المؤلفون: 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, Stephen C Ekker

المصدر: eLife, Vol 9 (2020)

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

الوصف: 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.

وصف الملف: electronic resource

العلاقة: https://elifesciences.org/articles/54572Test; https://doaj.org/toc/2050-084XTest

الوصول الحر: https://doaj.org/article/304fac055ae14ae1937ee0f79c8cd7bcTest

المؤلفون: 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, Stephen C. Ekker

المصدر: The CRISPR Journal. 5:40-52

مصطلحات موضوعية: Genetics, Biotechnology

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_________::6cbaee14b19044019f47dbbc8ea184afTest
https://doi.org/10.1089/crispr.2021.0087Test

المؤلفون: 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, Stephen C, Ekker

المصدر: The CRISPR journal. 5(1)

مصطلحات موضوعية: Gene Editing, Humans, RNA, DNA Breaks, Double-Stranded, DNA, CRISPR-Cas Systems

الوصف: 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

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=pmid________::2c9f39575e116a6e2fd2df5b9df6f473Test
https://pubmed.ncbi.nlm.nih.gov/34935462Test

المؤلفون: 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, Hirotaka Ata

مصطلحات موضوعية: 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

الوصف: 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.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_________::6c46d8446d52816bbe565304406968c4Test
https://doi.org/10.1101/2021.05.28.446234Test

المؤلفون: 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, Hsin-kai Liao

مصطلحات موضوعية: Trap (computing), Evolutionary biology, biology.animal, Vertebrate, Biology, Gene

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_________::d31bb60ac105227278f22463adaaf56cTest
https://doi.org/10.7554/elife.54572.sa2Test

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

مصطلحات موضوعية: Genetics, Transposable element, 0303 health sciences, medicine.medical_specialty, Positional cloning, biology, Locus (genetics), biology.organism_classification, 03 medical and health sciences, 0302 clinical medicine, Molecular genetics, Proteome, medicine, Allele, Gene, Zebrafish, 030217 neurology & neurosurgery, 030304 developmental biology

الوصف: The zebrafish is a powerful model to explore the molecular genetics and expression of the vertebrate genome. The gene break transposon (GBT) is a unique insertional mutagen that reports the expression of the tagged member of the proteome while generating Cre-revertible genetic alleles. This 1000+ locus collection represents novel codex expression data from the illuminated mRFP protein trap, with 36% and 87% of the cloned lines showcasing to our knowledge the first described expression of these genes at day 2 and day 4 of development, respectively. Analyses of 183 molecularly characterized loci indicate a rich mix of genes involved in diverse cellular processes from cell signaling to DNA repair. The mutagenicity of the GBT cassette is very high as assessed using both forward and reverse genetic approaches. Sampling over 150 lines for visible phenotypes after 5dpf shows a similar rate of discovery of embryonic phenotypes as ENU and retroviral mutagenesis. Furthermore, five cloned insertions were in loci with previously described phenotypes; embryos homozygous for each of the corresponding GBT alleles displayed strong loss of function phenotypes comparable to published mutants using other mutagenesis strategies (ryr1b,fras1,tnnt2a, edarandhmcn1). Using molecular assessment after positional cloning, to date nearly all alleles cause at least a 99+% knockdown of the tagged gene. Interestingly, over 35% of the cloned loci represent 68 mutants in zebrafish orthologs of human disease loci, including nervous, cardiovascular, endocrine, digestive, musculoskeletal, immune and integument systems. The GBT protein trapping system enabled the construction of a comprehensive protein codex including novel expression annotation, identifying new functional roles of the vertebrate genome and generating a diverse collection of potential models of human disease.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::e8c3c1b145de69fd747d3468ba17b824Test

المؤلفون: Randall G. Krug, Camden L. Daby, Dakota C. Jacobs, Bethany C. Berry, Rhianna M. Urban, Ashley N. Sigafoos, Ryan P. Cotter, Biswadeep Das, Ma Kayla R. Serres, Tanya L. Schwab, Jennifer L. Gauerke, Morgan O. Petersen, Han B. Lee, Karl J. Clark

مصطلحات موضوعية: Exon, medicine.anatomical_structure, Mineralocorticoid receptor, Glucocorticoid receptor, biology, Adrenal gland, Gene expression, medicine, ACTH receptor, Receptor, biology.organism_classification, Zebrafish, Cell biology

الوصف: When vertebrates face acute stressors, their bodies rapidly undergo a repertoire of physiological and behavioral adaptations, which is termed the stress response (SR). Rapid physiological changes in heart rate and blood sugar levels occur via the interaction of glucocorticoids and their cognate receptors following hypothalamic-pituitary-adrenal (HPA) axis activation. These physiological changes are observed within minutes of encountering a stressor and the rapid time domain rules out genomic responses that require gene expression changes. Although behavioral changes corresponding to physiological changes are commonly observed, it is not clearly understood to what extent HPA axis activation dictates adaptive behavior. We hypothesized that rapid locomotor response to acute stressors in zebrafish requires HPI axis activation. In teleost fish, interrenal cells (I) are functionally homologous to the adrenal gland cortical layer. We derived 8 frameshift mutants in genes involved in HPI axis function: two mutants in exon 2 of mc2r (adrenocorticotropic hormone receptor), two in each of exon 2 and exon 5 of nr3c1 (glucocorticoid receptor), and two in exon 2 of nr3c2 (mineralocorticoid receptor). Exposing larval zebrafish to mild environmental stressors, acute changes in salinity or light illumination, results in a rapid locomotor response. We show here that this locomotor response requires a functioning HPI axis via the action of mc2r (adrenocorticotropic hormone receptor) and the canonical glucocorticoid receptor encoded by nr3c1 gene, but not mineralocorticoid receptor (nr3c2). Our rapid behavioral assay paradigm based on HPI axis biology may prove useful to screen for genetic, pharmacological, or environmental modifiers of the HPA axis.SignificanceAltered HPA axis activity is acknowledged as a causative and critical prognostic factor in many psychiatric disorders including depression. Nonetheless, genome wide association studies (GWAS) on depression have revealed conflicting findings about susceptibility loci, while identifying several genetic loci that warrant further investigations in the process. Such findings indicate that psychiatric disorders with complex genetic foundations require functional studies as well as genetic analyses. We developed a sensitive behavioral assay paradigm that leverages the genetic amenability and rapid development of zebrafish and demonstrated that our assay system reliably detects changes in HPA axis responsiveness. Our functional genetics and behavioral assay approach provides a useful platform to discover novel genetic, pharmacological, or environmental modifiers of the HPA axis.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::094f2bec75df8123320171eb47372cc5Test

المؤلفون: Han B. Lee, Camden L. Daby, Roberto Lopez Cervera, Alaa Koleilat, Melissa S. McNulty, Karl J. Clark, Hannah S. Bostwick, Tanya L. Schwab, Hirotaka Ata

المصدر: Human Gene Therapy

مصطلحات موضوعية: 0301 basic medicine, Genotype, Laboratory Method, Sequence analysis, Cost-Benefit Analysis, Biology, Polymerase Chain Reaction, law.invention, 03 medical and health sciences, symbols.namesake, law, Genetics, CRISPR, Animals, Molecular Biology, Genotyping, Polymerase chain reaction, Alleles, Zebrafish, DNA Primers, Sanger sequencing, Transcription activator-like effector nuclease, Sequence Analysis, DNA, Zebrafish Proteins, 030104 developmental biology, symbols, Molecular Medicine, Restriction fragment length polymorphism

الوصف: Customizable endonucleases such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) enable rapid generation of mutant strains at genomic loci of interest in animal models and cell lines. With the accelerated pace of generating mutant alleles, genotyping has become a rate-limiting step to understanding the effects of genetic perturbation. Unless mutated alleles result in distinct morphological phenotypes, mutant strains need to be genotyped using standard methods in molecular biology. Classic restriction fragment length polymorphism (RFLP) or sequencing is labor-intensive and expensive. Although simpler than RFLP, current versions of allele-specific PCR may still require post-polymerase chain reaction (PCR) handling such as sequencing, or they are more expensive if allele-specific fluorescent probes are used. Commercial genotyping solutions can take weeks from assay design to result, and are often more expensive than assembling reactions in-house. Key components of commercial assay systems are often proprietary, which limits further customization. Therefore, we developed a one-step open-source genotyping method based on quantitative PCR. The allele-specific qPCR (ASQ) does not require post-PCR processing and can genotype germline mutants through either threshold cycle (Ct) or end-point fluorescence reading. ASQ utilizes allele-specific primers, a locus-specific reverse primer, universal fluorescent probes and quenchers, and hot start DNA polymerase. Individual laboratories can further optimize this open-source system as we completely disclose the sequences, reagents, and thermal cycling protocol. We have tested the ASQ protocol to genotype alleles in five different genes. ASQ showed a 98-100% concordance in genotype scoring with RFLP or Sanger sequencing outcomes. ASQ is time-saving because a single qPCR without post-PCR handling suffices to score genotypes. ASQ is cost-effective because universal fluorescent probes negate the necessity of designing expensive probes for each locus.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::a65a6cbb8a698e715af16d327f80d144Test
https://pubmed.ncbi.nlm.nih.gov/26986823Test