Title

Using an Inducible CRISPR-dCas9-KRAB Effector System to Dissect Transcriptional Regulation in Human Embryonic Stem Cells

UMMS Affiliation

Program in Molecular Medicine; Diabetes Center of Excellence

Publication Date

2016-11-11

Document Type

Book Chapter

Disciplines

Cell Biology | Molecular Biology

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.

Keywords

CRISPR-Cas9, Gene regulation, Gene silencing and activation, Human ES cells, Transcription, dCas9-KRAB

DOI of Published Version

10.1007/978-1-4939-6518-2_16

Source

Parsi KM, Hennessy E, Kearns N, Maehr R. Using an Inducible CRISPR-dCas9-KRAB Effector System to Dissect Transcriptional Regulation in Human Embryonic Stem Cells. Methods Mol Biol. 2017;1507:221-233. doi: 10.1007/978-1-4939-6518-2_16. PMID: 27832543. Link to article on publisher's site

Journal/Book/Conference Title

Methods in molecular biology (Clifton, N.J.)

Related Resources

Link to Article in PubMed

PubMed ID

27832543

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