RESUMEN
Genetic variants in ZNF536 contribute to the risk for neuropsychiatric disorders such as schizophrenia, autism, and others. The role of this putative transcriptional repressor in brain development and function is, however, largely unknown. We generated znf536 knockout (KO) zebrafish and studied their behavior, brain anatomy, and brain function. Larval KO zebrafish showed a reduced ability to compete for food, resulting in decreased total body length and size. This phenotype can be rescued by segregating the homozygous KO larvae from their wild-type and heterozygous siblings, enabling studies of adult homozygous KO animals. In adult KO zebrafish, we observed significant reductions in anxiety-like behavior and social interaction. These znf536 KO zebrafish have decreased cerebellar volume, corresponding to decreased populations of specific neuronal cells, especially in the valvular cerebelli (Va). Finally, using a Tg[mbp:mgfp] line, we identified a previously undetected myelin structure located bilaterally within the Va, which also displayed a reduction in volume and disorganization in KO zebrafish. These findings indicate an important role for ZNF536 in brain development and implicate the cerebellum in the pathophysiology of neuropsychiatric disorders.
Asunto(s)
Cerebelo , Pez Cebra , Animales , Pez Cebra/genética , Pez Cebra/metabolismo , Animales Modificados Genéticamente/metabolismo , Cerebelo/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Encéfalo/metabolismoRESUMEN
DISC1 is a genetic risk factor for multiple psychiatric disorders. Compared to the dozens of murine Disc1 models, there is a paucity of zebrafish disc1 models-an organism amenable to high-throughput experimentation. We conducted the longitudinal neurobehavioral analysis of disc1 mutant zebrafish across key stages of life. During early developmental stages, disc1 mutants exhibited abrogated behavioral responses to sensory stimuli across multiple testing platforms. Moreover, during exposure to an acoustic sensory stimulus, loss of disc1 resulted in the abnormal activation of neurons in the pallium, cerebellum, and tectum-anatomical sites involved in the integration of sensory perception and motor control. In adulthood, disc1 mutants exhibited sexually dimorphic reduction in anxiogenic behavior in novel paradigms. Together, these findings implicate disc1 in sensorimotor processes and the genesis of anxiogenic behaviors, which could be exploited for the development of novel treatments in addition to investigating the biology of sensorimotor transformation in the context of disc1 deletion.
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CRISPR-associated transposons (CASTs) are natural RNA-directed transposition systems. We demonstrate that transposon protein TniQ plays a central role in promoting R-loop formation by RNA-guided DNA-targeting modules. TniQ residues, proximal to CRISPR RNA (crRNA), are required for recognizing different crRNA categories, revealing an unappreciated role of TniQ to direct transposition into different classes of crRNA targets. To investigate adaptations allowing CAST elements to utilize attachment sites inaccessible to CRISPR-Cas surveillance complexes, we compared and contrasted PAM sequence requirements in both I-F3b CAST and I-F1 CRISPR-Cas systems. We identify specific amino acids that enable a wider range of PAM sequences to be accommodated in I-F3b CAST elements compared with I-F1 CRISPR-Cas, enabling CAST elements to access attachment sites as sequences drift and evade host surveillance. Together, this evidence points to the central role of TniQ in facilitating the acquisition of CRISPR effector complexes for RNA-guided DNA transposition.
Asunto(s)
Proteínas Asociadas a CRISPR , ARN , ADN/genética , Sistemas CRISPR-Cas , Proteínas Asociadas a CRISPR/genéticaRESUMEN
The zebrafish telencephalon is composed of highly specialized subregions that regulate complex behaviors such as learning, memory, and social interactions. The transcriptional signatures of the neuronal cell types in the telencephalon and the timeline of their emergence from larva to adult remain largely undescribed. Using an integrated analysis of single-cell transcriptomes of approximately 64,000 cells obtained from 6-day-postfertilization (dpf), 15-dpf, and adult telencephalon, we delineated nine main neuronal cell types in the pallium and eight in the subpallium and nominated novel marker genes. Comparing zebrafish and mouse neuronal cell types revealed both conserved and absent types and marker genes. Mapping of cell types onto a spatial larval reference atlas created a resource for anatomical and functional studies. Using this multiage approach, we discovered that although most neuronal subtypes are established early in the 6-dpf fish, some emerge or expand in number later in development. Analyzing the samples from each age separately revealed further complexity in the data, including several cell types that expand substantially in the adult forebrain and do not form clusters at the larval stages. Together, our work provides a comprehensive transcriptional analysis of the cell types in the zebrafish telencephalon and a resource for dissecting its development and function.
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Transcriptoma , Pez Cebra , Animales , Ratones , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Neuronas/metabolismo , Telencéfalo/metabolismoRESUMEN
BACKGROUND: In the past decade, the zebrafish community has widely embraced targeted mutagenesis technologies, resulting in an abundance of mutant lines. While many lines have proven to be useful for investigating gene function, many have also shown no apparent phenotype, or phenotypes not of interest to the originating lab. In order for labs to document and share information about these lines, we have created ZebraShare as a new resource offered within ZFIN. METHODS: ZebraShare involves a form-based submission process generated by ZFIN. The ZebraShare interface (https://zfin.org/action/zebrashare) can be accessed on ZFIN under "Submit Data". Users download the Submission Workbook and complete the required fields, then submit the completed workbook with associated images and captions, generating a new ZFIN publication record. ZFIN curators add the submitted phenotype and mutant information to the ZFIN database, provide mapping information about mutations, and cross reference this information across the appropriate ZFIN databases. We present here examples of ZebraShare submissions, including phf21aa, kdm1a, ctnnd1, snu13a, and snu13b mutant lines. RESULTS: Users can find ZebraShare submissions by searching ZFIN for specific alleles or line designations, just as for alleles submitted through the normal process. We present several potential examples of submission types to ZebraShare including a phenotypic mutants, mildly phenotypic, and early lethal mutants. Mutants for kdm1a show no apparent skeletal phenotype, and phf21aa mutants show only a mild skeletal phenotype, yet these genes have specific human disease relevance and therefore may be useful for further studies. The p120-catenin encoding gene, ctnnd1, was knocked out to investigate a potential role in brain development or function. The homozygous ctnnd1 mutant disintegrates during early somitogenesis and the heterozygote has localized defects, revealing vital roles in early development. Two snu13 genes were knocked out to investigate a role in muscle formation. The snu13a;snu13b double mutant has an early embryonic lethal phenotype, potentially related to a proposed role in the core splicing complex. In each example, the mutants submitted to ZebraShare display phenotypes that are not ideally suited to their originating lab's project directions but may be of great relevance to other researchers. CONCLUSION: ZebraShare provides an opportunity for researchers to directly share information about mutant lines within ZFIN, which is widely used by the community as a central database of information about zebrafish lines. Submissions of alleles with a phenotypic or unexpected phenotypes is encouraged to promote collaborations, disseminate lines, reduce redundancy of effort and to promote efficient use of time and resources. We anticipate that as submissions to ZebraShare increase, they will help build an ultimately more complete picture of zebrafish genetics and development.
RESUMEN
High-throughput behavioral phenotyping is critical to genetic or chemical screening approaches. Zebrafish larvae are amenable to high-throughput behavioral screening because of their rapid development, small size, and conserved vertebrate brain architecture. Existing commercial behavioral phenotyping systems are expensive and not easily modified for new assays. Here, we describe a modular, highly adaptable, and low-cost system. Along with detailed assembly and operation instructions, we provide data acquisition software and a robust, parallel analysis pipeline. We validate our approach by analyzing stimulus response profiles in larval zebrafish, confirming prepulse inhibition phenotypes of two previously isolated mutants, and highlighting best practices for growing larvae prior to behavioral testing. Our new design thus allows rapid construction and streamlined operation of many large-scale behavioral setups with minimal resources and fabrication expertise, with broad applications to other aquatic organisms.
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Genomic studies have identified hundreds of candidate genes near loci associated with risk for schizophrenia. To define candidates and their functions, we mutated zebrafish orthologs of 132 human schizophrenia-associated genes. We created a phenotype atlas consisting of whole-brain activity maps, brain structural differences, and profiles of behavioral abnormalities. Phenotypes were diverse but specific, including altered forebrain development and decreased prepulse inhibition. Exploration of these datasets identified promising candidates in more than 10 gene-rich regions, including the magnesium transporter cnnm2 and the translational repressor gigyf2, and revealed shared anatomical sites of activity differences, including the pallium, hypothalamus, and tectum. Single-cell RNA sequencing uncovered an essential role for the understudied transcription factor znf536 in the development of forebrain neurons implicated in social behavior and stress. This phenotypic landscape of schizophrenia-associated genes prioritizes more than 30 candidates for further study and provides hypotheses to bridge the divide between genetic association and biological mechanism.
Asunto(s)
Esquizofrenia/genética , Esquizofrenia/fisiopatología , Animales , Encéfalo , Corteza Cerebral , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/genética , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Fenotipo , Polimorfismo de Nucleótido Simple/genética , Pez Cebra/genéticaRESUMEN
Copy-number variants of chromosome 16 region 16p11.2 are linked to neuropsychiatric disorders and are among the most prevalent in autism spectrum disorders. Of many 16p11.2 genes, Kctd13 has been implicated as a major driver of neurodevelopmental phenotypes. The function of KCTD13 in the mammalian brain, however, remains unknown. Here we delete the Kctd13 gene in mice and demonstrate reduced synaptic transmission. Reduced synaptic transmission correlates with increased levels of Ras homolog gene family, member A (RhoA), a KCTD13/CUL3 ubiquitin ligase substrate, and is reversed by RhoA inhibition, suggesting increased RhoA as an important mechanism. In contrast to a previous knockdown study, deletion of Kctd13 or kctd13 does not increase brain size or neurogenesis in mice or zebrafish, respectively. These findings implicate Kctd13 in the regulation of neuronal function relevant to neuropsychiatric disorders and clarify the role of Kctd13 in neurogenesis and brain size. Our data also reveal a potential role for RhoA as a therapeutic target in disorders associated with KCTD13 deletion.
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Encéfalo/metabolismo , Proteínas Portadoras/metabolismo , Eliminación de Gen , Transmisión Sináptica/genética , Proteínas de Pez Cebra/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Animales , Trastorno del Espectro Autista/genética , Trastorno del Espectro Autista/psicología , Trastorno Autístico/genética , Trastorno Autístico/psicología , Encéfalo/anatomía & histología , Encéfalo/citología , Encéfalo/patología , Región CA1 Hipocampal/metabolismo , Región CA1 Hipocampal/patología , Proteínas Portadoras/genética , Deleción Cromosómica , Trastornos de los Cromosomas/genética , Trastornos de los Cromosomas/psicología , Cromosomas Humanos Par 16/genética , Proteínas Cullin/metabolismo , Femenino , Discapacidad Intelectual/genética , Discapacidad Intelectual/psicología , Masculino , Ratones , Herencia Multifactorial/genética , Neurogénesis/genética , Tamaño de los Órganos/genética , Reproducibilidad de los Resultados , Transmisión Sináptica/efectos de los fármacos , Complejos de Ubiquitina-Proteína Ligasa , Pez Cebra , Proteínas de Pez Cebra/genética , Proteínas de Unión al GTP rho/antagonistas & inhibidores , Proteína de Unión al GTP rhoARESUMEN
The CRISPR/Cas system uses guide RNAs (gRNAs) to direct sequence-specific DNA cleavage. Not every gRNA elicits cleavage and the mechanisms that govern gRNA activity have not been resolved. Low activity could result from either failure to form a functional Cas9-gRNA complex or inability to recognize targets in vivo. Here we show that both phenomena influence Cas9 activity by comparing mutagenesis rates in zebrafish embryos with in vitro cleavage assays. In vivo, our results suggest that genomic factors such as CTCF inhibit mutagenesis. Comparing near-identical gRNA sequences with different in vitro activities reveals that internal gRNA interactions reduce cleavage. Even though gRNAs containing these structures do not yield cleavage-competent complexes, they can compete with active gRNAs for binding to Cas9. These results reveal that both genomic context and internal gRNA interactions can interfere with Cas9-mediated cleavage and illuminate previously uncharacterized features of Cas9-gRNA complex formation.
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Proteínas Asociadas a CRISPR/metabolismo , ARN Guía de Kinetoplastida/metabolismo , Animales , Secuencia de Bases , Genoma , Conformación de Ácido Nucleico , ARN Guía de Kinetoplastida/química , Pez CebraRESUMEN
Error-prone repair of DNA double-strand breaks (DSBs) has been postulated to occur through classical non-homologous end joining (NHEJ) in systems ranging from nematode somatic tissues to zebrafish embryos. Contrary to this model, we show that zebrafish embryos mutant for DNA polymerase theta (Polq), a critical component of alternative end joining (alt-EJ), cannot repair DSBs induced by CRISPR/Cas9 or ionizing radiation. In the absence of DSBs, polq mutants are phenotypically normal, but they do not survive mutagenesis and display dramatic differences in the mutation profiles compared with the wild-type. These results show that alt-EJ repair is essential and dominant during the early development of a vertebrate.
RESUMEN
In just 3 years CRISPR genome editing has transformed biology, and its popularity and potency continue to grow. New CRISPR effectors and rules for locating optimum targets continue to be reported, highlighting the need for computational CRISPR targeting tools to compile these rules and facilitate target selection and design. CHOPCHOP is one of the most widely used web tools for CRISPR- and TALEN-based genome editing. Its overarching principle is to provide an intuitive and powerful tool that can serve both novice and experienced users. In this major update we introduce tools for the next generation of CRISPR advances, including Cpf1 and Cas9 nickases. We support a number of new features that improve the targeting power, usability and efficiency of CHOPCHOP. To increase targeting range and specificity we provide support for custom length sgRNAs, and we evaluate the sequence composition of the whole sgRNA and its surrounding region using models compiled from multiple large-scale studies. These and other new features, coupled with an updated interface for increased usability and support for a continually growing list of organisms, maintain CHOPCHOP as one of the leading tools for CRISPR genome editing. CHOPCHOP v2 can be found at http://chopchop.cbu.uib.no.
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Proteínas Bacterianas/genética , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Endonucleasas/genética , Genoma , ARN Guía de Kinetoplastida/síntesis química , Programas Informáticos , Animales , Proteínas Bacterianas/metabolismo , Proteína 9 Asociada a CRISPR , Desoxirribonucleasa I/genética , Desoxirribonucleasa I/metabolismo , Endonucleasas/metabolismo , Edición Génica , Humanos , Almacenamiento y Recuperación de la Información , Internet , Motivos de Nucleótidos , ARN Guía de Kinetoplastida/genética , Nucleasas de los Efectores Tipo Activadores de la Transcripción/genética , Nucleasas de los Efectores Tipo Activadores de la Transcripción/metabolismoRESUMEN
We describe the identification and characterization of novel homing endonucleases using genome database mining to identify putative target sites, followed by high throughput activity screening in a bacterial selection system. We characterized the substrate specificity and kinetics of these endonucleases by monitoring DNA cleavage events with deep sequencing. The endonuclease specificities revealed by these experiments can be partially recapitulated using 3D structure-based computational models. Analysis of these models together with genome sequence data provide insights into how alternative endonuclease specificities were generated during natural evolution.
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Endodesoxirribonucleasas/metabolismo , Secuencia de Bases , Simulación por Computador , ADN/química , División del ADN , Endodesoxirribonucleasas/química , Secuenciación de Nucleótidos de Alto Rendimiento , Modelos Moleculares , Análisis de Secuencia de ADN , Especificidad por SustratoRESUMEN
The CRISPR/Cas9 system has been implemented in a variety of model organisms to mediate site-directed mutagenesis. A wide range of mutation rates has been reported, but at a limited number of genomic target sites. To uncover the rules that govern effective Cas9-mediated mutagenesis in zebrafish, we targeted over a hundred genomic loci for mutagenesis using a streamlined and cloning-free method. We generated mutations in 85% of target genes with mutation rates varying across several orders of magnitude, and identified sequence composition rules that influence mutagenesis. We increased rates of mutagenesis by implementing several novel approaches. The activities of poor or unsuccessful single-guide RNAs (sgRNAs) initiating with a 5' adenine were improved by rescuing 5' end homogeneity of the sgRNA. In some cases, direct injection of Cas9 protein/sgRNA complex further increased mutagenic activity. We also observed that low diversity of mutant alleles led to repeated failure to obtain frame-shift mutations. This limitation was overcome by knock-in of a stop codon cassette that ensured coding frame truncation. Our improved methods and detailed protocols make Cas9-mediated mutagenesis an attractive approach for labs of all sizes.
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Mutagénesis , Oligonucleótidos/genética , ARN Guía de Kinetoplastida/genética , Alelos , Animales , Frecuencia de los Genes , Humanos , Mutación INDEL , Tasa de Mutación , ARN Guía de Kinetoplastida/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
Homing endonucleases (HEs) can be used to induce targeted genome modification to reduce the fitness of pathogen vectors such as the malaria-transmitting Anopheles gambiae and to correct deleterious mutations in genetic diseases. We describe the creation of an extensive set of HE variants with novel DNA cleavage specificities using an integrated experimental and computational approach. Using computational modeling and an improved selection strategy, which optimizes specificity in addition to activity, we engineered an endonuclease to cleave in a gene associated with Anopheles sterility and another to cleave near a mutation that causes pyruvate kinase deficiency. In the course of this work we observed unanticipated context-dependence between bases which will need to be mechanistically understood for reprogramming of specificity to succeed more generally.
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Evolución Molecular Dirigida/métodos , Endodesoxirribonucleasas/química , Endodesoxirribonucleasas/metabolismo , Ingeniería de Proteínas/métodos , Animales , Anopheles/genética , Bacterias/genética , Biología Computacional , División del ADN , Endodesoxirribonucleasas/genética , Genes de Insecto , Modelos Moleculares , Especificidad por SustratoRESUMEN
Combinatorial sequence optimization for protein design requires libraries of discrete side-chain conformations. The discreteness of these libraries is problematic, particularly for long, polar side chains, since favorable interactions can be missed. Previously, an approach to loop remodeling where protein backbone movement is directed by side-chain rotamers predicted to form interactions previously observed in native complexes (termed "motifs") was described. Here, we show how such motif libraries can be incorporated into combinatorial sequence optimization protocols and improve native complex recapitulation. Guided by the motif rotamer searches, we made improvements to the underlying energy function, increasing recapitulation of native interactions. To further test the methods, we carried out a comprehensive experimental scan of amino acid preferences in the I-AniI protein-DNA interface and found that many positions tolerated multiple amino acids. This sequence plasticity is not observed in the computational results because of the fixed-backbone approximation of the model. We improved modeling of this diversity by introducing DNA flexibility and reducing the convergence of the simulated annealing algorithm that drives the design process. In addition to serving as a benchmark, this extensive experimental data set provides insight into the types of interactions essential to maintain the function of this potential gene therapy reagent.
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Proteínas de Unión al ADN/química , ADN/química , Estructura Terciaria de Proteína , Algoritmos , Aminoácidos/química , Simulación por Computador , ADN/metabolismo , Modelos Moleculares , Conformación de Ácido Nucleico , Unión Proteica , Conformación Proteica , Proteínas/química , Proteínas/metabolismoRESUMEN
Homing endonucleases have great potential as tools for targeted gene therapy and gene correction, but identifying variants of these enzymes capable of cleaving specific DNA targets of interest is necessary before the widespread use of such technologies is possible. We identified homologues of the LAGLIDADG homing endonuclease I-AniI and their putative target insertion sites by BLAST searches followed by examination of the sequences of the flanking genomic regions. Amino acid substitutions in these homologues that were located close to the target site DNA, and thus potentially conferring differences in target specificity, were grafted onto the I-AniI scaffold. Many of these grafts exhibited novel and unexpected specificities. These findings show that the information present in genomic data can be exploited for endonuclease specificity redesign.
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ADN/genética , Endodesoxirribonucleasas , Genoma/genética , Mapeo RestrictivoRESUMEN
Genetic methods of manipulating or eradicating disease vector populations have long been discussed as an attractive alternative to existing control measures because of their potential advantages in terms of effectiveness and species specificity. The development of genetically engineered malaria-resistant mosquitoes has shown, as a proof of principle, the possibility of targeting the mosquito's ability to serve as a disease vector. The translation of these achievements into control measures requires an effective technology to spread a genetic modification from laboratory mosquitoes to field populations. We have suggested previously that homing endonuclease genes (HEGs), a class of simple selfish genetic elements, could be exploited for this purpose. Here we demonstrate that a synthetic genetic element, consisting of mosquito regulatory regions and the homing endonuclease gene I-SceI, can substantially increase its transmission to the progeny in transgenic mosquitoes of the human malaria vector Anopheles gambiae. We show that the I-SceI element is able to invade receptive mosquito cage populations rapidly, validating mathematical models for the transmission dynamics of HEGs. Molecular analyses confirm that expression of I-SceI in the male germline induces high rates of site-specific chromosomal cleavage and gene conversion, which results in the gain of the I-SceI gene, and underlies the observed genetic drive. These findings demonstrate a new mechanism by which genetic control measures can be implemented. Our results also show in principle how sequence-specific genetic drive elements like HEGs could be used to take the step from the genetic engineering of individuals to the genetic engineering of populations.
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Anopheles/genética , Ingeniería Genética , Insectos Vectores/genética , Control de Mosquitos/métodos , Animales , Animales Modificados Genéticamente , Desoxirribonucleasas de Localización Especificada Tipo II/genética , Femenino , Genes Reporteros/genética , Genotipo , Masculino , Datos de Secuencia Molecular , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Enzymes use substrate-binding energy both to promote ground-state association and to stabilize the reaction transition state selectively. The monomeric homing endonuclease I-AniI cleaves with high sequence specificity in the centre of a 20-base-pair (bp) DNA target site, with the amino (N)-terminal domain of the enzyme making extensive binding interactions with the left (-) side of the target site and the similarly structured carboxy (C)-terminal domain interacting with the right (+) side. Here we show that, despite the approximate twofold symmetry of the enzyme-DNA complex, there is almost complete segregation of interactions responsible for substrate binding to the (-) side of the interface and interactions responsible for transition-state stabilization to the (+) side. Although single base-pair substitutions throughout the entire DNA target site reduce catalytic efficiency, mutations in the (-) DNA half-site almost exclusively increase the dissociation constant (K(D)) and the Michaelis constant under single-turnover conditions (K(M)*), and those in the (+) half-site primarily decrease the turnover number (k(cat)*). The reduction of activity produced by mutations on the (-) side, but not mutations on the (+) side, can be suppressed by tethering the substrate to the endonuclease displayed on the surface of yeast. This dramatic asymmetry in the use of enzyme-substrate binding energy for catalysis has direct relevance to the redesign of endonucleases to cleave genomic target sites for gene therapy and other applications. Computationally redesigned enzymes that achieve new specificities on the (-) side do so by modulating K(M)*, whereas redesigns with altered specificities on the (+) side modulate k(cat)*. Our results illustrate how classical enzymology and modern protein design can each inform the other.