Using an Inducible CRISPR-dCas9-KRAB Effector System to Dissect Transcriptional Regulation in Human Embryonic Stem Cells.
Methods Mol Biol
; 1507: 221-233, 2017.
Article
em En
| MEDLINE
| ID: mdl-27832543
ABSTRACT
CRISPR-Cas9 effector systems have wide applications for the stem cell and regenerative medicine field. The ability to dissect the functional gene regulatory networks in pluripotency and potentially in differentiation intermediates of all three germ layers makes this a valuable tool for the stem cell community. Catalytically inactive Cas9 fused to transcriptional/chromatin effector domains allows for silencing or activation of a genomic region of interest. Here, we describe the application of an inducible, RNA-guided, nuclease-deficient (d) Cas9-KRAB system (adapted from Streptococcus pyogenes) to silence target gene expression in human embryonic stem cells, via KRAB repression at the promoter region. This chapter outlines a detailed protocol for generation of a stable human embryonic stem cell line containing both Sp-dCas9-KRAB and sgRNA, followed by inducible expression of Sp-dCas9-KRAB to analyze functional effects of dCas9-KRAB at target loci in human embryonic stem cells.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Transcrição Gênica
/
Ativação Transcricional
/
Técnicas de Silenciamento de Genes
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Sistemas CRISPR-Cas
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Células-Tronco Embrionárias Humanas
Limite:
Humans
Idioma:
En
Ano de publicação:
2017
Tipo de documento:
Article