RESUMO
Identifying genes involved in biological processes is critical for understanding the molecular building blocks of life. We used engineered CRISPR (clustered regularly interspaced short palindromic repeats) to efficiently mutate specific loci in zebrafish (Danio rerio) and screen for genes involved in vertebrate biological processes. We found that increasing CRISPR efficiency by injecting optimized amounts of Cas9-encoding mRNA and multiplexing single guide RNAs (sgRNAs) allowed for phenocopy of known mutants across many phenotypes in embryos. We performed a proof-of-concept screen in which we used intersecting, multiplexed pool injections to examine 48 loci and identified two new genes involved in electrical-synapse formation. By deep sequencing target loci, we found that 90% of the genes were effectively screened. We conclude that CRISPR can be used as a powerful reverse genetic screening strategy in vivo in a vertebrate system.
Assuntos
Sistemas CRISPR-Cas , Embrião não Mamífero/fisiologia , Testes Genéticos/métodos , Peixe-Zebra/genética , Animais , Regulação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Loci Gênicos , Pigmentação/genética , Pigmentação/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Epitélio Pigmentado da Retina/embriologia , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/genéticaRESUMO
Neural network function is based upon the patterns and types of connections made between neurons. Neuronal synapses are adhesions specialized for communication and they come in two types, chemical and electrical. Communication at chemical synapses occurs via neurotransmitter release whereas electrical synapses utilize gap junctions for direct ionic and metabolic coupling. Electrical synapses are often viewed as symmetrical structures, with the same components making both sides of the gap junction. By contrast, we show that a broad set of electrical synapses in zebrafish, Danio rerio, require two gap-junction-forming Connexins for formation and function. We find that one Connexin functions presynaptically while the other functions postsynaptically in forming the channels. We also show that these synapses are required for the speed and coordination of escape responses. Our data identify a genetic basis for molecular asymmetry at vertebrate electrical synapses and show they are required for appropriate behavioral performance.