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1.
Cell ; 177(2): 478-491.e20, 2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30929901

RESUMO

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.


Assuntos
Esquizofrenia/genética , Esquizofrenia/fisiopatologia , Animais , Encéfalo , Córtex Cerebral , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Peixe-Zebra/genética
2.
Mol Cell ; 83(11): 1827-1838.e6, 2023 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-37267904

RESUMO

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.


Assuntos
Proteínas Associadas a CRISPR , RNA , DNA/genética , Sistemas CRISPR-Cas , Proteínas Associadas a CRISPR/genética
3.
Genome Res ; 33(4): 658-671, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-37072188

RESUMO

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.


Assuntos
Transcriptoma , Peixe-Zebra , Animais , Camundongos , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Neurônios/metabolismo , Telencéfalo/metabolismo
4.
Mol Psychiatry ; 28(9): 3769-3781, 2023 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-37794116

RESUMO

Microdeletion of a 3Mb region encompassing 45 protein-coding genes at chromosome 22q11.2 (22q11.2DS) predisposes individuals to multiple neurodevelopmental disorders and is one of the greatest genetic risk factors for schizophrenia. Defective mitochondrial function has been hypothesized to contribute to 22q11.2DS pathogenesis; however, which of the six mitochondrial genes contribute to neurodevelopmental phenotypes and their underlying mechanisms remain unresolved. To systematically test 22q11.2DS genes for functional roles in neurodevelopment and behavior, we generated genetic mutants for each of the 37 conserved zebrafish orthologs and performed high throughput behavioral phenotyping using seven behavioral assays. Through this unbiased approach, we identified five single-gene mutants with partially overlapping behavioral phenotypes. Two of these genes, mrpl40 and prodha, encode for mitochondrial proteins and, similar to what we observed in mrpl40 and prodha mutants, pharmacologic inhibition of mitochondrial function during development results in microcephaly. Single mutant analysis shows that both mrpl40 and prodha mutants display aberrant neural stem and progenitor cell proliferation, with each gene regulating distinct cell populations. Finally, double mutants for both mrpl40 and prodha display aggravated behavioral phenotypes and neural stem and progenitor cell analysis reveals a previously unrecognized partially redundant role for mrpl40 and prodha in regulating radial glia-like cell proliferation. Combined, our results demonstrate a critical role for mitochondrial function in neural stem and progenitor cell populations in the developing vertebrate brain and provide compelling evidence that mitochondrial dysfunction during neurodevelopment is linked to brain volume and behavioral phenotypes observed in models of 22q11.2DS.


Assuntos
Síndrome de DiGeorge , Esquizofrenia , Animais , Humanos , Síndrome de DiGeorge/genética , Síndrome de DiGeorge/patologia , Proteínas Mitocondriais , Peixe-Zebra , Esquizofrenia/genética , Encéfalo/patologia
5.
Nature ; 551(7679): 227-231, 2017 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-29088697

RESUMO

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.


Assuntos
Encéfalo/metabolismo , Proteínas de Transporte/metabolismo , Deleção de Genes , Transmissão Sináptica/genética , Proteínas de Peixe-Zebra/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Transtorno do Espectro Autista/genética , Transtorno do Espectro Autista/psicologia , Transtorno Autístico/genética , Transtorno Autístico/psicologia , Encéfalo/anatomia & histologia , Encéfalo/citologia , Encéfalo/patologia , Região CA1 Hipocampal/metabolismo , Região CA1 Hipocampal/patologia , Proteínas de Transporte/genética , Deleção Cromossômica , Transtornos Cromossômicos/genética , Transtornos Cromossômicos/psicologia , Cromossomos Humanos Par 16/genética , Proteínas Culina/metabolismo , Feminino , Deficiência Intelectual/genética , Deficiência Intelectual/psicologia , Masculino , Camundongos , Herança Multifatorial/genética , Neurogênese/genética , Tamanho do Órgão/genética , Reprodutibilidade dos Testes , Transmissão Sináptica/efeitos dos fármacos , Complexos Ubiquitina-Proteína Ligase , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Proteína rhoA de Ligação ao GTP
6.
Neurobiol Dis ; 169: 105738, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35460869

RESUMO

Epilepsy is one of the most common neurological disorders. The X-linked gene PCDH19 is associated with sporadic and familial epilepsy in humans, typically with early-onset clustering seizures and intellectual disability in females but not in so-called 'carrier' males, suggesting that mosaic PCDH19 expression is required to produce epilepsy. To characterize the role of loss of PCDH19 function in epilepsy, we generated zebrafish with truncating pcdh19 variants. Evaluating zebrafish larvae for electrophysiological abnormalities, we observed hyperexcitability phenotypes in both mosaic and non-mosaic pcdh19+/- and pcdh19-/- mutant larvae. Thus, we demonstrate that the key feature of epilepsy-network hyperexcitability-can be modeled effectively in zebrafish, even though overt spontaneous seizure-like swim patterns were not observed. Further, zebrafish with non-mosaic pcdh19 mutation displayed reduced numbers of inhibitory interneurons suggesting a potential cellular basis for the observed hyperexcitability. Our findings in both mosaic and non-mosaic pcdh19 mutant zebrafish challenge the prevailing theory that mosaicism governs all PCDH19-related phenotypes and point to interneuron-mediated mechanisms underlying these phenotypes.


Assuntos
Epilepsia , Peixe-Zebra , Animais , Caderinas/genética , Epilepsia/genética , Feminino , Masculino , Mutação/genética , Protocaderinas
8.
Nucleic Acids Res ; 44(W1): W272-6, 2016 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-27185894

RESUMO

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.


Assuntos
Proteínas de Bactérias/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Endonucleases/genética , Genoma , RNA Guia de Cinetoplastídeos/síntese química , Software , Animais , Proteínas de Bactérias/metabolismo , Proteína 9 Associada à CRISPR , Desoxirribonuclease I/genética , Desoxirribonuclease I/metabolismo , Endonucleases/metabolismo , Edição de Genes , Humanos , Armazenamento e Recuperação da Informação , Internet , Motivos de Nucleotídeos , RNA Guia de Cinetoplastídeos/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismo
9.
Nature ; 473(7346): 212-5, 2011 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-21508956

RESUMO

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.


Assuntos
Anopheles/genética , Engenharia Genética , Insetos Vetores/genética , Controle de Mosquitos/métodos , Animais , Animais Geneticamente Modificados , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Feminino , Genes Reporter/genética , Genótipo , Masculino , Dados de Sequência Molecular , Proteínas de Saccharomyces cerevisiae/genética
10.
Nucleic Acids Res ; 42(22): 13839-52, 2014 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-25389263

RESUMO

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.


Assuntos
Endodesoxirribonucleases/metabolismo , Sequência de Bases , Simulação por Computador , DNA/química , Clivagem do DNA , Endodesoxirribonucleases/química , Sequenciamento de Nucleotídeos em Larga Escala , Modelos Moleculares , Análise de Sequência de DNA , Especificidade por Substrato
11.
Nucleic Acids Res ; 42(10): 6463-75, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24682825

RESUMO

LAGLIDADG homing endonucleases (LHEs) are compact endonucleases with 20-22 bp recognition sites, and thus are ideal scaffolds for engineering site-specific DNA cleavage enzymes for genome editing applications. Here, we describe a general approach to LHE engineering that combines rational design with directed evolution, using a yeast surface display high-throughput cleavage selection. This approach was employed to alter the binding and cleavage specificity of the I-Anil LHE to recognize a mutation in the mouse Bruton tyrosine kinase (Btk) gene causative for mouse X-linked immunodeficiency (XID)-a model of human X-linked agammaglobulinemia (XLA). The required re-targeting of I-AniI involved progressive resculpting of the DNA contact interface to accommodate nine base differences from the native cleavage sequence. The enzyme emerging from the progressive engineering process was specific for the XID mutant allele versus the wild-type (WT) allele, and exhibited activity equivalent to WT I-AniI in vitro and in cellulo reporter assays. Fusion of the enzyme to a site-specific DNA binding domain of transcription activator-like effector (TALE) resulted in a further enhancement of gene editing efficiency. These results illustrate the potential of LHE enzymes as specific and efficient tools for therapeutic genome engineering.


Assuntos
Endodesoxirribonucleases/química , Proteínas Tirosina Quinases/genética , Tirosina Quinase da Agamaglobulinemia , Animais , Células Cultivadas , Clivagem do DNA , Proteínas de Ligação a DNA/química , Evolução Molecular Direcionada , Endodesoxirribonucleases/metabolismo , Loci Gênicos , Genômica , Células HEK293 , Recombinação Homóloga , Humanos , Indicadores e Reagentes , Camundongos , Mutação , Estrutura Terciária de Proteína
12.
Nucleic Acids Res ; 42(4): 2564-76, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24270794

RESUMO

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.


Assuntos
Evolução Molecular Direcionada/métodos , Endodesoxirribonucleases/química , Endodesoxirribonucleases/metabolismo , Engenharia de Proteínas/métodos , Animais , Anopheles/genética , Bactérias/genética , Biologia Computacional , Clivagem do DNA , Endodesoxirribonucleases/genética , Genes de Insetos , Modelos Moleculares , Especificidade por Substrato
13.
Nature ; 461(7268): 1300-4, 2009 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-19865174

RESUMO

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.


Assuntos
Biocatálise , Simulação por Computador , Endonucleases/metabolismo , DNA Polimerase Dirigida por RNA/metabolismo , Termodinâmica , Sítios de Ligação , Biologia Computacional , DNA/química , DNA/metabolismo , Endonucleases/química , Cinética , Modelos Moleculares , Ligação Proteica , Conformação Proteica , DNA Polimerase Dirigida por RNA/química , Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato
14.
Transl Psychiatry ; 14(1): 82, 2024 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-38331943

RESUMO

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.


Assuntos
Cerebelo , Peixe-Zebra , Animais , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Animais Geneticamente Modificados/metabolismo , Cerebelo/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Encéfalo/metabolismo
15.
bioRxiv ; 2023 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-36711666

RESUMO

Microdeletion of a 3Mbp region encompassing 45 protein-coding genes at chromosome 22q11.2 (22q11.2DS) predisposes to multiple neurodevelopmental disorders and is one of the greatest genetic risk factors for schizophrenia. Defective mitochondrial function has been hypothesized to contribute to 22q11.2DS pathogenesis; however, which of the six mitochondrial genes contribute to neurodevelopmental phenotypes and their underlying mechanisms remain unresolved. To systematically test 22q11.2DS genes for functional roles in neurodevelopment and behavior, we generated genetic mutants for each of the 37 conserved zebrafish orthologs and performed high throughput behavioral phenotyping using seven behavioral assays. Through this unbiased approach, we identified five single-gene mutants with partially overlapping behavioral phenotypes. Two of these genes, mrpl40 and prodha , encode for mitochondrial proteins and, similar to what we observed in mrpl40 and prodha mutants, pharmacologic inhibition of mitochondrial function during development results in microcephaly. Finally, we show that both mrpl40 and prodha mutants display neural stem and progenitor cell phenotypes, with each gene regulating different neural stem cell populations. Combined, our results demonstrate a critical role for mitochondrial function in neural stem and progenitor cell populations in the developing vertebrate brain and provide compelling evidence that mitochondrial dysfunction during neurodevelopment is linked to brain volume and behavioral phenotypes observed in models of 22q11.2DS.

16.
iScience ; 26(7): 107099, 2023 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-37416451

RESUMO

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.

17.
J Biol Chem ; 286(37): 32617-27, 2011 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-21778233

RESUMO

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.


Assuntos
DNA/genética , Endodesoxirribonucleases , Genoma/genética , Mapeamento por Restrição
18.
PeerJ ; 9: e11007, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33954026

RESUMO

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.

19.
Front Behav Neurosci ; 14: 606900, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33536882

RESUMO

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.

20.
Methods Mol Biol ; 1414: 265-83, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27094297

RESUMO

Predicting the outcome of engineered and naturally occurring sequence perturbations to protein-DNA interfaces requires accurate computational modeling technologies. It has been well established that computational design to accommodate small numbers of DNA target site substitutions is possible. This chapter details the basic method of design used in the Rosetta macromolecular modeling program that has been successfully used to modulate the specificity of DNA-binding proteins. More recently, combining computational design and directed evolution has become a common approach for increasing the success rate of protein engineering projects. The power of such high-throughput screening depends on computational methods producing multiple potential solutions. Therefore, this chapter describes several protocols for increasing the diversity of designed output. Lastly, we describe an approach for building comparative models of protein-DNA complexes in order to utilize information from homologous sequences. These models can be used to explore how nature modulates specificity of protein-DNA interfaces and potentially can even be used as starting templates for further engineering.


Assuntos
Biologia Computacional , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo
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