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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

Silenciando a faísca: Edição do genoma CRISPR/Cas9 em peixes fraca elétricos

Published: October 27th, 2019

DOI:

10.3791/60253

1Department of Integrative Biology, Michigan State University, 2Faculty of Life Sciences, Unit of Biology and Ecology of Fishes, Humboldt University, 3Department of Biology, Cape Breton University

Aqui, um protocolo é apresentado para produzir e traseira CRISPR / Cas9 genoma nocaute peixeelétrico. Descritos em detalhes são os requisitos necessários de biologia molecular, reprodução e criação para um ginásio e um mormyrid, e técnicas de injeção para produzir larvas DelF 0 induzidas por Cas9.

A eletrorecepção e a eletrogênese mudaram na história evolutiva dos vertebrados. Há um grau impressionante de convergência nesses fenótipos derivados independentemente, que compartilham uma arquitetura genética comum. Isto é talvez melhor exemplificado pelas numerosas características convergentes de gymnotiformes e mormyrids, dois folheados teleost ricos em espécies que produzem e detectam campos elétricos fracos e são chamados de peixes fracamente elétricos. Nos 50 anos desde a descoberta de que peixes elétricos fracos usam eletricidade para sentir seu entorno e se comunicar, uma crescente comunidade de cientistas ganhou enormes insights sobre a evolução do desenvolvimento, sistemas e circuitos de neurociência, fisiologia celular, ecologia, biologia evolutiva e comportamento. Mais recentemente, tem havido uma proliferação de recursos genômicos para peixes elétricos. O uso desses recursos já facilitou insights importantes no que diz respeito à conexão entre genótipo e fenótipo nessas espécies. Um grande obstáculo à integração de dados genômicos com dados phenotípicos de peixes elétricos fracos é a falta atual de ferramentas genômicas funcionais. Relatamos aqui um protocolo completo para a realização de mutagênese CRISPR/Cas9 que utiliza mecanismos de reparo de DNA endógenos em peixes fracamente elétricos. Demonstramos que este protocolo é igualmente eficaz tanto na espécie mormyrid Brienomyrus brachyistius e no gymnotiform Brachyhypopomus gauderio usando CRISPR/Cas9 para atingir indels e mutações pontuais na primeira exon do sódio canal gene scn4aa. Usando este protocolo, embriões de ambas as espécies foram obtidos e genotipados para confirmar que as mutações previstas no primeiro exon do canal de sódio scn4aa estavam presentes. O fenótipo de sucesso knock-out foi confirmado com gravações mostrando amplitudes reduzidas de descarga de órgãos elétricos quando comparadas a controles não injetados combinados com o tamanho.

A eletrorecepção e a eletrogênese mudaram na história evolutiva dos vertebrados. Duas linhagens de peixes teleost, osteoglossiformes e siluriformes, evoluíram o electroreception em paralelo, e cinco linhagens de teleosts (gymnotiformes, mormyrids, e os Astros generacopus, Malapterurus, e Synodontis) evoluiu a eletrogênese em paralelo. Há um grau impressionante de convergência nestes fenótipos derivados independentemente, que compartilham uma arquitetura genética comum1,2,3.

Isto é talvez melhor exemplificado pelas numeros....

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Todos os métodos descritos aqui foram aprovados pelo Institutional Animal Care and Use Committee (IACUC) da Michigan State University.

1. Seleção de alvos sgRNA

Nota: Um protocolo é fornecido para o projeto manual de sgRNAs na etapa 1.1. Isso foi utilizado para a seleção de alvos scn4aa. Um protocolo adicional é fornecido para facilitar este processo (passo 1.2) usando o portal web EFISHGENOMICS. É aconselhável que os usuários selec.......

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Os locais-alvo sgRNA foram identificados dentro do exon 1 de scn4aa em B. gauderio e B. brachyistius como descrito na Seção 1. Os sgRNAs foram gerados conforme descrito na Seção 2. Após a seleção e síntese bem sucedida sgRNA (Figura 1), clivagem in vitro foi testado(Figura 2). Os sgRNAs que demonstram o corte in vitro foram selecionados então para microinjections da única pilha.

Os peixes adultos f.......

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A riqueza phenotípica de peixes fracamente elétricos, juntamente com uma recente proliferação de recursos genômicos, motiva uma forte necessidade de ferramentas genômicas funcionais no modelo de peixe seleto fraco. Este sistema é particularmente atraente devido à evolução convergente de numerosas características phenotípicas em linhagens paralelas de peixes, que são facilmente mantidos em laboratório.

O protocolo aqui descrito demonstra a eficácia da técnica CRISPR/Cas9 em linh.......

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Os autores reconhecem os esforços heróicos de Monica Lucas, Katherine Shaw, Ryan Taylor, Jared Thompson, Nicole Robichaud e Hope Healey para ajudar com a criação de peixes, coleta de dados e desenvolvimento precoce do protocolo. Gostaríamos também de agradecer aos três revisores por suas sugestões ao manuscrito. Acreditamos que o produto final seja de melhor qualidade depois de abordar seus comentários. Este trabalho foi financiado pelo apoio da National Science Foundation #1644965 e #1455405 ao JRG, e da bolsa DG do Conselho de Pesquisa de Ciências Naturais e Engenharia para o VLS.

....

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NameCompanyCatalog NumberComments
20 mg/mL RNA grade GlycogenThermo ScientificR0551
50 bp DNA ladderNEBN3236L
borosilicate glass capillary with filamentSutter InstrumentBF100-58-10(O.D. 1.0mm, I.D. 0.58 mm, 10 cm length)
Cas9 protein with NLS; 1 mg/mLPNA BiologyCP01
Dneasy Blood & Tissue KitQiagen69506
Eppendorf FemptoJet 4i MicroinjectorFisher ScientificE5252000021
Eppendorf Microloader Pipette TipsFisher Scientific10289651
Hamilton syringeFisher Scientific14-824-654referred to as "precision glass syringe" in the protocol
KimwipeFisher Scientific06-666referred to as "delicate task wipe" in the protocol
MEGAscript T7 Transcription KitInvitrogenAM1334
NEBuffer 3NEBB7003Sused for in vitro cleavage assay
OneTaq DNA kitNEBM0480L
OvaprimSyndel USAhttps://www.syndel.com/ovaprim-ovammmlu010.htmlreferred to as "spawning agent" in the protocol
ParafilmFisher ScientificS37440referred to as "thermoplastic" in the protocol
Pipette pullerWPISU-P97sutter brand
QIAquick PCR Purification KitQiagen28106
Reusable needle- requires customizationFisher Scientific7803-02Customize to 0.7 inches long; point style 4 and angle 25
T4 DNA polymeraseNEBM0203LUse with the 10X NEB buffer that is included
Teflon coated toolsbonefolder.comT-SPATULA4PIECEreferred to as "polytetrafluoroethene" in the protocol

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