Efficient targeted integration directed by short homology in zebrafish and mammalian cells.
Elife
; 92020 05 15.
Article
em En
| MEDLINE
| ID: mdl-32412410
ABSTRACT
Efficient precision genome engineering requires high frequency and specificity of integration at the genomic target site. Here, we describe a set of resources to streamline reporter gene knock-ins in zebrafish and demonstrate the broader utility of the method in mammalian cells. Our approach uses short homology of 24-48 bp to drive targeted integration of DNA reporter cassettes by homology-mediated end joining (HMEJ) at high frequency at a double strand break in the targeted gene. Our vector series, pGTag (plasmids for Gene Tagging), contains reporters flanked by a universal CRISPR sgRNA sequence which enables in vivo liberation of the homology arms. We observed high rates of germline transmission (22-100%) for targeted knock-ins at eight zebrafish loci and efficient integration at safe harbor loci in porcine and human cells. Our system provides a straightforward and cost-effective approach for high efficiency gene targeting applications in CRISPR and TALEN compatible systems.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Peixe-Zebra
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Genes Reporter
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Proteínas de Fluorescência Verde
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Técnicas de Introdução de Genes
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Proteínas Associadas a CRISPR
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Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas
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Sistemas CRISPR-Cas
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Nucleases dos Efetores Semelhantes a Ativadores de Transcrição
Limite:
Animals
/
Humans
Idioma:
En
Ano de publicação:
2020
Tipo de documento:
Article