title: Cas9 effector-mediated regulation of transcription and differentiation in human pluripotent stem cells.
abstract: The identification of the trans-acting factors and cis-regulatory modules that are involved in human pluripotent stem cell (hPSC) maintenance and differentiation is necessary to dissect the operating regulatory networks in these processes and thereby identify nodes where signal input will direct desired cell fate decisions in vitro or in vivo. To deconvolute these networks, we established a method to influence the differentiation state of hPSCs with a CRISPR-associated catalytically inactive dCas9 fused to an effector domain. In human embryonic stem cells, we find that the dCas9 effectors can exert positive or negative regulation on the expression of developmentally relevant genes, which can influence cell differentiation status when impinging on a key node in the regulatory network that governs the cell state. This system provides a platform for the interrogation of the underlying regulators governing specific differentiation decisions, which can then be employed to direct cellular differentiation down desired pathways.
authors.affiliation: Diabetes Center of Excellence, University of Massachusetts Medical School, Worcester, MA 01605, USA.
authors.forename: Nicola A
authors.initials: NA
authors.lastname: Kearns
authors.forename: Ryan M J
authors.initials: RM
authors.lastname: Genga
authors.forename: Metewo S
authors.initials: MS
authors.lastname: Enuameh
authors.forename: Manuel
authors.initials: M
authors.lastname: Garber
authors.forename: Scot A
authors.initials: SA
authors.lastname: Wolfe
authors.forename: René
authors.initials: R
authors.lastname: Maehr
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D000076987:CRISPR-Associated Protein 9; D064130:CRISPR-Associated Proteins / Q000235:genetics*; D064113:CRISPR-Cas Systems; D002454:Cell Differentiation / Q000235:genetics*; D019070:Cell Lineage / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism; D004720:Endonucleases / Q000235:genetics*; D053263:Gene Regulatory Networks / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D050814:Octamer Transcription Factor-3 / Q000235:genetics; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D055760:SOXF Transcription Factors / Q000096:biosynthesis; D014158:Transcription, Genetic; D015533:Transcriptional Activation / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D064130:CRISPR-Associated Proteins; D004268:DNA-Binding Proteins; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; C109188:SOX17 protein, human; D055760:SOXF Transcription Factors; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases
keywords: CRISPR; Cas9; Differentiation; Gene activation; Pluripotent stem cell; Transcriptional repression