RESUMEN
Germ line loss-of-function heterozygous mutations in the RUNX1 gene cause familial platelet disorder with associated myeloid malignancies (FPDMM) characterized by thrombocytopenia and a life-long risk of hematological malignancies. Although gene therapies are being considered as promising therapeutic options, current preclinical models do not recapitulate the human phenotype and are unable to elucidate the relative fitness of mutation-corrected and RUNX1-heterozygous mutant hematopoietic stem and progenitor cells (HSPCs) in vivo long term. We generated a rhesus macaque with an FPDMM competitive repopulation model using CRISPR/Cas9 nonhomologous end joining editing in the RUNX1 gene and the AAVS1 safe-harbor control locus. We transplanted mixed populations of edited autologous HSPCs and tracked mutated allele frequencies in blood cells. In both animals, RUNX1-edited cells expanded over time compared with AAVS1-edited cells. Platelet counts remained below the normal range in the long term. Bone marrows developed megakaryocytic dysplasia similar to human FPDMM, and CD34+ HSPCs showed impaired in vitro megakaryocytic differentiation, with a striking defect in polyploidization. In conclusion, the lack of a competitive advantage for wildtype or control-edited HSPCs over RUNX1 heterozygous-mutated HSPCs long term in our preclinical model suggests that gene correction approaches for FPDMM will be challenging, particularly to reverse myelodysplastic syndrome/ acute myeloid leukemia predisposition and thrombopoietic defects.
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Subunidad alfa 2 del Factor de Unión al Sitio Principal , Leucemia Mieloide Aguda , Animales , Humanos , Macaca mulatta , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Leucemia Mieloide Aguda/patología , Trombopoyesis , FenotipoRESUMEN
BACKGROUND: Genome editing using CRISPR/Cas9 has become a powerful tool in zebrafish to generate targeted gene knockouts models. However, its use for targeted knock-in remains challenging due to inefficient homology directed repair (HDR) pathway in zebrafish, highlighting the need for efficient and cost-effective screening methods. RESULTS: Here, we present our fluorescent PCR and capillary electrophoresis based screening approach for knock-in using a single-stranded oligodeoxynucleotide donor (ssODN) as a repair template for the targeted insertion of epitope tags, or single nucleotide changes to recapitulate pathogenic human alleles. For the insertion of epitope tags, we took advantage of the expected change in size of the PCR product. For point mutations, we combined fluorescent PCR with restriction fragment length polymorphism (RFLP) analysis to distinguish the fish with the knock-in allele. As a proof-of-principle, we present our data on the generation of fish lines with insertion of a FLAG tag at the tcnba locus, an HA tag at the gata2b locus, and a point mutation observed in Gaucher disease patients in the gba gene. Despite the low number of germline transmitting founders (1-5%), combining our screening methods with prioritization of founder fish by fin biopsies allowed us to establish stable knock-in lines by screening 12 or less fish per gene. CONCLUSIONS: We have established a robust pipeline for the generation of zebrafish models with precise integration of small DNA sequences and point mutations at the desired sites in the genome. Our screening method is very efficient and easy to implement as it is PCR-based and only requires access to a capillary sequencer.
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Mutación Puntual , Pez Cebra , Humanos , Animales , Pez Cebra/genética , Epítopos , InvestigaciónRESUMEN
Progressive pancytopenia is a common feature observed in DNA crosslink repair deficiency disorder, Fanconi anemia (FA). However, this phenotype has not been recapitulated in single FA gene knockout animal models. In this study, we analyzed hematological characteristics in zebrafish null mutants for two FA genes, fanca and fanco. In adult mutants, we demonstrate age-associated reduction in blood cell counts for all lineages, resembling progressive pancytopenia in FA patients. In larval mutants, we demonstrate vascular injury-induced thrombosis defects, particularly upon treatment with crosslinking agent diepoxybutane (DEB), indicating DNA damage induced inefficiency of thrombocytes.
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Anemia de Fanconi , Pancitopenia , Trombosis , Animales , Daño del ADN , Anemia de Fanconi/genética , Humanos , Pancitopenia/genética , Trombosis/genética , Pez CebraRESUMEN
ZRSR2 (zinc finger CCCH-type, RNA binding motif and serine/arginine rich 2) is an essential splicing factor involved in 3' splice-site recognition as a component of both the major and minor spliceosomes that mediate the splicing of U2-type (major) and U12-type (minor) introns, respectively. Studies of ZRSR2-depleted cell lines and ZRSR2-mutated patient samples revealed its essential role in the U12-dependent minor spliceosome. However, the role of ZRSR2 during embryonic development is not clear, as its function is compensated for by Zrsr1 in mice. Here, we utilized the zebrafish model to investigate the role of zrsr2 during embryonic development. Using CRISPR/Cas9 technology, we generated a zrsr2-knockout zebrafish line, termed zrsr2hg129/hg129 (p.Trp167Argfs*9) and examined embryo development in the homozygous mutant embryos. zrsr2hg129/hg129 embryos displayed multiple developmental defects starting at 4 days post fertilization (dpf) and died after 8 dpf, suggesting that proper Zrsr2 function is required during embryonic development. The global transcriptome analysis of 3 dpf zrsr2hg129/hg129 embryos revealed that the loss of Zrsr2 results in the downregulation of essential metabolic pathways and the aberrant retention of minor introns in about one-third of all minor intron-containing genes in zebrafish. Overall, our study has demonstrated that the role of Zrsr2 as a component of the minor spliceosome is conserved and critical for proper embryonic development in zebrafish.
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Empalme del ARN , Ribonucleoproteínas , Pez Cebra , Animales , Ratones , Desarrollo Embrionario , Intrones/genética , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Sitios de Empalme de ARN , Empalme del ARN/genética , Factores de Empalme de ARN/genética , Empalmosomas/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
DHX15, a DEAH box containing RNA helicase, is a splicing factor required for the last step of splicing. Recent studies identified a recurrent mutational hotspot, R222G, in DHX15 in â¼ 6% of acute myeloid leukemia (AML) patients that carry the fusion protein RUNX1-RUNX1T1 produced by t (8;21) (q22;q22). Studies using yeast mutants showed that substitution of G for the residue equivalent to R222 leads to loss of its helicase function, suggesting that it is a loss-of-function mutation. To elucidate the role of DHX15 during development, we established the first vertebrate knockout model with CRISPR/Cas9 in zebrafish. Our data showed that dhx15 expression is enriched in the brain, eyes, pectoral fin primordia, liver and intestinal bulb during embryonic development. Dhx15 deficiency leads to pleiotropic morphological phenotypes in homozygous mutant embryos starting at 3 days post fertilization (dpf) that result in lethality by 7 dpf, revealing an essential role during embryonic development. RNA-seq analysis suggested important roles of Dhx15 in chromatin and nucleosome assembly and regulation of the Mdm2-p53 pathway. Interestingly, exons corresponding to the alternate transcriptional start sites for tp53 and mdm2 were preferentially expressed in the mutant embryos, leading to significant upregulation of their alternate isoforms, Δ113p53 (orthologous to Δ133p53 isoform in human) and mdm2-P2 (isoform using distal promoter P2), respectively. We speculate that these alterations in the Mdm2-p53 pathway contribute to the development of AML in patients with t(8;21) and somatically mutated DHX15.
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Proteínas Proto-Oncogénicas c-mdm2/genética , ARN Helicasas/genética , Proteína p53 Supresora de Tumor/genética , Proteínas de Pez Cebra/genética , Empalme Alternativo , Animales , Animales Modificados Genéticamente , Humanos , Regiones Promotoras Genéticas , Isoformas de Proteínas , Proteínas Proto-Oncogénicas c-mdm2/biosíntesis , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , ARN Helicasas/metabolismo , Sitios de Empalme de ARN , Empalme del ARN , Factores de Empalme de ARN/genética , Sitio de Iniciación de la Transcripción , Activación Transcripcional , Proteína p53 Supresora de Tumor/metabolismo , Pez Cebra , Proteínas de Pez Cebra/metabolismoRESUMEN
KCTD7 is a member of the potassium channel tetramerization domain-containing protein family and has been associated with progressive myoclonic epilepsy (PME), characterized by myoclonus, epilepsy, and neurological deterioration. Here we report four affected individuals from two unrelated families in which we identified KCTD7 compound heterozygous single nucleotide variants through exome sequencing. RNAseq was used to detect a non-annotated splicing junction created by a synonymous variant in the second family. Whole-cell patch-clamp analysis of neuroblastoma cells overexpressing the patients' variant alleles demonstrated aberrant potassium regulation. While all four patients experienced many of the common clinical features of PME, they also showed variable phenotypes not previously reported, including dysautonomia, brain pathology findings including a significantly reduced thalamus, and the lack of myoclonic seizures. To gain further insight into the pathogenesis of the disorder, zinc finger nucleases were used to generate kctd7 knockout zebrafish. Kctd7 homozygous mutants showed global dysregulation of gene expression and increased transcription of c-fos, which has previously been correlated with seizure activity in animal models. Together these findings expand the known phenotypic spectrum of KCTD7-associated PME, report a new animal model for future studies, and contribute valuable insights into the disease.
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Epilepsias Mioclónicas Progresivas/genética , Canales de Potasio/genética , Animales , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Mutación , Epilepsias Mioclónicas Progresivas/fisiopatología , Linaje , Fenotipo , Pez CebraRESUMEN
Fanconi Anemia (FA) is a genomic instability syndrome resulting in aplastic anemia, developmental abnormalities, and predisposition to hematological and other solid organ malignancies. Mutations in genes that encode proteins of the FA pathway fail to orchestrate the repair of DNA damage caused by DNA interstrand crosslinks. Zebrafish harbor homologs for nearly all known FA genes. We used multiplexed CRISPR/Cas9-mediated mutagenesis to generate loss-of-function mutants for 17 FA genes: fanca, fancb, fancc, fancd1/brca2, fancd2, fance, fancf, fancg, fanci, fancj/brip1, fancl, fancm, fancn/palb2, fanco/rad51c, fancp/slx4, fancq/ercc4, fanct/ube2t, and two genes encoding FA-associated proteins: faap100 and faap24. We selected two indel mutations predicted to cause premature truncations for all but two of the genes, and a total of 36 mutant lines were generated for 19 genes. Generating two independent mutant lines for each gene was important to validate their phenotypic consequences. RT-PCR from homozygous mutant fish confirmed the presence of transcripts with indels in all genes. Interestingly, 4 of the indel mutations led to aberrant splicing, which may produce a different protein than predicted from the genomic sequence. Analysis of RNA is thus critical in proper evaluation of the consequences of the mutations introduced in zebrafish genome. We used fluorescent reporter assay, and western blots to confirm loss-of-function for several mutants. Additionally, we developed a DEB treatment assay by evaluating morphological changes in embryos and confirmed that homozygous mutants from all the FA genes that could be tested (11/17), displayed hypersensitivity and thus were indeed null alleles. Our multiplexing strategy helped us to evaluate 11 multiple gene knockout combinations without additional breeding. Homozygous zebrafish for all 19 single and 11 multi-gene knockouts were adult viable, indicating FA genes in zebrafish are generally not essential for early development. None of the mutant fish displayed gross developmental abnormalities except for fancp-/- fish, which were significantly smaller in length than their wildtype clutch mates. Complete female-to-male sex reversal was observed in knockouts for 12/17 FA genes, while partial sex reversal was seen for the other five gene knockouts. All adult females were fertile, and among the adult males, all were fertile except for the fancd1 mutants and one of the fancj mutants. We report here generation and characterization of zebrafish knockout mutants for 17 FA disease-causing genes, providing an integral resource for understanding the pathophysiology associated with the disrupted FA pathway.
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Anemia de Fanconi/genética , Pez Cebra/genética , Animales , Sistemas CRISPR-Cas , Daño del ADN , Anemia de Fanconi/fisiopatología , Femenino , Fertilidad/genética , Fertilidad/fisiología , Mutación del Sistema de Lectura , Técnicas de Inactivación de Genes , Humanos , Masculino , Fenotipo , Empalme del ARN/genética , Procesos de Determinación del Sexo/genética , Procesos de Determinación del Sexo/fisiología , Desarrollo Sexual/genética , Desarrollo Sexual/fisiología , Pez Cebra/crecimiento & desarrollo , Pez Cebra/fisiología , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/fisiologíaRESUMEN
Holoprosencephaly (HPE) is the most common congenital anomaly affecting the forebrain and face in humans and occurs as frequently as 1:250 conceptions or 1:10,000 livebirths. Sonic Hedgehog signaling molecule is one of the best characterized HPE genes that plays crucial roles in numerous developmental processes including midline neural patterning and craniofacial development. The Frizzled class G-protein coupled receptor Smoothened (SMO), whose signaling activity is tightly regulated, is the sole obligate transducer of Hedgehog-related signals. However, except for previous reports of somatic oncogenic driver mutations in human cancers (or mosaic tumors in rare syndromes), any potential disease-related role of SMO genetic variation in humans is largely unknown. To our knowledge, ours is the first report of a human hypomorphic variant revealed by functional testing of seven distinct nonsynonymous SMO variants derived from HPE molecular and clinical data. Here we describe several zebrafish bioassays developed and guided by a systems biology analysis. This analysis strategy, and detection of hypomorphic variation in human SMO, demonstrates the necessity of integrating the genomic variant findings in HPE probands with other components of the Hedgehog gene regulatory network in overall medical interpretations.
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Holoprosencefalia/genética , Holoprosencefalia/patología , Mutación/genética , Receptor Smoothened/química , Receptor Smoothened/genética , Secuencia de Aminoácidos , Animales , Mutación con Ganancia de Función/genética , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Mutación con Pérdida de Función/genética , Modelos Biológicos , Morfolinos/farmacología , Mutagénesis/genética , Fenotipo , Dominios Proteicos , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
Genetic variation in the highly conserved Sonic Hedgehog (SHH) gene is one of the most common genetic causes for the malformations of the brain and face in humans described as the holoprosencephaly clinical spectrum. However, only a minor fraction of known SHH variants have been experimentally proven to lead to abnormal function. Employing a phenotypic rescue assay with synthetic human messenger RNA variant constructs in shha-/- knockout zebrafish, we evaluated 104 clinically reported in-frame and missense SHH variants. Our data helped us to classify them into loss of function variants (31), hypomorphic variants (33), and nonpathogenic variants (40). We discuss the strengths and weaknesses of currently accepted predictors of variant deleteriousness and the American College of Medical Genetics and Genomics guidelines for variant interpretation in the context of this functional model; furthermore, we demonstrate the robustness of model systems such as zebrafish as a rapid method to resolve variants of uncertain significance.
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Sistemas CRISPR-Cas/genética , Variación Genética , Proteínas Hedgehog/genética , Holoprosencefalia/genética , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Alelos , Animales , Modelos Animales de Enfermedad , Familia , Estudios de Asociación Genética , Guías como Asunto , Humanos , Mutación con Pérdida de Función/genética , Mutación/genética , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Sociedades CientíficasRESUMEN
The use of CRISPR/Cas9 as a genome-editing tool in various model organisms has radically changed targeted mutagenesis. Here, we present a high-throughput targeted mutagenesis pipeline using CRISPR/Cas9 technology in zebrafish that will make possible both saturation mutagenesis of the genome and large-scale phenotyping efforts. We describe a cloning-free single-guide RNA (sgRNA) synthesis, coupled with streamlined mutant identification methods utilizing fluorescent PCR and multiplexed, high-throughput sequencing. We report germline transmission data from 162 loci targeting 83 genes in the zebrafish genome, in which we obtained a 99% success rate for generating mutations and an average germline transmission rate of 28%. We verified 678 unique alleles from 58 genes by high-throughput sequencing. We demonstrate that our method can be used for efficient multiplexed gene targeting. We also demonstrate that phenotyping can be done in the F1 generation by inbreeding two injected founder fish, significantly reducing animal husbandry and time. This study compares germline transmission data from CRISPR/Cas9 with those of TALENs and ZFNs and shows that efficiency of CRISPR/Cas9 is sixfold more efficient than other techniques. We show that the majority of published "rules" for efficient sgRNA design do not effectively predict germline transmission rates in zebrafish, with the exception of a GG or GA dinucleotide genomic match at the 5' end of the sgRNA. Finally, we show that predicted off-target mutagenesis is of low concern for in vivo genetic studies.
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Sistemas CRISPR-Cas , Marcación de Gen , Ensayos Analíticos de Alto Rendimiento , Fenotipo , Alelos , Animales , Técnicas de Inactivación de Genes , Marcación de Gen/métodos , Estudio de Asociación del Genoma Completo , Genómica , Células Germinativas/inmunología , Humanos , Mutagénesis , Sitios de Carácter Cuantitativo , ARN Guía de Kinetoplastida/genética , Eliminación de Secuencia , Pez CebraRESUMEN
OBJECTIVES: To characterise the clinical features, immune manifestations and molecular mechanisms in a recently described autoinflammatory disease caused by mutations in TRNT1, a tRNA processing enzyme, and to explore the use of cytokine inhibitors in suppressing the inflammatory phenotype. METHODS: We studied nine patients with biallelic mutations in TRNT1 and the syndrome of congenital sideroblastic anaemia with immunodeficiency, fevers and developmental delay (SIFD). Genetic studies included whole exome sequencing (WES) and candidate gene screening. Patients' primary cells were used for deep RNA and tRNA sequencing, cytokine profiling, immunophenotyping, immunoblotting and electron microscopy (EM). RESULTS: We identified eight mutations in these nine patients, three of which have not been previously associated with SIFD. Three patients died in early childhood. Inflammatory cytokines, mainly interleukin (IL)-6, interferon gamma (IFN-γ) and IFN-induced cytokines were elevated in the serum, whereas tumour necrosis factor (TNF) and IL-1ß were present in tissue biopsies of patients with active inflammatory disease. Deep tRNA sequencing of patients' fibroblasts showed significant deficiency of mature cytosolic tRNAs. EM of bone marrow and skin biopsy samples revealed striking abnormalities across all cell types and a mix of necrotic and normal-appearing cells. By immunoprecipitation, we found evidence for dysregulation in protein clearance pathways. In 4/4 patients, treatment with a TNF inhibitor suppressed inflammation, reduced the need for blood transfusions and improved growth. CONCLUSIONS: Mutations of TRNT1 lead to a severe and often fatal syndrome, linking protein homeostasis and autoinflammation. Molecular diagnosis in early life will be crucial for initiating anti-TNF therapy, which might prevent some of the severe disease consequences.
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Anemia Sideroblástica/genética , Antiinflamatorios/uso terapéutico , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Síndromes de Inmunodeficiencia/genética , Mutación , Nucleotidiltransferasas/genética , ARN de Transferencia/genética , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Adulto , Anemia Sideroblástica/sangre , Niño , Preescolar , Citocinas/sangre , Citocinas/genética , Discapacidades del Desarrollo/genética , Femenino , Enfermedades Genéticas Ligadas al Cromosoma X/sangre , Humanos , Inmunofenotipificación , Masculino , Linaje , Fenotipo , Factor de Necrosis Tumoral alfa/análisis , Secuenciación del ExomaRESUMEN
CRISPRz (http://research.nhgri.nih.gov/CRISPRz/) is a database of CRISPR/Cas9 target sequences that have been experimentally validated in zebrafish. Programmable RNA-guided CRISPR/Cas9 has recently emerged as a simple and efficient genome editing method in various cell types and organisms, including zebrafish. Because the technique is so easy and efficient in zebrafish, the most valuable asset is no longer a mutated fish (which has distribution challenges), but rather a CRISPR/Cas9 target sequence to the gene confirmed to have high mutagenic efficiency. With a highly active CRISPR target, a mutant fish can be quickly replicated in any genetic background anywhere in the world. However, sgRNA's vary widely in their activity and models for predicting target activity are imperfect. Thus, it is very useful to collect in one place validated CRISPR target sequences with their relative mutagenic activities. A researcher could then select a target of interest in the database with an expected activity. Here, we report the development of CRISPRz, a database of validated zebrafish CRISPR target sites collected from published sources, as well as from our own in-house large-scale mutagenesis project. CRISPRz can be searched using multiple inputs such as ZFIN IDs, accession number, UniGene ID, or gene symbols from zebrafish, human and mouse.
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Sistemas CRISPR-Cas , Bases de Datos Genéticas , ARN , Pez Cebra/genética , Animales , Marcación de Gen , Humanos , Ratones , Mutagénesis , Pez Cebra/embriologíaRESUMEN
Craniosynostosis presents either as a nonsyndromic congenital anomaly or as a finding in nearly 200 genetic syndromes. Our previous genome-wide association study of sagittal nonsyndromic craniosynostosis identified associations with variants downstream from BMP2 and intronic in BBS9. Because no coding variants in BMP2 were identified, we hypothesized that conserved non-coding regulatory elements may alter BMP2 expression. In order to identify and characterize noncoding regulatory elements near BMP2, two conserved noncoding regions near the associated region on chromosome 20 were tested for regulatory activity with a Renilla luciferase assay. For a 711 base pair noncoding fragment encompassing the most strongly associated variant, rs1884302, the luciferase assay showed that the risk allele (C) of rs1884302 drives higher expression of the reporter than the common allele (T). When this same DNA fragment was tested in zebrafish transgenesis studies, a strikingly different expression pattern of the green fluorescent reporter was observed depending on whether the transgenic fish had the risk (C) or the common (T) allele at rs1884302. The in vitro results suggest that altered BMP2 regulatory function at rs1884302 may contribute to the etiology of sagittal nonsyndromic craniosynostosis. The in vivo results indicate that differences in regulatory activity depend on the presence of a C or T allele at rs1884302.
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Proteína Morfogenética Ósea 2/genética , Anomalías Congénitas/genética , Craneosinostosis/genética , Predisposición Genética a la Enfermedad , Alelos , Animales , Animales Modificados Genéticamente/genética , Anomalías Congénitas/fisiopatología , Secuencia Conservada , Regulación de la Expresión Génica/genética , Estudio de Asociación del Genoma Completo , Humanos , Polimorfismo de Nucleótido Simple , Secuencias Reguladoras de Ácidos Nucleicos/genética , Pez Cebra/genéticaRESUMEN
CRISPR/Cas9 has emerged as a versatile genome-engineering tool that relies on a single guide RNA (sgRNA) and the Cas9 enzyme for genome editing. Simple, fast and economical methods to generate sgRNAs have made targeted mutagenesis routine in cultured cells, mice, zebrafish and other model systems. Pre-screening of sgRNAs for target efficacy is desirable both for successful mutagenesis and minimizing wasted animal husbandry on targets with poor activity. Here, we describe an easy, quick and cost-effective fluorescent polymerase chain reaction (PCR)-based method, CRISPR Somatic Tissue Activity Test (CRISPR-STAT), to determine target-specific efficiency of sgRNA. As a proof of principle, we validated our method using 28 sgRNAs with known and varied levels of germline transmission efficiency in zebrafish by analysis of their somatic activity in injected embryos. Our data revealed a strong positive correlation between the fluorescent PCR profiles of the injected embryos and the germline transmission efficiency. Furthermore, the assay was sensitive enough to evaluate multiplex gene targeting. This method is easy to implement by laboratories with access to a capillary sequencer. Although we validated the method using CRISPR/Cas9 and zebrafish, it can be applied to other model systems and other genome targeting nucleases.
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Sistemas CRISPR-Cas , Mutación INDEL , Reacción en Cadena de la Polimerasa/métodos , ARN/metabolismo , Animales , Fluorescencia , Pez Cebra/genéticaRESUMEN
BACKGROUND: Neutrophils are the predominant phagocytes that provide protection against bacterial and fungal infections. Genetically determined neutrophil disorders confer a predisposition to severe infections and reveal novel mechanisms that control vesicular trafficking, hematopoiesis, and innate immunity. METHODS: We clinically evaluated seven children from five families who had neutropenia, neutrophil dysfunction, bone marrow fibrosis, and nephromegaly. To identify the causative gene, we performed homozygosity mapping using single-nucleotide polymorphism arrays, whole-exome sequencing, immunoblotting, immunofluorescence, electron microscopy, a real-time quantitative polymerase-chain-reaction assay, immunohistochemistry, flow cytometry, fibroblast motility assays, measurements of apoptosis, and zebrafish models. Correction experiments were performed by transfecting mutant fibroblasts with the nonmutated gene. RESULTS: All seven affected children had homozygous mutations (Thr224Asn or Glu238Lys, depending on the child's ethnic origin) in VPS45, which encodes a protein that regulates membrane trafficking through the endosomal system. The level of VPS45 protein was reduced, as were the VPS45 binding partners rabenosyn-5 and syntaxin-16. The level of ß1 integrin was reduced on the surface of VPS45-deficient neutrophils and fibroblasts. VPS45-deficient fibroblasts were characterized by impaired motility and increased apoptosis. A zebrafish model of vps45 deficiency showed a marked paucity of myeloperoxidase-positive cells (i.e., neutrophils). Transfection of patient cells with nonmutated VPS45 corrected the migration defect and decreased apoptosis. CONCLUSIONS: Defective endosomal intracellular protein trafficking due to biallelic mutations in VPS45 underlies a new immunodeficiency syndrome involving impaired neutrophil function. (Funded by the National Human Genome Research Institute and others.).
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Síndromes de Inmunodeficiencia/genética , Neutropenia/congénito , Proteínas de Transporte Vesicular/genética , Animales , Niño , Endosomas/metabolismo , Homocigoto , Humanos , Síndromes de Inmunodeficiencia/congénito , Síndromes de Inmunodeficiencia/inmunología , Mutación , Neutropenia/genética , Neutrófilos/fisiología , Fenotipo , Transporte de Proteínas , Proteínas de Transporte Vesicular/metabolismo , Pez CebraRESUMEN
CBFß and RUNX1 form a DNA-binding heterodimer and are both required for hematopoietic stem cell (HSC) generation in mice. However, the exact role of CBFß in the production of HSCs remains unclear. Here, we generated and characterized 2 zebrafish cbfb null mutants. The cbfb(-/-) embryos underwent primitive hematopoiesis and developed transient erythromyeloid progenitors, but they lacked definitive hematopoiesis. Unlike runx1 mutants, in which HSCs are not formed, nascent, runx1(+)/c-myb(+) HSCs were formed in cbfb(-/-) embryos. However, the nascent HSCs were not released from the aorta-gonad-mesonephros (AGM) region, as evidenced by the accumulation of runx1(+) cells in the AGM that could not enter circulation. Moreover, wild-type embryos treated with an inhibitor of RUNX1-CBFß interaction, Ro5-3335, phenocopied the hematopoietic defects in cbfb(-/-) mutants, rather than those in runx1(-/-) mutants. Finally, we found that cbfb was downstream of the Notch pathway during HSC development. Our data suggest that runx1 and cbfb are required at 2 different steps during early HSC development. CBFß is not required for nascent HSC emergence but is required for the release of HSCs from AGM into circulation. Our results also indicate that RUNX1 can drive the emergence of nascent HSCs in the AGM without its heterodimeric partner CBFß.
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Factor de Unión a CCAAT/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Hematopoyesis/fisiología , Células Madre Hematopoyéticas/metabolismo , Proteínas de Pez Cebra/genética , Animales , Factor de Unión a CCAAT/metabolismo , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Técnicas de Inactivación de Genes , Hibridación in Situ , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Pez Cebra , Proteínas de Pez Cebra/metabolismoRESUMEN
Since the beginnings of domestication, the craniofacial architecture of the domestic dog has morphed and radiated to human whims. By beginning to define the genetic underpinnings of breed skull shapes, we can elucidate mechanisms of morphological diversification while presenting a framework for understanding human cephalic disorders. Using intrabreed association mapping with museum specimen measurements, we show that skull shape is regulated by at least five quantitative trait loci (QTLs). Our detailed analysis using whole-genome sequencing uncovers a missense mutation in BMP3. Validation studies in zebrafish show that Bmp3 function in cranial development is ancient. Our study reveals the causal variant for a canine QTL contributing to a major morphologic trait.
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Proteína Morfogenética Ósea 3/genética , Craneosinostosis/genética , Perros/genética , Variación Genética , Sitios de Carácter Cuantitativo , Cráneo/metabolismo , Animales , Evolución Biológica , Cruzamiento , Mapeo Cromosómico , Estudio de Asociación del Genoma Completo , Genotipo , Humanos , Mutación Missense , Mascotas , Fenotipo , Cráneo/anatomía & histología , Pez Cebra/genéticaRESUMEN
Generation of zebrafish (Danio rerio) models with targeted insertion of epitope tags and point mutations is highly desirable for functional genomics and disease modeling studies. Currently, CRISPR/Cas9-mediated knock-in is the method of choice for insertion of exogeneous sequences by providing a repair template for homology-directed repair (HDR). A major hurdle in generating knock-in models is the labor and cost involved in screening of injected fish to identify the precise knock-in events due to low efficiency of the HDR pathway in zebrafish. Thus, we developed fluorescent PCR-based high-throughput screening methods for precise knock-in of epitope tags and point mutations in zebrafish. Here, we provide a step-by-step guide that describes selection of an active sgRNA near the intended knock-in site, design of single-stranded oligonucleotide (ssODN) templates for HDR, quick validation of somatic knock-in using injected embryos, and screening for germline transmission of precise knock-in events to establish stable lines. Our screening method relies on the size-based separation of all fragments in an amplicon by fluorescent PCR and capillary electrophoresis, thus providing a robust and cost-effective strategy. Although we present the use of this protocol for insertion of epitope tags and point mutations, it can be used for insertion of any small DNA fragments (e.g., LoxP sites, in-frame codons). Furthermore, the screening strategy described here can be used to screen for precise knock-in of small DNA sequences in any model system, as PCR amplification of the target region is its only requirement. Key features This protocol expands the use of fluorescent PCR and CRISPR-STAT for screening of precise knock-in of small insertions and point mutations in zebrafish. Allows validation of selected sgRNA and HDR template within two weeks by somatic knock-in screening. Allows robust screening of point mutations by combining restriction digest with CRISPR-STAT. Graphical overview Overview of the three-phase knock-in pipeline in zebrafish (created with BioRender.com).
RESUMEN
Distinguishing individuals or small groups is essential for many experiments. The regenerative properties of zebrafish make traditional marking methods for rodent models (e.g., tattoos, tissue clipping) ineffective. Fluorescent Visible Implant Elastomer (VIE) can permanently mark juvenile and adult zebrafish but to date no marking technique has been described for larval stage zebrafish. This protocol, Zebrafish Injectable Plastic for Identification Tagging (ZIP IT), utilizes VIE in zebrafish as early as 2 days post fertilization (dpf) using standard microinjection methods and direct injection using an insulin syringe at one month. Larval zebrafish between 2 and 7 dpf were injected in the dorsal musculature. At one month, retention and visibility of the VIE was observed in 72% of the injected fish with no effect on growth; however, a variable change in mortality was observed, generally higher than the uninjected fish. This demonstrates that VIE can be used in very early stages of fish development, providing the first procedure to track individuals or groups within a larger population. Subcutaneous injection of juvenile zebrafish starting at one month has greater than 99%-mark retention and visibility with very low mortality. The combination of larvae and juvenile VIE injections also provide a powerful tool to track and gather data from marked fish throughout their lifespan.â¢We present a method for tagging individuals or groups of zebrafish at most life stages (larvae or juvenile/adult) with Visible Implant Elastomer (VIE).â¢The larvae and juvenile injection procedures can be combined so that an individual fish can be tagged for its entire lifetime. Larvae injections become significantly less visible after one month; however, the fish can be reinjected using the juvenile procedure, thus allowing continuity of the visible mark.â¢This protocol was empirically built on the "Evaluation of VIE tags in Zebrafish (Danio rerio)" by Hohn and Petrie-Hanson (2013) and expanded to include larvae and a small batch elastomer mixing technique.
RESUMEN
Pigment patterns and skin appendages are prominent features of vertebrate skin. In zebrafish, regularly patterned pigment stripes and an array of calcified scales form simultaneously in the skin during post-embryonic development. Understanding the mechanisms that regulate stripe patterning and scale morphogenesis may lead to the discovery of fundamental mechanisms that govern the development of animal form. To learn about cell types and signaling interactions that govern skin patterning and morphogenesis, we generated and analyzed single-cell transcriptomes of skin from wild-type fish as well as fish having genetic or transgenically induced defects in squamation or pigmentation. These data reveal a previously undescribed population of epidermal cells that express transcripts encoding enamel matrix proteins, suggest hormonal control of epithelial-mesenchymal signaling, clarify the signaling network that governs scale papillae development, and identify a critical role for the hypodermis in supporting pigment cell development. Additionally, these comprehensive single-cell transcriptomic data representing skin phenotypes of biomedical relevance should provide a useful resource for accelerating the discovery of mechanisms that govern skin development and homeostasis.