Using an Inducible CRISPR-dCas9-KRAB Effector System to Dissect Transcriptional Regulation in Human Embryonic Stem Cells.
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.
Program in Molecular Medicine, Diabetes Center of Excellence, University of Massachusetts Medical School, Worcester, MA, 01605, USA.
Krishna Mohan
KM
Parsi
Program in Molecular Medicine, Diabetes Center of Excellence, University of Massachusetts Medical School, Worcester, MA, 01605, USA.
Erica
E
Hennessy
Program in Molecular Medicine, Diabetes Center of Excellence, University of Massachusetts Medical School, Worcester, MA, 01605, USA.
Nicola
N
Kearns
Program in Molecular Medicine, Diabetes Center of Excellence, University of Massachusetts Medical School, Worcester, MA, 01605, USA. rene.maehr@umassmed.edu.
René
R
Maehr
D064113:CRISPR-Cas Systems; D002460:Cell Line; D055785:Gene Knockdown Techniques; D000066449:Human Embryonic Stem Cells / Q000378:metabolism*; D006801:Humans; D011401:Promoter Regions, Genetic; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D000094704:RNA, Guide, CRISPR-Cas Systems; D012097:Repressor Proteins; C422407:ZNF350 protein, human
CRISPR-Cas9; Gene regulation; Gene silencing and activation; Human ES cells; Transcription; dCas9-KRAB