An efficient and scalable pipeline for epitope tagging in mammalian stem cells using Cas9 ribonucleoprotein.
Elife
; 72018 04 11.
Article
em En
| MEDLINE
| ID: mdl-29638216
ABSTRACT
CRISPR/Cas9 can be used for precise genetic knock-in of epitope tags into endogenous genes, simplifying experimental analysis of protein function. However, Cas9-assisted epitope tagging in primary mammalian cell cultures is often inefficient and reliant on plasmid-based selection strategies. Here, we demonstrate improved knock-in efficiencies of diverse tags (V5, 3XFLAG, Myc, HA) using co-delivery of Cas9 protein pre-complexed with two-part synthetic modified RNAs (annealed crRNAtracrRNA) and single-stranded oligodeoxynucleotide (ssODN) repair templates. Knock-in efficiencies of ~5-30%, were achieved without selection in embryonic stem (ES) cells, neural stem (NS) cells, and brain-tumor-derived stem cells. Biallelic-tagged clonal lines were readily derived and used to define Olig2 chromatin-bound interacting partners. Using our novel web-based design tool, we established a 96-well format pipeline that enabled V5-tagging of 60 different transcription factors. This efficient, selection-free and scalable epitope tagging pipeline enables systematic surveys of protein expression levels, subcellular localization, and interactors across diverse mammalian stem cells.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Ribonucleoproteínas
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Células-Tronco
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Fatores de Transcrição
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Mapeamento de Epitopos
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Ensaios de Triagem em Larga Escala
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Sistemas CRISPR-Cas
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Proteína 9 Associada à CRISPR
Limite:
Animals
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Humans
Idioma:
En
Ano de publicação:
2018
Tipo de documento:
Article