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.
Author contributions
R.M. and S.A.W. developed the concepts and approach, performed experiments and data analysis. N.A.K., R.M.J.G. and M.S.E. developed the approach, performed experiments and data analysis. M.G. developed the sgRNA target analysis approach. All authors prepared and edited the manuscript prior to submission.
Funding
This research was supported by the US National Institutes of Health (NIH) [R01GM068110 to S.A.W.]; and by the Helmsley Charitable Trust and the Glass Family Charitable Foundation (R.M.). Deposited in PMC for release after 12 months.
