachary, WareJoncas, Christoph, Seiler, Eiko, Nakamaru-Ogiso, Karl J, Clark, and Stephen C, Ekker

Subjects
Gene Editing, Animals, Humans, CRISPR-Cas Systems, DNA, Mitochondrial, Zebrafish, Research Articles, Mitochondria
Abstract

Functional analyses of mitochondria have been hampered by few effective approaches to manipulate mitochondrial DNA (mtDNA) and a lack of existing animal models. Recently a TALE-derived base editor was shown to induce C-to-T (or G-to-A) sequence changes in mtDNA. We report here the FusX TALE Base Editor (FusXTBE) to facilitate broad-based access to TALE mitochondrial base editing technology. TALE Writer is a de novo in silico design tool to map potential mtDNA base editing sites. FusXTBE was demonstrated to function with comparable activity to the initial base editor in human cells in vitro. Zebrafish embryos were used as a pioneering in vivo test system, with FusXTBE inducing 90+% editing efficiency in mtDNA loci as an example of near-complete induction of mtDNA heteroplasmy in vivo. Gene editing specificity as precise as a single nucleotide was observed for a protein-coding gene. Nondestructive genotyping enables single-animal mtDNA analyses for downstream biological functional genomic applications. FusXTBE is a new gene editing toolkit for exploring important questions in mitochondrial biology and genetics.

Published
2021

31. Protein-Trap Insertional Mutagenesis Uncovers New Genes Involved in Zebrafish Skin Development, Including a Neuregulin 2a-Based ErbB Signaling Pathway Required during Median Fin Fold Morphogenesis.

Author

Stephanie E Westcot, Julia Hatzold, Mark D Urban, Stefânia K Richetti, Kimberly J Skuster, Rhianna M Harm, Roberto Lopez Cervera, Noriko Umemoto, Melissa S McNulty, Karl J Clark, Matthias Hammerschmidt, and Stephen C Ekker

Subjects
Medicine, Science
Abstract

Skin disorders are widespread, but available treatments are limited. A more comprehensive understanding of skin development mechanisms will drive identification of new treatment targets and modalities. Here we report the Zebrafish Integument Project (ZIP), an expression-driven platform for identifying new skin genes and phenotypes in the vertebrate model Danio rerio (zebrafish). In vivo selection for skin-specific expression of gene-break transposon (GBT) mutant lines identified eleven new, revertible GBT alleles of genes involved in skin development. Eight genes--fras1, grip1, hmcn1, msxc, col4a4, ahnak, capn12, and nrg2a--had been described in an integumentary context to varying degrees, while arhgef25b, fkbp10b, and megf6a emerged as novel skin genes. Embryos homozygous for a GBT insertion within neuregulin 2a (nrg2a) revealed a novel requirement for a Neuregulin 2a (Nrg2a)-ErbB2/3-AKT signaling pathway governing the apicobasal organization of a subset of epidermal cells during median fin fold (MFF) morphogenesis. In nrg2a mutant larvae, the basal keratinocytes within the apical MFF, known as ridge cells, displayed reduced pAKT levels as well as reduced apical domains and exaggerated basolateral domains. Those defects compromised proper ridge cell elongation into a flattened epithelial morphology, resulting in thickened MFF edges. Pharmacological inhibition verified that Nrg2a signals through the ErbB receptor tyrosine kinase network. Moreover, knockdown of the epithelial polarity regulator and tumor suppressor lgl2 ameliorated the nrg2a mutant phenotype. Identifying Lgl2 as an antagonist of Nrg2a-ErbB signaling revealed a significantly earlier role for Lgl2 during epidermal morphogenesis than has been described to date. Furthermore, our findings demonstrated that successive, coordinated ridge cell shape changes drive apical MFF development, making MFF ridge cells a valuable model for investigating how the coordinated regulation of cell polarity and cell shape changes serves as a crucial mechanism of epithelial morphogenesis.

Published
2015
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32. A Convolutional Neural Network based Cascade Reconstruction for the IceCube Neutrino Observatory

Author

J. Werthebach, A. Franckowiak, Chujie Chen, Ioana Codrina Maris, K. Hoshina, A. Steuer, A. Weindl, Maryon Ahrens, P. Schlunder, Saskia Philippen, Glenn Spiczak, G. Momenté, Segev BenZvi, Spencer Klein, M. Rameez, Sam De Ridder, Frederik Tenholt, R. Nagai, D. F. Cowen, Martin Unland Elorrieta, Stephen L. Hauser, Christoph Raab, Nadège Iovine, Barbara Skrzypek, Kara Hoffman, Sarah Mancina, N. van Eijndhoven, U. Naumann, Raamis Hussain, D. Berley, G. C. Hill, Tobias Hoinka, Vedant Basu, Steve Sclafani, J. Kiryluk, B. J. P. Jones, Andres Medina, D. Mockler, Bunheng Ty, Ralph Engel, Moritz Kellermann, U. Katz, Emily Dvorak, Timo Karg, K. Andeen, D. R. Nygren, Marek Kowalski, D. Kang, Giovanni Renzi, K. D. de Vries, M. Stamatikos, Hershal Pandya, Wolfgang Rhode, Robert Stein, A.A. Alves, Pablo Correa, D. Seckel, S. Fahey, Javier Gonzalez, Roxanne Turcotte, Maximilian Karl Scharf, Alexander Trettin, P. B. Price, I. Taboada, Gisela Anton, Michael O. Wolf, Lenka Tomankova, Max Renschler, Y. Makino, Matthias Vraeghe, Tyce DeYoung, K. Tollefson, D. J. Koskinen, K. Rawlins, Nathan Whitehorn, C. Weaver, T. Anderson, B. A. Clark, Francesco Lucarelli, Brandom Pries, L. Schumacher, Devyn Rysewyk Cantu, P. Peiffer, J. Kim, Daria Pankova, E. Friedman, M. Kauer, Alan Coleman, John Hardin, Shefali Shefali, Nora Valtonen-Mattila, M. E. Huber, D. B. Fox, Alexander Fritz, D. Z. Besson, T. O. B. Schmidt, E. Bourbeau, Suyong Choi, M. Silva, Najia Moureen Binte Amin, J. P. Lazar, Sebastian Böser, R. Morse, Spencer Griswold, Jakob Bottcher, T. Montaruli, David A. Williams, A. Goldschmidt, K. Mase, Abdul Rehman, David Kappesser, Kurt Woschnagg, Paul Evenson, Clara E. Hill, Le Viet Nguyen, Sebastian Sanchez Herrera, Marcos Santander, Carsten Rott, L. Gerhardt, Y. Popovych, J. Evans, Maria Tselengidou, D. Soldin, J. B. Tjus, Chiara Bellenghi, Alexander A. Harnisch, Benjamin Bastian, Anastasia Maria Barbano, Markus Ackermann, S. W. Barwick, Matti Jansson, Immacolata Carmen Rea, P. Eller, Simone Garrappa, H. Schieler, Dmitry Chirkin, Kael Hanson, A. Kyriacou, A. Olivas, Hermann Kolanoski, Simeon Reusch, A. O. Pollmann, Elisa Bernardini, K. Meagher, S. Hickford, J. C. Gallagher, Kirill Filimonov, Mehmet Gunduz, Agnieszka Leszczyńska, R. C. Bay, Alexander Kappes, Pranav Dave, Jan Soedingrekso, K. Krings, Xianwu Xu, J. Sandroos, James Madsen, Lu Lu, M. J. Weiss, Stef Verpoest, H. Dujmovic, Austin Schneider, Juan Carlos Diaz-Velez, Jenni Adams, K.-H. Becker, Yiqian Xu, M. Plum, A. Wallace, Katharina Morik, J. Stettner, Daniel Bindig, Ramesh Koirala, Gerrit Wrede, C.P. de los Heros, Thomas Huber, Ek Narayan Paudel, Allan Hallgren, Matt Dunkman, R. Joppe, T. Stezelberger, Colin Turley, S. Kopper, Christian Glaser, Paolo Desiati, S. Sarker, C. Alispach, Damian Pieloth, R. G. Stokstad, Ali Kheirandish, Sreetama Goswami, Minjin Jeong, Martin Ha Minh, T. Glüsenkamp, Janet Conrad, G. Krückl, Jannis Necker, Sebastian Baur, R. Cross, Sebastian Fiedlschuster, Surujhdeo Seunarine, Ken'ichi Kin, Jannes Brostean-Kaiser, Chunfai Tung, Alejandro Diaz, M. J. Larson, M. U. Nisa, Tim Ruhe, E. O'Sullivan, René Reimann, Chad Finley, Federica Bradascio, A. V. Balagopal, Alexander Burgman, M. Meier, Michael Campana, F. Huang, Christoph Tönnis, Benjamin Hokanson-Fasig, E. Blaufuss, Leander Fischer, Gerrit Roellinghoff, Mirco Hunnefeld, Marie Oehler, Thomas Stuttard, William Luszczak, G. H. Collin, Sarah Pieper, Dirk Ryckbosch, S. Robertson, R. Snihur, A. Ludwig, Wing Yan Ma, Yang Lyu, Grant Parker, L. Köpke, Karl J. Clark, Juanan Aguilar, I. Safa, Merlin Schaufel, James DeLaunay, Marjon Moulai, Olga Botner, Cristian Jesus Lozano Mariscal, Kendall Mahn, L. Classen, Johan Wulff, K. Wiebe, Timothyblake Watson, J. J. Beatty, Tianlu Yuan, Stephan Meighen-Berger, S. Yoshida, Kayla Leonard, A. Ishihara, V. Baum, H. Niederhausen, Won Nam Kang, Joshua Hignight, A. Sharma, Sukeerthi Dharani, Francis Halzen, J. Merz, Paras Koundal, Mike Richman, X. Bai, Konstancja Satalecka, F. McNally, Emma C. Hettinger, I. Ansseau, Michael Kovacevich, Theo Glauch, Juliana Stachurska, H. Dembinski, Reina H. Maruyama, Frank G. Schröder, Summer Blot, Thomas Ehrhardt, D. Tosi, D. van Eijk, Mark Weyrauch, Spencer Axani, Elisa Lohfink, Aaron Fienberg, Amirreza Raissi, S. Tilav, Michael DuVernois, Carlos Arguelles, Jessie Micallef, Stephanie Bron, R. S. Busse, Y. L. Li, J. P. Yanez, Gerald Przybylski, Christian Haack, Christopher Wiebusch, R. Halliday, Kunal Deoskar, K. Helbing, Alexander Sandrock, N. Kurahashi, Todor Stanev, Benedikt Riedel, Cristina Lagunas Gualda, C. Wendt, G. W. Sullivan, A. Karle, James E. Braun, Philipp Furst, D. Hebecker, G. de Wasseige, George Japaridze, Roger Moore, Paul Coppin, Andrea Turcati, T. Gregoire, J. van Santen, R. Hoffmann, Alex Pizzuto, Gary Binder, J. Bourbeau, Richard Naab, Zhedong Zhang, C. Walck, Thomas K. Gaisser, Ava Ghadimi, Franziska Tischbein, Seongjin In, Elisa Resconi, Alessio Porcelli, P. Mallik, Catherine De Clercq, M. Rongen, Qinrui Liu, Martina Karl, Jochen M. Schneider, Christian Spiering, Justin Vandenbroucke, Maria Prado Rodriguez, Abhishek Desai, Xinyue Kang, Claudio Kopper, S. C. Nowicki, Subir Sarkar, T. Kintscher, Frederik Hermann Lauber, Justin Lanfranchi, David Vannerom, Ben Smithers, Jean Pierre Twagirayezu, A. Haungs, Rui An, Nahee Park, K. Hultqvist, Darren Grant, T. Stürwald, Z. Griffith, Felix Henningsen, A. R. Fazely, Simona Toscano, J. L. Kelley, Markus Ahlers, Erik Ganster, Samvel Ter-Antonyan, Rasha Abbasi, B. J. Whelan, Jan Weldert, L. Halve, Physics, Faculty of Sciences and Bioengineering Sciences, Vriendenkring VUB, Elementary Particle Physics, and collaboration, The IceCube

Subjects
FOS: Computer and information sciences, Computer Science - Machine Learning, Astrophysics::High Energy Astrophysical Phenomena, cs.LG, Data analysis, FOS: Physical sciences, Fitting methods, 01 natural sciences, Convolutional neural network, Calibration, Cluster finding, Neutrino detectors, Pattern recognition, High Energy Physics - Experiment, IceCube Neutrino Observatory, Machine Learning (cs.LG), High Energy Physics - Experiment (hep-ex), 0103 physical sciences, 010303 astronomy & astrophysics, Instrumentation, Mathematical Physics, 010308 nuclear & particles physics, business.industry, hep-ex, Deep learning, Detector, Neutrino detector, Computer engineering, Orders of magnitude (time), 13. Climate action, Cascade, Pattern recognition (psychology), Artificial intelligence, business
Abstract

Continued improvements on existing reconstruction methods are vital to the success of high-energy physics experiments, such as the IceCube Neutrino Observatory. In IceCube, further challenges arise as the detector is situated at the geographic South Pole where computational resources are limited. However, to perform real-time analyses and to issue alerts to telescopes around the world, powerful and fast reconstruction methods are desired. Deep neural networks can be extremely powerful, and their usage is computationally inexpensive once the networks are trained. These characteristics make a deep learning-based approach an excellent candidate for the application in IceCube. A reconstruction method based on convolutional architectures and hexagonally shaped kernels is presented. The presented method is robust towards systematic uncertainties in the simulation and has been tested on experimental data. In comparison to standard reconstruction methods in IceCube, it can improve upon the reconstruction accuracy, while reducing the time necessary to run the reconstruction by two to three orders of magnitude., Comment: 39 pages, 15 figures, submitted to Journal of Instrumentation; added references

Published
2021

33. Cre

Author

Fang, Liu, Sekhar, Kambakam, Maira P, Almeida, Zhitao, Ming, Jordan M, Welker, Wesley A, Wierson, Laura E, Schultz-Rogers, Stephen C, Ekker, Karl J, Clark, Jeffrey J, Essner, and Maura, McGrail

Subjects
Integrases, Animals, CRISPR-Cas Systems, Alleles, Zebrafish
Abstract

The ability to regulate gene activity spatially and temporally is essential to investigate cell-type-specific gene function during development and in postembryonic processes and disease models. The Cre/

Published
2021

34. Zebrafish Cre/loxregulated UFlip alleles generated by CRISPR/Cas targeted integration provide cell-type specific conditional gene inactivation

Author

Sekhar Kambakam, Karl J. Clark, Jeffrey J. Essner, Schultz-Rogers Le, Zhitao Ming, Wesley A. Wierson, Maira P. Almeida, Maura McGrail, Jordan M. Welker, Liu F, and Stephen C. Ekker

Subjects
Gene knockdown, Reporter gene, Mutant, Wild type, Cre recombinase, RBBP4, Allele, Biology, Gene, Cell biology
Abstract

The ability to regulate gene activity spatially and temporally is essential to investigate cell type specific gene function during development and in postembryonic processes and disease models. The Cre/loxsystem has been widely used for performing cell and tissue-specific conditional analysis of gene function in zebrafish, but simple and efficient methods for isolation of stable, Cre/loxregulated alleles are lacking. Here we applied our GeneWeld CRISPR/Cas9 short homology-directed targeted integration strategy to generate floxed conditional alleles that provide robust gene knockdown and strong loss of function phenotypes. A universal targeting vector, UFlip, with sites for cloning short 24-48 bp homology arms flanking a floxed mRFP gene trap plus secondary reporter cassette, was integrated into an intron inhdac1, rbbp4, andrb1. Active, gene off orientationhdac1-UFlip-Offandrb1-UFlip-Offintegration alleles result in >99% reduction of gene expression in homozygotes and recapitulate known indel loss of function phenotypes. Passive, gene on orientationrbbp4-UFlip-Onandrb1-UFlip-Onintegration alleles do not cause phenotypes in trans-heterozygous combination with an indel mutation. Cre recombinase injection leads to recombination at alternating pairs ofloxPandlox2272sites, inverting and locking the cassette into the active, gene off orientation, and the expected mutant phenotypes. In combination with our endogenous neural progenitor Cre drivers we demonstraterbbp4-UFlip-Onandrb1-UFlip-Ongene inactivation phenotypes can be restricted to specific neural cell populations. Replacement of the UFlip mRFP primary reporter gene trap with a 2A-RFP inrbbp4-UFlip-Off, or 2A-KalTA4 inrb1-UFlip-Off, shows strong RFP expression in wild type or UAS:RFP injected embryos, respectively. Together these results validate a simplified approach for efficient isolation of highly mutagenic Cre/loxresponsive conditional gene alleles to advance zebrafish Cre recombinase genetics.

Published
2021

35. Biallelic variants in PROZ as a cause of hypercoagulability and livedo racemosa

Author

Michael T. Zimmermann, Karl J. Clark, Sarah A. Kroc, Ashley N. Sigafoos, Pavel N. Pichurin, Eric W. Klee, Nicole L. Bertsch, Filippo Vairo, Nikita R. Dsouza, and Han B. Lee

Subjects
medicine.medical_specialty, business.industry, Protein Z, Hematology, Livedo racemosa, Thrombophilia, medicine.disease, Dermatology, medicine, Humans, medicine.symptom, business, Livedo Reticularis
Published
2020

36. A Primer Genetic Toolkit for Exploring Mitochondrial Biology and Disease Using Zebrafish

Author

Ankit Sabharwal, Jarryd M. Campbell, Tanya L. Schwab, Zachary WareJoncas, Mark D. Wishman, Hirotaka Ata, Wiebin Liu, Noriko Ichino, Danielle E. Hunter, Jake D. Bergren, Mark D. Urban, Rhianna M. Urban, Shannon R. Holmberg, Bibekananda Kar, Alex Cook, Yonghe Ding, Xiaolei Xu, Karl J. Clark, and Stephen C. Ekker

Subjects
Genes, Mitochondrial, Genome, Mitochondrial, Genetics, Animals, Humans, mitochondria, mitochondrial disorders, zebrafish, gene editing, TALEN, Gene Breaking Transposon, Zebrafish, Genetics (clinical), Mitochondria
Abstract

Mitochondria are a dynamic eukaryotic innovation that play diverse roles in biology and disease. The mitochondrial genome is remarkably conserved in all vertebrates, encoding the same 37-gene set and overall genomic structure, ranging from 16,596 base pairs (bp) in the teleost zebrafish (Danio rerio) to 16,569 bp in humans. Mitochondrial disorders are amongst the most prevalent inherited diseases, affecting roughly 1 in every 5000 individuals. Currently, few effective treatments exist for those with mitochondrial ailments, representing a major unmet patient need. Mitochondrial dysfunction is also a common component of a wide variety of other human illnesses, ranging from neurodegenerative disorders such as Huntington’s disease and Parkinson’s disease to autoimmune illnesses such as multiple sclerosis and rheumatoid arthritis. The electron transport chain (ETC) component of mitochondria is critical for mitochondrial biology and defects can lead to many mitochondrial disease symptoms. Here, we present a publicly available collection of genetic mutants created in highly conserved, nuclear-encoded mitochondrial genes in Danio rerio. The zebrafish system represents a potentially powerful new opportunity for the study of mitochondrial biology and disease due to the large number of orthologous genes shared with humans and the many advanced features of this model system, from genetics to imaging. This collection includes 15 mutant lines in 13 different genes created through locus-specific gene editing to induce frameshift or splice acceptor mutations, leading to predicted protein truncation during translation. Additionally, included are 11 lines created by the random insertion of the gene-breaking transposon (GBT) protein trap cassette. All these targeted mutant alleles truncate conserved domains of genes critical to the proper function of the ETC or genes that have been implicated in human mitochondrial disease. This collection is designed to accelerate the use of zebrafish to study many different aspects of mitochondrial function to widen our understanding of their role in biology and human disease.

Published
2022

37. Chimeric RNA:DNA Donorguide Improves HDR in vitro and in vivo

Author

Camden L. Daby, Karl J. Clark, Brandon W. Simone, Gabriel Martínez-Gálvez, William A.C. Gendron, Han B. Lee, Santiago Restrepo-Castillo, Stephen C. Ekker, and Hirotaka Ata

Subjects
Homology directed repair, Trans-activating crRNA, chemistry.chemical_compound, Transcription activator-like effector nuclease, chemistry, Genome editing, DNA repair, Chimeric RNA, Computational biology, Gene conversion, Biology, DNA
Abstract

Introducing small genetic changes to study specific mutations or reverting clinical mutations to wild type has been an area of interest in precision genomics for several years. In fact, it has been found that nearly 90% of all human pathogenic mutations are caused by small genetic variations, and the methods to efficiently and precisely correct these errors are critically important. One common way to make these small DNA changes is to provide a single stranded DNA (ssDNA) donor containing the desired alteration together with a targeted double-strand break (DSB) at the genomic target. The donor is typically flanked by regions of homology that are often ~30-100bp in length to leverage the homology directed repair (HDR) pathway. Coupling a ssDNA donor with a CRISPR-Cas9 to produce a targeted DSB is one of the most streamlined approaches to introduce small changes. However, in many cell types this approach results in a low rate of incorporation of the desired alteration and has undesired imprecise repair at the 5’ or 3’ junction due to artifacts of the DNA repair process. We herein report a technology that couples the spatial temporal localization of an ssDNA repair template and leverages the nucleic acid components of the CRISPR-Cas9 system. We show that by direct fusion of an ssDNA template to the trans activating RNA (tracrRNA) to generate an RNA-DNA chimera, termed Donorguide, we recover precise integration of genetic alterations, with both increased integration rates and decreased imprecision at the 5’ or 3’ junctions relative to an ssODN donor in vitro in HEK293T cells as well as in vivo in zebrafish. Further, we show that this technology can be used to enhance gene conversion with other gene editing tools such as TALENs.

Published
2021

38. Endogenous zebrafish proneural Cre drivers generated by CRISPR/Cas9 short homology directed targeted integration

Author

Stephen C. Ekker, Maura McGrail, Maira P. Almeida, Jordan M. Welker, Sahiba Siddiqui, Karl J. Clark, Jon Luiken, and Jeffrey J. Essner

Subjects
Science, Transgene, Cre recombinase, Proneural genes, Article, Animals, Genetically Modified, Genetics, Recombinase, Animals, CRISPR, Gene Knock-In Techniques, Homologous Recombination, Promoter Regions, Genetic, Zebrafish, Gene, Multidisciplinary, Integrases, biology, Cas9, Functional genomics, biology.organism_classification, Cell biology, Medicine, CRISPR-Cas Systems
Abstract

We previously reported efficient precision targeted integration of reporter DNA in zebrafish and human cells using CRISPR/Cas9 and short regions of homology. Here, we apply this strategy to isolate zebrafish Cre recombinase drivers whose spatial and temporal restricted expression mimics endogenous genes. A 2A-Cre recombinase transgene with 48 bp homology arms was targeted into proneural genes ascl1b, olig2 and neurod1. We observed high rates of germline transmission ranging from 10 to 100% (2/20 olig2; 1/5 neurod1; 3/3 ascl1b). The transgenic lines Tg(ascl1b-2A-Cre)is75, Tg(olig2-2A-Cre)is76, and Tg(neurod1-2A-Cre)is77 expressed functional Cre recombinase in the expected proneural cell populations. Somatic targeting of 2A-CreERT2 into neurod1 resulted in tamoxifen responsive recombination in the nervous system. The results demonstrate Cre recombinase expression is driven by the native promoter and regulatory elements of the targeted genes. This approach provides a straightforward, efficient, and cost-effective method to generate cell type specific zebrafish Cre and CreERT2 drivers, overcoming challenges associated with promoter-BAC and transposon mediated transgenics.

Published
2021

39. GeneWeld: Efficient Targeted Integration Directed by Short Homology in Zebrafish

Author

Maira P. Almeida, Carla M. Mann, Zhitao Ming, Maura McGrail, Karl J. Clark, Stephen C. Ekker, Jeffrey J. Essner, Wesley A. Wierson, Jordan M. Welker, Melanie E. Torrie, and Drena Dobbs

Subjects
biology, Cas9, Strategy and Management, Mechanical Engineering, Metals and Alloys, Gene targeting, Computational biology, biology.organism_classification, Industrial and Manufacturing Engineering, Genome engineering, Genome editing, Gene knockin, Methods Article, CRISPR, Zebrafish, Gene
Abstract

Efficient precision genome engineering requires high frequency and specificity of integration at the genomic target site. Multiple design strategies for zebrafish gene targeting have previously been reported with widely varying frequencies for germline recovery of integration alleles. The GeneWeld protocol and pGTag (plasmids for Gene Tagging) vector series provide a set of resources to streamline precision gene targeting in zebrafish. Our approach uses short homology of 24-48 bp to drive targeted integration of DNA reporter cassettes by homology-mediated end joining (HMEJ) at a CRISPR/Cas induced DNA double-strand break. The pGTag vectors contain reporters flanked by a universal CRISPR sgRNA sequence to liberate the targeting cassette in vivo and expose homology arms for homology-driven integration. Germline transmission rates for precision-targeted integration alleles range 22-100%. Our system provides a streamlined, straightforward, and cost-effective approach for high-efficiency gene targeting applications in zebrafish. Graphic abstract: [Image: see text] GeneWeld method for CRISPR/Cas9 targeted integration.

Published
2021

40. Contributors

Author

Rida Ansari, Lior Appelbaum, W. Philip Bartel, Carla Denise Bonan, Caroline H. Brennan, Edward A. Burton, Michael J. Carvan III, Karl J. Clark, Rosane Souza Da Silva, Konstantin A. Demin, Flavia De Santis, Vincenzo Di Donato, Elena Dreosti, Marc Ekker, Amanda Facciol, Gert Flik, Robert T. Gerlai, Marnix Gorissen, Fabrizio Grieco, Su Guo, Amanda A. Heeren, Dennis M. Higgs, Ellen J. Hoffman, Kerstin Howe, Khang Hua, Arnim Jenett, Allan V. Kalueff, Konstantinos Kalyviotis, Justin W. Kenney, Anton M. Lakstygal, Han B. Lee, Emmanuel Marquez-Legorreta, Noam Miller, Rodsy Modhurima, Stephan C.F. Neuhauss, Lucas P.J.J. Noldus, William HJ. Norton, Periklis Pantazis, David M. Parichy, Marielle Piber, John H. Postlethwait, David Pritchett, Hanyu Qin, Jason Rihel, Domino K. Schlegel, Ethan K. Scott, Alexander Stankiewicz, S.J. Stednitz, Ruud A.J. Tegelenbosch, Javier Terriente, Steven Tran, Benjamin Tsang, Sachiko Tsuda, Ruud van den Bos, Victor S. Van Laar, Monica Ryff Moreira Roca Vianna, P. Washbourne, Svante Winberg, Jiale Xu, David Zada, and Irina V. Zhdanova

Published
2021

41. Genetic therapy in a mitochondrial disease model suggests a critical role for liver dysfunction in mortality

Author

Roberto Lopez Cervera, Mark D Wishman, Ankit Sabharwal, MaKayla R Serres, Jennifer L Anderson, Shannon R Holmberg, Bibekananda Kar, Anthony J Treichel, Noriko Ichino, Weibin Liu, Jingchun Yang, Yonghe Ding, Yun Deng, Jean M Lacey, William J Laxen, Perry R Loken, Devin Oglesbee, Steven A Farber, Karl J Clark, Xiaolei Xu, and Stephen C Ekker

Subjects
General Immunology and Microbiology, General Neuroscience, General Medicine, General Biochemistry, Genetics and Molecular Biology
Abstract

The clinical and largely unpredictable heterogeneity of phenotypes in patients with mitochondrial disorders demonstrates the ongoing challenges in the understanding of this semi-autonomous organelle in biology and disease. Previously, we used the gene-breaking transposon to create 1200 transgenic zebrafish strains tagging protein-coding genes (Ichino et al., 2020), including the lrpprc locus. Here, we present and characterize a new genetic revertible animal model that recapitulates components of Leigh Syndrome French Canadian Type (LSFC), a mitochondrial disorder that includes diagnostic liver dysfunction. LSFC is caused by allelic variations in the LRPPRC gene, involved in mitochondrial mRNA polyadenylation and translation. lrpprc zebrafish homozygous mutants displayed biochemical and mitochondrial phenotypes similar to clinical manifestations observed in patients, including dysfunction in lipid homeostasis. We were able to rescue these phenotypes in the disease model using a liver-specific genetic model therapy, functionally demonstrating a previously under-recognized critical role for the liver in the pathophysiology of this disease.

Published
2020

42. High efficiency In Vivo genome engineering with a simplified 15-RVD GoldyTALEN design.

Author

Alvin C Ma, Han B Lee, Karl J Clark, and Stephen C Ekker

Subjects
Medicine, Science
Abstract

Transcription activator-like effector nucleases (TALENs) enable genome engineering in cell culture and many organisms. Recently, the GoldyTALEN scaffold was shown to readily introduce mutations in zebrafish (Danio rerio) and livestock through non-homologous end joining (NHEJ) and homology-directed repair (HDR). To deploy the GoldyTALEN system for high-throughput mutagenesis in model organisms, a simple design with high efficacy is desirable. We tested the in vivo efficacy of a simplified 15-RVD GoldyTALEN design (spacer between 13-20 bp and T nucleotide preceding each TALEN binding site) in zebrafish. All 14 tested TALEN pairs (100%) introduced small insertions and deletions at somatic efficacy ranging from 24 to 86%, and mutations were inheritable at high frequencies (18-100%). By co-injecting two GoldyTALEN pairs, inheritable deletions of a large genomic fragment up to 18 kb were successfully introduced at two different loci. In conclusion, these high efficiency 15-RVD GoldyTALENs are useful for high-throughput mutagenesis in diverse application including hypothesis testing from basic science to precision medicine.

Published
2013
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43. EV-Scale Sterile Neutrino Search Using Eight Years of Atmospheric Muon Neutrino Data from the IceCube Neutrino Observatory

Author

K. Tollefson, Albrecht Karle, U. Katz, R. S. Busse, G. H. Collin, Christopher Wiebusch, Kunal Deoskar, Thomas Ehrhardt, P. Schlunder, R. Hellauer, Spencer Axani, D. B. Fox, Kayla Leonard, R. Maunu, Alan Coleman, Sukeerthi Dharani, Francis Halzen, J. Merz, S. Hickford, R. Joppe, A. Wallace, Delia Tosi, Sebastian Baur, M. G. Aartsen, Carlos Arguelles, David Vannerom, I. Ansseau, Hermann Kolanoski, Juliana Stachurska, H. Dembinski, Stef Verpoest, Jessie Micallef, Matthias Vraeghe, George Japaridze, Roger Moore, Tim Ruhe, N. Kurahashi, Todor Stanev, C. Wendt, Ioana Codrina Maris, H. Bagherpour, Johan Wulff, Sebastian Böser, Olga Botner, J. Kim, Elisa Bernardini, A. Ludwig, Christoph Raab, Nadège Iovine, Timothyblake Watson, D. Heereman, Gisela Anton, M. Stamatikos, J. Bourbeau, K. Rawlins, G. de Wasseige, Won Nam Kang, V. Baum, Jenni Adams, D. Berley, G. C. Hill, H. Niederhausen, A. Steuer, C. Alispach, Bunheng Ty, Stephen L. Hauser, Raamis Hussain, Janet Conrad, B. Relethford, Jan Soedingrekso, Ralph Engel, Moritz Kellermann, Z. Griffith, Y. Makino, Seongjin In, R. Hoffmann, James Madsen, Konstancja Satalecka, Benjamin Bastian, Theo Glauch, Anastasia Maria Barbano, Saskia Philippen, F. Jonske, R. Cross, M. J. Larson, Lu Lu, T. Kittler, X. Bai, G. Neer, Alexander Fritz, A. Goldschmidt, D. van Eijk, Lenka Tomankova, Giovanni Renzi, D. Seckel, S. Fahey, Elisa Resconi, Alessio Porcelli, Claudio Kopper, Daniel Bindig, G. Krückl, Alejandro Diaz, C. Pérez de los Heros, E. O’Sullivan, Le Viet Nguyen, Wing Yan Ma, James DeLaunay, Marjon Moulai, R. G. Stokstad, K. Krings, M. Plum, P. B. Price, William Luszczak, M. Kauer, Suyong Choi, David Kappesser, S. E. Sanchez Herrera, Francesco Lucarelli, T. O. B. Schmidt, Tyce DeYoung, Chad Finley, Nathan Whitehorn, Immacolata Carmen Rea, Matti Jansson, D. Z. Besson, C. Walck, Thomas K. Gaisser, G. B. Yodh, A. Kyriacou, M. Silva, Najia Moureen Binte Amin, K.-H. Becker, Yiqian Xu, Qinrui Liu, J. Becker Tjus, T. Glüsenkamp, Subir Sarkar, Martina Karl, Matt Dunkman, Kendall Mahn, Christoph Tönnis, F. Huang, K. Meagher, Jochen M. Schneider, J. Stettner, T. R. Wood, Kurt Woschnagg, Paul Evenson, T. Palczewski, S. De Ridder, Y. L. Li, G. W. Sullivan, J. J. Beatty, Tianlu Yuan, Jeffrey Lazar, Chunfai Tung, James E. Braun, Alexander Trettin, E. Cheung, J. Werthebach, Thomas Stuttard, Mirco Hunnefeld, Marie Oehler, Alexander Sandrock, Elisa Lohfink, Thomas Meures, N. L. Strotjohann, M. A. Unland Elorrieta, M. Wallraff, Michael DuVernois, Benjamin Hokanson-Fasig, Felix Henningsen, Karl J. Clark, Simone Garrappa, Dmitry Chirkin, Sarah Pieper, D. Hebecker, Anatoli Fedynitch, K. Helbing, Chujie Chen, Dirk Ryckbosch, Joshua Hignight, M. Zöcklein, T. Grégoire, Timo Sturwald, J. C. Gallagher, Timo Karg, Gerald Przybylski, Federica Bradascio, Yang Lyu, Alexander Kappes, Alexander Burgman, M. Song, T. L. Carver, Paras Koundal, Mike Richman, Sarah Mancina, Paul Coppin, Anna Franckowiak, Tobias Hoinka, Vedant Basu, Steve Sclafani, F. McNally, A. R. Fazely, M. Usner, Grant Parker, R. Halliday, Stephan Meighen-Berger, K. D. de Vries, Gerrit Wrede, Andrea Turcati, J. van Santen, K. Hoshina, Glenn Spiczak, Thomas Huber, G. Momenté, Merlin Schaufel, A. Stößl, J. Kiryluk, K. Andeen, D. R. Nygren, Marek Kowalski, A. Weindl, Maryon Ahrens, Frederik Tenholt, R. Nagai, M. Medici, M. Rameez, Javier Gonzalez, Roxanne Turcotte, Alex Pizzuto, D. F. Cowen, Reina H. Maruyama, E. Pinat, N. Kulacz, U. Naumann, Martin Rongen, Ek Narayan Paudel, Allan Hallgren, Giorgio Maggi, K. Wiebe, A. Balagopal, Gary Binder, Zhedong Zhang, E. Unger, Simona Toscano, J. Auffenberg, Pablo Correa, Summer Blot, Brian Clark, C. De Clercq, P. Heix, K. Mase, P. Mallik, M. E. Huber, A. O'Murchadha, Marcos Santander, Ramesh Koirala, David A. Williams, L. Schumacher, Benedikt Riedel, S. Tilav, Max Renschler, D. Kang, D. J. Koskinen, Markus Ackermann, S. W. Barwick, L. Gerhardt, M. J. Weiss, E. Bourbeau, J. P. Yanez, Ben Smithers, Colin Turley, A. Olivas, S. Kopper, Spencer Griswold, I. Taboada, Michael O. Wolf, René Reimann, A. Haungs, M. Prado Rodriguez, Maria Tselengidou, D. Soldin, Nahee Park, Pranav Dave, K. Hultqvist, Darren Grant, E. Blaufuss, Mehmet Gunduz, Agnieszka Leszczyńska, R. C. Bay, M. Günder, Austin Schneider, Steven Robertson, Juan Carlos Diaz-Velez, Shefali Shefali, L. Classen, T. Stezelberger, Abdul Rehman, Carsten Rott, Y. Popovych, T. Kintscher, Frederik Hermann Lauber, Xianwu Xu, A. Terliuk, J. Sandroos, C. Bohm, Justin Lanfranchi, Christian Spiering, Justin Vandenbroucke, Abhishek Desai, S. C. Nowicki, Rasha Abbasi, B. J. Whelan, Jan Weldert, L. Halve, Philipp Eller, J. Felde, J. Buscher, D. Rysewyk Cantu, A. Obertacke Pollmann, J. L. Kelley, Markus Ahlers, Erik Ganster, Samvel Ter-Antonyan, Frank G. Schröder, J. Lünemann, N. van Eijndhoven, C. Haack, Emily Dvorak, Aaron Fienberg, Amirreza Raissi, Minjin Jeong, Chris Weaver, Stephanie Bron, C. J. Lozano Mariscal, H. Dujmovic, L. Rauch, S. Yoshida, Surujhdeo Seunarine, R. Snihur, Juanan Aguilar, Kael Hanson, A. Ishihara, Maximilian Meier, Paolo Desiati, Segev BenZvi, Spencer Klein, B. J. P. Jones, Andres Medina, Kara Hoffman, D. Mockler, Hershal Pandya, Daria Pankova, E. Friedman, Wolfgang Rhode, Robert Stein, L. Wills, P. Muth, R. Morse, Jakob Bottcher, T. Montaruli, Maximilian Karl Scharf, P. Peiffer, Ibrahim Safa, Damian Pieloth, Ali Kheirandish, W. Van Driessche, Jannes Brostean-Kaiser, M. U. Nisa, L. Köpke, T. Anderson, John Hardin, H. Schieler, Kirill Filimonov, Physics, Faculty of Sciences and Bioengineering Sciences, Vriendenkring VUB, and Elementary Particle Physics

Subjects
Sterile neutrino, Physics::Instrumentation and Detectors, General Physics and Astronomy, sterile [neutrino], 01 natural sciences, Cosmology, IceCube, High Energy Physics - Experiment, Subatomär fysik, High Energy Physics - Experiment (hep-ex), High Energy Physics - Phenomenology (hep-ph), Astronomi, astrofysik och kosmologi, Subatomic Physics, TOOL, Astronomy, Astrophysics and Cosmology, atmosphere [muon], Muon neutrino, Physics, oscillation [neutrino], Astrophysics::Instrumentation and Methods for Astrophysics, hep-ph, neutrino: sterile, mass difference [neutrino], ddc, muon: atmosphere, observatory, High Energy Physics - Phenomenology, Physique des particules élémentaires, signature, Particle physics, data analysis method, Scale (ratio), Astrophysics::High Energy Astrophysical Phenomena, neutrino: mass difference, FOS: Physical sciences, IceCube Neutrino Observatory, statistical analysis, 0103 physical sciences, OSCILLATIONS, ddc:530, 010306 general physics, hep-ex, ICE, High Energy Physics::Phenomenology, neutrino: mixing angle, CONVERSION, Physics and Astronomy, COSMOLOGY, High Energy Physics::Experiment, neutrino: oscillation, BAYESIAN-INFERENCE, mixing angle [neutrino], experimental results
Abstract

Physical review letters 125(14), 141801 (1-11) (2020). doi:10.1103/PhysRevLett.125.141801, The results of a 3+1 sterile neutrino search using eight years of data from the IceCube Neutrino Observatory are presented. A total of 305 735 muon neutrino events are analyzed in reconstructed energy-zenith space to test for signatures of a matter-enhanced oscillation that would occur given a sterile neutrino state with a mass-squared differences between 0.01 and 100 eV$^2$. The best-fit point is found to be at sin$^2$($2θ_{24}$)=0.10 and $Δm_{41}^2$=4.5 eV$^2$, which is consistent with the no sterile neutrino hypothesis with a $p$ value of 8.0%., Published by APS, College Park, Md.

Published
2020

44. IceCube-Gen2: The Window to the Extreme Universe

Author

J. Kim, A. Gartner, A. Obertacke Pollmann, B. Hoffmann, J. L. Kelley, Markus Ahlers, Jenni Adams, Akimichi Taketa, D. van Eijk, Daniel Bindig, G. H. Collin, T. C. Arlen, Robert Lahmann, J. J. Beatty, Tianlu Yuan, U. Nauman, Azadeh Keivani, Y. L. Li, Karl J. Clark, T. Kintscher, M. Song, T. L. Carver, Paras Koundal, Michael Kovacevich, Christian Glaser, Frederik Hermann Lauber, T. Huege, Erik Ganster, Benedikt Riedel, D. Seckel, Anna Nelles, R. G. Stokstad, F. McNally, R. Maunu, M. Prado Rodriguez, Kayla Leonard, N. Kurahashi, James E. Braun, Amy Connolly, Samvel Ter-Antonyan, A. Terliuk, Justin Lanfranchi, Pranav Dave, Jannis Necker, C. Wendt, S. Wren, A. Sharma, Sukeerthi Dharani, David Vannerom, M. H. Shaevitz, Alexander Trettin, Reina H. Maruyama, Francis Halzen, J. Merz, K. Krings, Rasha Abbasi, Ben Smithers, Qinrui Liu, M. J. Larson, T. Anderson, Summer Blot, G. de Wasseige, I. Ansseau, D. Hebecker, Jorge Torres, Killian Holzapfel, Martina Karl, C. J. Lozano Mariscal, John Hardin, Jean Pierre Twagirayezu, Daniel García-Fernández, T. Stezelberger, S. Mandalia, E. O'Sullivan, R. Hoffmann, M. Plum, Juliana Stachurska, H. Dembinski, C. Pérez de los Heros, S. Márka, G. WSullivan, A. Haungs, Paul Coppin, Z. Griffith, J. C. Gallagher, Saskia Philippen, Federica Bradascio, Jochen M. Schneider, Srubabati Goswami, Andrea Turcati, Wing Yan Ma, Nahee Park, Y. Makino, Sarah Mancina, C. Walck, M. Kauer, Suyong Choi, Anna Franckowiak, Tobias Hoinka, Chad Finley, David Kappesser, T. Gregoire, Ella Roberts, J. van Santen, S. De Ridder, Jan Weldert, Chunfai Tung, F. Jonske, Ken'ichi Kin, L. Halve, Philipp Eller, K. Hultqvist, N. L. Strotjohann, H. Dujmovic, Vedant Basu, M. A. Unland Elorrieta, Alex Pizzuto, K. D. de Vries, I. CMariş, Gerrit Wrede, R. Gernhaeuser, Thomas Huber, Matti Jansson, Thomas K. Gaisser, M. Richman, Christoph Tönnis, James DeLaunay, Gary Binder, K.-H. Becker, Ek Narayan Paudel, Allan Hallgren, U. Latif, Hiroyuki Tanaka, I. Safa, Steve Sclafani, J. Kiryluk, K. Andeen, P. B. Price, H. Schieler, Kirill Filimonov, Segev BenZvi, Alexander Fritz, D. Z. Besson, Darren Grant, Marjon Moulai, Yiqian Xu, Felix Henningsen, S. Robertson, Aswathi Balagopal, Francesco Lucarelli, Pisin Chen, Matt Dunkman, Merlin Schaufel, Patrick Allison, Spencer Klein, Cosmin Deaconu, Tim Ruhe, A. Ludwig, George Japaridze, Javier Gonzalez, C. B. Krauss, Roxanne Turcotte, T. O. B. Schmidt, Simona Toscano, Roger Moore, P. Heix, R. S. Busse, Chujie Chen, Pablo Correa, L. Gerhard, S. De Kockere, J. Felde, Surujhdeo Seunarine, R. Snihur, J. Buscher, D. Rysewyk Cantu, A. Weindl, R. Hellauer, Giorgio Maggi, H. Niederhausen, Mauricio Bustamante, D. Southall, Julia Böttcher, J. Bourbeau, Lenka Tomankova, Maryon Ahrens, A. Burgman, Christopher Wiebusch, Darko Veberič, Juanan Aguilar, T. R. Wood, Christian Spiering, Frederik Tenholt, R. Nagai, L. Schumacher, C. De Clercq, Benjamin Hokanson-Fasig, L. V. Nguyen, C. Lagunas Gualda, K. Hughes, Kara Hoffman, C. Alispach, J. M. LoSecco, Joshua Hignight, K. Helbing, Timo Sturwald, Xinyue Kang, Richard Naab, Kunal Deoskar, Janet Conrad, Zackary Meyers, K. Meagher, Mehmet Gunduz, Agnieszka Leszczyńska, R. C. Bay, David A. Williams, Kurt Woschnagg, M. Zöcklein, M. Silva, Claudio Kopper, Eric Oberla, Ramesh Koirala, E. Cheung, Thomas Stuttard, Martin Rongen, Najia Moureen Binte Amin, R. Cross, Paul Evenson, A. Karle, Sebastian Böser, Seongjin In, Johannes Werthebach, J. P. Lazar, Markus Ackermann, Austin Schneider, Yang Lyu, Justin Vandenbroucke, Juan Carlos Diaz-Velez, Beverley A. Clark, Timo Karg, Sarah Pieper, Hershal Pandya, Wolfgang Rhode, Zhedong Zhang, P. Schlunder, A. Ishihara, Elisa Resconi, Subir Sarkar, William Luszczak, Clara E. Hill, Ava Ghadimi, Alessio Porcelli, Alan Coleman, J. Auffenberg, Grant Parker, Robert Stein, Dirk Ryckbosch, Benjamin Bastian, Anastasia Maria Barbano, Abhishek Desai, T. Kittler, J. Nam, P. Mallik, E. Blaufuss, S. Zierke, T. Stanev, M. Bohmer, Stephan Meighen-Berger, Simone Garrappa, P. Muth, Dmitry Chirkin, M. E. Huber, Marcos Santander, Christoph Raab, Nadège Iovine, J. Becker Tjus, L. Classen, Colin Turley, S. C. Nowicki, K. Farrag, M. Kleifges, O. Kalekin, A. Olivas, Alexander Kappes, D. Berley, G. C. Hill, Abigail G. Vieregg, Frank G. Schröder, D. Heinen, Erin Carnie-Bronca, N. Kulacz, D. Tosi, J. C. Hanson, Bunheng Ty, Ralph Engel, Moritz Kellermann, Gisela Anton, Elisa Lohfink, Elisa Bernardini, Damian Pieloth, Ali Kheirandish, Jan Soedingrekso, Giovanni Renzi, Michael DuVernois, Jannes Brostean-Kaiser, K. Wiebe, S. Fahey, A. R. Fazely, Tyce DeYoung, J. Lünemann, Thomas Ehrhardt, Nathan Whitehorn, Immacolata Carmen Rea, M. U. Nisa, Aaron Fienberg, Gerald Przybylski, G. Krückl, L. Papp, Amirreza Raissi, L. Köpke, Chris Weaver, R. Halliday, Alejandro Diaz, Stephanie Bron, S. Söldner-Rembold, James Pinfold, Ryan Burley, M. Riegel, H. Bagherpour, Stephanie Wissel, Olga Botner, Y. Pan, A. Steuer, S. Tilav, D. Kang, M. deWith, V. Baum, J. P. Yanez, I. Taboada, Stephen L. Hauser, Raamis Hussain, Michael O. Wolf, M. Stamatikos, John Evans, Shefali Shefali, Christoph Welling, Abdul Rehman, Carsten Rott, K. Tollefson, A. Goldschmidt, Y. Popovych, Troels Petersen, S. E. Sanchez Herrera, Simeon Reusch, S. Hickford, Xianwu Xu, J. Sandroos, C. Bohm, R. Joppe, Spencer Axani, Sebastian Baur, Carlos Arguelles, Jessie Micallef, Teppei Katori, K. Rawlins, James Madsen, Lu Lu, M. G. Aartsen, T. Glüsenkamp, M. Meier, Michael Campana, P. Sandstrom, Kendall Mahn, B. J. P. Jones, K. Hoshina, Glenn Spiczak, Andres Medina, G. Momenté, D. Mockler, M. Rameez, D. F. Cowen, Daria Pankova, E. Friedman, R. Morse, T. Montaruli, E. Unger, Maximilian Karl Scharf, P. Peiffer, J. Stettner, Gerrit Roellinghoff, Mirco Hunnefeld, Marie Oehler, Max Renschler, D. J. Koskinen, E. Bourbeau, Spencer Griswold, Maria Tselengidou, D. Soldin, Z. Márka, Minjin Jeong, L. Rauch, S. Yoshida, N. van Eijndhoven, C. Haack, Emily Dvorak, D. R. Nygren, Marek Kowalski, Kael Hanson, Paolo Desiati, J. Haugen, Lars Steffen Weinstock, Johan Wulff, Won Nam Kang, Timothyblake Watson, X. Bai, G. Neer, Konstancja Satalecka, Theo Glauch, U. Katz, Alexander Sandrock, Ilse Plaisier, K. Mase, D. B. Fox, Hermann Kolanoski, M. J. Weiss, Stef Verpoest, Imre Bartos, David J. Smith, S. Kopper, René Reimann, Leander Fischer, F. Huang, Matthias Vraeghe, Physics, Faculty of Sciences and Bioengineering Sciences, Vriendenkring VUB, and Elementary Particle Physics

Subjects
Physics, High Energy Astrophysical Phenomena (astro-ph.HE), astro-ph.HE, Nuclear and High Energy Physics, Active galactic nucleus, 010308 nuclear & particles physics, High-energy astronomy, Gravitational wave, media_common.quotation_subject, Astrophysics::High Energy Astrophysical Phenomena, Astrophysics::Instrumentation and Methods for Astrophysics, Astronomy, FOS: Physical sciences, Cosmic ray, 01 natural sciences, Universe, Neutron star, 0103 physical sciences, Neutrino, Neutrino astronomy, Astrophysics - High Energy Astrophysical Phenomena, 010303 astronomy & astrophysics, media_common
Abstract

The observation of electromagnetic radiation from radio to $\gamma$-ray wavelengths has provided a wealth of information about the universe. However, at PeV (10$^{15}$ eV) energies and above, most of the universe is impenetrable to photons. New messengers, namely cosmic neutrinos, are needed to explore the most extreme environments of the universe where black holes, neutron stars, and stellar explosions transform gravitational energy into non-thermal cosmic rays. The discovery of cosmic neutrinos with IceCube has opened this new window on the universe. In this white paper, we present an overview of a next-generation instrument, IceCube-Gen2, which will sharpen our understanding of the processes and environments that govern the universe at the highest energies. IceCube-Gen2 is designed to: 1) Resolve the high-energy neutrino sky from TeV to EeV energies; 2) Investigate cosmic particle acceleration through multi-messenger observations; 3) Reveal the sources and propagation of the highest energy particles in the universe; 4) Probe fundamental physics with high-energy neutrinos. IceCube-Gen2 will increase the annual rate of observed cosmic neutrinos by a factor of ten compared to IceCube, and will be able to detect sources five times fainter than its predecessor. Furthermore, through the addition of a radio array, IceCube-Gen2 will extend the energy range by several orders of magnitude compared to IceCube. Construction will take 8 years and cost about \$350M. The goal is to have IceCube-Gen2 fully operational by 2033. IceCube-Gen2 will play an essential role in shaping the new era of multi-messenger astronomy, fundamentally advancing our knowledge of the high-energy universe. This challenging mission can be fully addressed only in concert with the new survey instruments across the electromagnetic spectrum and gravitational wave detectors which will be available in the coming years., Comment: 56 pages, 29 figures

Published
2020

45. A homozygous missense variant in UBE2T is associated with a mild Fanconi anemia phenotype

Author

Agata Smogorzewska, Alejandro Ferrer, Eric W. Klee, Christopher T. Schmitz, Nikita R. Dsouza, Mrinal M. Patnaik, Michael T. Zimmermann, Karl J. Clark, Laura Schultz-Rogers, Nicole Jacobi, Mark R. Litzow, Francis P. Lach, Tanya L. Schwab, Abhishek A. Mangaonkar, and Kimberly A. Rickman

Subjects
Extramural, business.industry, Bone marrow failure, Hematology, Ubiquitin-conjugating enzyme, medicine.disease, Phenotype, Fanconi Anemia Complementation Group Proteins, Haematopoiesis, Fanconi Anemia, Fanconi anemia, Ubiquitin-Conjugating Enzymes, medicine, Cancer research, Missense mutation, Humans, business, Letters to the Editor
Published
2020

46. IceCube Search for High-Energy Neutrino Emission from TeV Pulsar Wind Nebulae

Author

C. Haack, Immacolata Carmen Rea, Emily Dvorak, K.-H. Becker, K. Hoshina, Yiqian Xu, Matt Dunkman, K. Mase, Yuya Makino, Minjin Jeong, Timothyblake Watson, Thomas Meures, B. Relethford, E. Cheung, Saskia Philippen, M. J. Weiss, Konstancja Satalecka, Theo Glauch, L. Rauch, U. Katz, Thomas Stuttard, S. De Ridder, Jeffrey Lazar, P. B. Price, D. Heereman, S. Yoshida, D. B. Fox, S. Kopper, Sarah Pieper, K. Jero, N. L. Strotjohann, Hermann Kolanoski, Dirk Ryckbosch, Stef Verpoest, Colin Turley, Felix Henningsen, M. Zöcklein, René Reimann, C. Alispach, Lenka Tomankova, T. Grégoire, Timo Karg, A. Steuer, Stephan Meighen-Berger, Janet Conrad, Thomas Ehrhardt, Frank G. Schröder, A. R. Fazely, M. Silva, Francesco Lucarelli, Raamis Hussain, R. Cross, A. Obertacke Pollmann, E. Pinat, T. O. B. Schmidt, Johannes Werthebach, M. Usner, Kael Hanson, Karl J. Clark, Maximilian Meier, S. Hickford, R. Joppe, Delia Tosi, Sebastian Baur, Chujie Chen, A. Weindl, Maryon Ahrens, Frederik Tenholt, R. Nagai, K. Helbing, Won Nam Kang, X. Bai, G. Neer, Simona Toscano, P. Heix, R. Maunu, J. Lünemann, William Luszczak, S. Tilav, M. Medici, Christian Spiering, L. Wills, P. Muth, P. Schlunder, Daria Pankova, E. Friedman, R. S. Busse, M. Song, T. L. Carver, Mike Richman, Amirreza Raissi, Federica Bradascio, Alexander Burgman, T. R. Wood, Ramesh Koirala, J. P. Yanez, Karim Ghorbani, Spencer Axani, Christopher Wiebusch, Kunal Deoskar, Paolo Desiati, Chris Weaver, Stephanie Bron, Kurt Woschnagg, C. J. Lozano Mariscal, G. Merino, Tim Ruhe, Justin Vandenbroucke, H. Bagherpour, Alexander Sandrock, J. L. Kelley, Markus Ahlers, Martin Rongen, George Japaridze, Roger Moore, A. Ludwig, Olga Botner, Paul Evenson, M. G. Aartsen, N. Wandkowsky, H. Dujmovic, Merlin Schaufel, M. Stamatikos, E. Blaufuss, Ioana Codrina Maris, L. Wille, H. Niederhausen, Carlos Arguelles, Ibrahim Safa, V. Baum, Jessie Micallef, L. Classen, Damian Pieloth, Ali Kheirandish, N. Kurahashi, J. Bourbeau, D. Kang, Todor Stanev, K. Tollefson, Surujhdeo Seunarine, Albrecht Karle, D. van Eijk, Christoph Raab, Nadège Iovine, P. Mallik, Simone Garrappa, Dmitry Chirkin, S. C. Nowicki, W. Van Driessche, C. Wendt, Alexander Kappes, Erik Ganster, K. Mallot, R. Morse, James W. Wood, Z. Griffith, Jakob Bottcher, D. Berley, G. C. Hill, Bunheng Ty, R. Snihur, T. Montaruli, Jannes Brostean-Kaiser, Juanan Aguilar, G. de Wasseige, G. W. Sullivan, K. Rawlins, Donglian Xu, Seongjin In, B. J. P. Jones, Ralph Engel, Moritz Kellermann, Elisa Resconi, I. Taboada, Samvel Ter-Antonyan, Elisa Lohfink, James E. Braun, Gisela Anton, James Madsen, Lu Lu, Alessio Porcelli, T. Kintscher, Frederik Hermann Lauber, Giovanni Renzi, A. Goldschmidt, D. Hebecker, Qinrui Liu, R. Hoffmann, T. Glüsenkamp, Le Viet Nguyen, L. Köpke, D. Seckel, S. Fahey, Michael O. Wolf, Alexander Fritz, D. Z. Besson, Martina Karl, A. Wallace, A. Terliuk, Paul Coppin, Andrea Turcati, Justin Lanfranchi, J. van Santen, Mehr Nisa, Jochen M. Schneider, B. J. Whelan, Claudio Kopper, A. Ishihara, Tyce DeYoung, Andres Medina, Alexander Trettin, Nathan Whitehorn, Alex Pizzuto, D. R. Nygren, Gerald Przybylski, S. E. Sanchez Herrera, M. A. Unland Elorrieta, Gary Binder, R. Halliday, A. Haungs, C. Pérez de los Heros, K. Krings, D. Mockler, Nahee Park, K. Hultqvist, M. Wallraff, Darren Grant, Kendall Mahn, C. Walck, Alan Coleman, E. O’Sullivan, T. Anderson, Marek Kowalski, J. C. Gallagher, M. Plum, Subir Sarkar, Jan Weldert, Thomas K. Gaisser, G. B. Yodh, A. Kyriacou, Gerrit Wrede, K. Meagher, L. Halve, Philipp Eller, Thomas Huber, Allan Hallgren, Benjamin Hokanson-Fasig, Yang Lyu, Elisa Bernardini, J. Auffenberg, Chunfai Tung, J. Becker Tjus, J. Felde, J. Buscher, D. Rysewyk Cantu, Jenni Adams, P. Peiffer, Joshua Hignight, Timo Sturwald, H. Schieler, Kirill Filimonov, K. Wiebe, Steven Robertson, R. G. Stokstad, Segev BenZvi, Shefali Shefali, Spencer Klein, Abdul Rehman, Carsten Rott, Kara Hoffman, Javier Gonzalez, Roxanne Turcotte, Giorgio Maggi, Matthias Vraeghe, C. De Clercq, Hershal Pandya, Wolfgang Rhode, David A. Williams, Xianwu Xu, Robert Stein, J. Sandroos, Markus Ackermann, S. W. Barwick, C. Bohm, J. Kim, Daniel Bindig, K. D. de Vries, N. Kulacz, Brian Clark, Chad Finley, Christoph Tönnis, Pranav Dave, M. Günder, T. Stezelberger, G. H. Collin, Sarah Mancina, Anna Franckowiak, Tobias Hoinka, Kayla Leonard, Steve Sclafani, Jan Soedingrekso, Francis Halzen, J. Kiryluk, K. Andeen, I. Ansseau, G. Krückl, Juliana Stachurska, Alejandro Diaz, Pablo Correa, H. Dembinski, M. Kauer, Suyong Choi, David Kappesser, R. Hellauer, Matti Jansson, T. Palczewski, Sebastian Böser, Benjamin Bastian, Anastasia Maria Barbano, T. Kittler, B. Eberhardt, M. E. Huber, Marcos Santander, L. Gerhardt, A. Olivas, M. J. Larson, Wing Yan Ma, James DeLaunay, Marjon Moulai, J. J. Beatty, Tianlu Yuan, F. McNally, A. Stößl, Reina H. Maruyama, Summer Blot, F. Jonske, L. Schumacher, F. Huang, Mehmet Gunduz, Agnieszka Leszczyńska, R. C. Bay, Austin Schneider, Juan Carlos Diaz-Velez, Glenn Spiczak, G. Momenté, M. Rameez, D. F. Cowen, U. Naumann, E. Unger, A. O'Murchadha, Max Renschler, D. J. Koskinen, E. Bourbeau, Spencer Griswold, Maria Tselengidou, D. Soldin, J. Stettner, Mirco Hunnefeld, Marie Oehler, N. van Eijndhoven, Physics, Faculty of Sciences and Bioengineering Sciences, Vriendenkring VUB, and Elementary Particle Physics

Subjects
010504 meteorology & atmospheric sciences, High-energy astronomy, Astrophysics::High Energy Astrophysical Phenomena, Neutrino astronomy, High energy astrophysics, FOS: Physical sciences, Cosmic ray, Astrophysics, Astrophysics::Cosmology and Extragalactic Astrophysics, 01 natural sciences, 7. Clean energy, Pulsar, 0103 physical sciences, 010303 astronomy & astrophysics, Astrophysics::Galaxy Astrophysics, 0105 earth and related environmental sciences, Physics, High Energy Astrophysical Phenomena (astro-ph.HE), astro-ph.HE, Astronomy and Astrophysics, Galactic plane, COSMIC-RAYS, CRAB-NEBULA, Crab Nebula, Physics and Astronomy, 13. Climate action, Space and Planetary Science, GALACTIC SOURCES, DISCOVERY, Physique des particules élémentaires, High Energy Physics::Experiment, Neutrino, Astrophysics - High Energy Astrophysical Phenomena, GAMMA-RAY EMISSION, Lepton
Abstract

Pulsar wind nebulae (PWNe) are the main gamma-ray emitters in the Galactic plane. They are diffuse nebulae that emit nonthermal radiation. Pulsar winds, relativistic magnetized outflows from the central star, shocked in the ambient medium produce a multiwavelength emission from the radio through gamma-rays. Although the leptonic scenario is able to explain most PWNe emission, a hadronic contribution cannot be excluded. A possible hadronic contribution to the high-energy gamma-ray emission inevitably leads to the production of neutrinos. Using 9.5 yr of all-sky IceCube data, we report results from a stacking analysis to search for neutrino emission from 35 PWNe that are high-energy gamma-ray emitters. In the absence of any significant correlation, we set upper limits on the total neutrino emission from those PWNe and constraints on hadronic spectral components., SCOPUS: ar.j, info:eu-repo/semantics/published

Published
2020

47. Author response: Building the vertebrate codex using the gene breaking protein trap library

Author

Ying Wang, Darius Balciunas, Shawn M. Burgess, Noriko Ichino, Xiaolei Xu, Yonghe Ding, MaKayla R Serres, Rhianna M. Urban, Sridhar Sivasubbu, Kimberly J. Skuster, Mark D. Urban, Jennifer L. Anderson, Matthias Hammerschmidt, Melissa S. McNulty, Anthony J. Treichel, Jeffrey J. Essner, Weibin Liu, Maura McGrail, Ankit Sabharwal, Lauren E Greif, Lisa A. Schimmenti, Xiao-Yan Wen, Stephen C. Ekker, Suzan El-Rass, Kyle J. Schaefbauer, Mark D Wishman, Camden L. Daby, Gaurav K. Varshney, Steven A. Farber, Karl J. Clark, and Hsin-kai Liao

Subjects
Trap (computing), Evolutionary biology, biology.animal, Vertebrate, Biology, Gene
Published
2020

48. Endogenous zebrafish proneural Cre drivers generated by CRISPR/Cas9 short homology directed targeted integration

Author

Jeffrey J. Essner, Maira P. Almeida, Maura McGrail, Jordan M. Welker, Stephen C. Ekker, and Karl J. Clark

Subjects
Cas9, Transgene, Recombinase, CRISPR, Cre recombinase, Biology, mCherry, biology.organism_classification, Gene, Zebrafish, Cell biology
Abstract

The Cre/lox recombinase system has been widely used for spatiotemporal control of gene expression in animal model systems, however, efficient methods to isolate zebrafish Cre drivers that reliably recapitulate endogenous expression patterns of known genes are in need. Here, we apply CRISPR/Cas9 double-strand breaks to direct targeted integration using short homology of a 2A-Cre recombinase transgene into the early coding region of proneural genes ascl1b, olig2 and neurod1. The pPRISM-2A-Cre targeting vector contains a linked lens-specific fluorescent secondary marker cassette for identification of transgenic embryos, and we observed high rates of germline transmission ranging from 10%-100% (2/20 olig2; 1/5 neurod1; 3/3 ascl1b). The established transgenic lines Tg(ascl1b-2A-Cre)is75, Tg(olig2-2A-Cre)is76, and Tg(neurod1-2ACre)is77 expressed functional Cre recombinase. in situ hybridization showed Cre expression reflected the pattern of the endogenous targeted proneural gene. In combination with Tg(ubi:loxP-EGFP-loxP-mCherry), each driver induced a switch from EGFP to mCherry expression in the expected proneural cell populations and their descendants, indicating efficient Cre-mediated recombination and excision of the floxed EGFP cassette. The results demonstrate Cre recombinase expression is driven by the native promoter and regulatory elements of the targeted genes. This approach provides a straightforward, efficient, and cost-effective method to generate cell type specific zebrafish Cre drivers whose spatial and temporal restricted expression mimics endogenous genes, surmounting the challenges associated with promoter BAC cloning and transposon mediated transgenesis.

Published
2020

49. Characterization of gene repression by designed transcription activator-like effector dimer proteins

Author

Jordan P. Wallace, Tanya L. Schwab, Nicole A. Becker, Karl J. Clark, William J. Phillips, Justin P. Peters, and L. James Maher

Subjects
DNA, Bacterial, Dimer, Biophysics, lac operon, Computational biology, Lac repressor, medicine.disease_cause, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Transcription (biology), Escherichia coli, Lac Repressors, medicine, Psychological repression, Transcription Activator-Like Effectors, 030304 developmental biology, Regulation of gene expression, 0303 health sciences, Chemistry, Effector, Escherichia coli Proteins, Articles, Gene Expression Regulation, Bacterial, Cell biology, Lac Operon, Nucleic Acid Conformation, 030217 neurology & neurosurgery, DNA
Abstract

Gene regulation by control of transcription initiation is a fundamental property of living cells. Much of our understanding of gene repression originated from studies of the E. coli lac operon switch, where DNA looping plays an essential role. To validate and generalize principles from lac for practical applications, we previously described artificial DNA looping driven by designed Transcription Activator-Like Effector Dimer (TALED) proteins. Because TALE monomers bind the idealized symmetrical lac operator sequence in two orientations, our prior studies detected repression due to multiple DNA loops. We now quantitatively characterize gene repression in living E. coli by a collection of individual TALED loops with systematic loop length variation. Fitting of a thermodynamic model allows unequivocal demonstration of looping and comparison of the engineered TALED repression system with the natural lac repressor system.Statement of SignificanceWe are designing and testing in living bacteria artificial DNA looping proteins engineered based on principles learned from studies of the E. coli lac repressor. The engineered proteins are based on artificial dimers of Transcription Activator-Like Effector (TALE) proteins that have programmable DNA binding specificities. The current work is the first to create unique DNA repression loops using this approach. Systematic study of repression as a function of loop size, with data fitting to a thermodynamic model, now allows this system to be compared in detail with lac repressor loops, and relevant biophysical parameters to be estimated. This approach has implications for the artificial regulation of gene expression.

Published
2020

50. A Genetic Model Therapy Proposes a Critical Role for Liver Dysfunction in Mitochondrial Biology and Disease

Author

Noriko Ichino, Yonghe Ding, Jingchun Yang, Xiaolei Xu, Ma Kayla R. Serres, Yun Deng, Anthony J. Treichel, Weibin Liu, Ankit Sabharwal, Karl J. Clark, Roberto Lopez Cervera, Stephen C. Ekker, Steven A. Farber, Mark D Wishman, and Jennifer L. Anderson

Subjects
Genetics, 0303 health sciences, Mitochondrial DNA, Mitochondrial disease, Mitochondrial mRNA polyadenylation, Biology, biology.organism_classification, medicine.disease, Phenotype, 3. Good health, 03 medical and health sciences, 0302 clinical medicine, Genetic model, medicine, Liver function, Allele, Zebrafish, 030217 neurology & neurosurgery, 030304 developmental biology
Abstract

The clinical and largely unpredictable heterogeneity of phenotypes in patients with mitochondrial disorders demonstrates the ongoing challenges in the understanding of this semi-autonomous organelle in biology and disease. Here we present a new animal model that recapitulates key components of Leigh Syndrome, French Canadian Type (LSFC), a mitochondrial disorder that includes diagnostic liver dysfunction. LSFC is caused by allelic variations in the Leucine Rich Pentatricopeptide repeat-containing motif (LRPPRC) gene. LRPPRC has native functions related to mitochondrial mRNA polyadenylation and translation as well as a role in gluconeogenesis. We used the Gene-Breaking Transposon (GBT) cassette to create a revertible, insertional mutant zebrafish line in the LRPPRC gene. lrpprc zebrafish homozygous mutants displayed impaired muscle development, liver function and lowered levels of mtDNA transcripts and are lethal by 12dpf, all outcomes similar to clinical phenotypes observed in patients. Investigations using an in vivo lipidomics approach demonstrated accumulation of non-polar lipids in these animals. Transcript profiling of the mutants revealed dysregulation of clinically important nuclearly encoded and mitochondrial transcripts. Using engineered liver-specific rescue as a genetic model therapy, we demonstrate survival past the initial larval lethality, as well as restored normal gut development, mitochondrial morphology and triglyceride levels functionally demonstrating a critical role for the liver in the pathophysiology of this model of mitochondrial disease. Understanding the molecular mechanism of the liver-mediated genetic rescue underscores the potential to improve the clinical diagnostic and therapeutic developments for patients suffering from these devastating disorders.

Published
2020

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