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  • Master Regulators and Cofactors of Human Neuronal Cell Fate Specification Identified by CRISPR Gene Activation Screens.

Master Regulators and Cofactors of Human Neuronal Cell Fate Specification Identified by CRISPR Gene Activation Screens.

Cell reports (2020-12-03)
Joshua B Black, Sean R McCutcheon, Shataakshi Dube, Alejandro Barrera, Tyler S Klann, Grayson A Rice, Shaunak S Adkar, Scott H Soderling, Timothy E Reddy, Charles A Gersbach
ABSTRACT

Technologies to reprogram cell-type specification have revolutionized the fields of regenerative medicine and disease modeling. Currently, the selection of fate-determining factors for cell reprogramming applications is typically a laborious and low-throughput process. Therefore, we use high-throughput pooled CRISPR activation (CRISPRa) screens to systematically map human neuronal cell fate regulators. We utilize deactivated Cas9 (dCas9)-based gene activation to target 1,496 putative transcription factors (TFs) in the human genome. Using a reporter of neuronal commitment, we profile the neurogenic activity of these factors in human pluripotent stem cells (PSCs), leading to a curated set of pro-neuronal factors. Activation of pairs of TFs reveals neuronal cofactors, including E2F7, RUNX3, and LHX8, that improve conversion efficiency, subtype specificity, and maturation of neuronal cell types. Finally, using multiplexed gene regulation with orthogonal CRISPR systems, we demonstrate improved neuronal differentiation with concurrent activation and repression of target genes, underscoring the power of CRISPR-based gene regulation for programming complex cellular phenotypes.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Anti-Microtubule-Associated Protein 2 (MAP2) Antibody, Chemicon®, from rabbit
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Doxycycline hyclate
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Bovine Serum Albumin, heat shock fraction, pH 7, ≥98%
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Puromycin dihydrochloride from Streptomyces alboniger, powder, BioReagent, suitable for cell culture
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Triton X-100, laboratory grade
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Fetal Bovine Serum, USA origin, sterile-filtered, suitable for cell culture, suitable for hybridoma
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Gentamicin solution, 50 mg/mL in deionized water, liquid, 0.1 μm filtered, BioReagent, suitable for cell culture
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Goat Serum Donor Herd, USA origin, sterile-filtered, suitable for cell culture
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Triton X-100, for molecular biology
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Ethylenediaminetetraacetic acid disodium salt solution, for molecular biology, 0.5 M in H2O, DNase, RNase, NICKase and protease, none detected