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
PubMed ID
27832543
Repository Citation
Parsi KM, Hennessy E, Kearns NA, Maehr R. (2016). Using an Inducible CRISPR-dCas9-KRAB Effector System to Dissect Transcriptional Regulation in Human Embryonic Stem Cells. Program in Molecular Medicine Publications. https://doi.org/10.1007/978-1-4939-6518-2_16. Retrieved from https://escholarship.umassmed.edu/pmm_pp/148