Massively parallel characterization of CRISPR activator efficacy in human induced pluripotent stem cells and neurons.
Por:
Wu Q, Wu J, Karim K, Chen X, Wang T, Iwama S, Carobbio S, Keen P, Vidal-Puig A, Kotter MR and Bassett A
Publicada:
6 abr 2023
Ahead of Print:
1 abr 2023
Resumen:
CRISPR activation (CRISPRa) is an important tool to perturb transcription, but its effectiveness varies between target genes. We employ human pluripotent stem cells with thousands of randomly integrated barcoded reporters to assess epigenetic features that influence CRISPRa efficacy. Basal expression levels are influenced by genomic context and dramatically change during differentiation to neurons. Gene activation by dCas9-VPR is successful in most genomic contexts, including developmentally repressed regions, and activation level is anti-correlated with basal gene expression, whereas dCas9-p300 is ineffective in stem cells. Certain chromatin states, such as bivalent chromatin, are particularly sensitive to dCas9-VPR, whereas constitutive heterochromatin is less responsive. We validate these rules at endogenous genes and show that activation of certain genes elicits a change in the stem cell transcriptome, sometimes showing features of differentiated cells. Our data provide rules to predict CRISPRa outcome and highlight its utility to screen for factors driving stem cell differentiation.
Filiaciones:
Wu Q:
Wellcome Sanger Institute, Hinxton, Cambridge CB10 1SA, UK
Wu J:
Wellcome Sanger Institute, Hinxton, Cambridge CB10 1SA, UK
Institute of Animal Science and Veterinary Medicine, Hubei Academy of Agricultural Sciences, Wuhan 430064, China
Karim K:
Department of Clinical Neurosciences, University of Cambridge, Cambridge CB2 0QQ, UK
Chen X:
Wellcome Sanger Institute, Hinxton, Cambridge CB10 1SA, UK
Southern University of Science and Technology, 1088 Xueyuan Ave, Nanshan, Shenzhen, Guangdong 518055, China
Wang T:
Department of Statistics, London School of Economics and Political Science, London WC2B 4RR, UK
Iwama S:
Wellcome Sanger Institute, Hinxton, Cambridge CB10 1SA, UK
:
Wellcome Sanger Institute, Hinxton, Cambridge CB10 1SA, UK
Metabolic Research Laboratories, Addenbrooke's Treatment Center, Institute of Metabolic Science, Addenbrooke's Hospital, University of Cambridge, Cambridge, UK
Centro de Investigación Príncipe Felipe, 46012 Valencia, Spain
Keen P:
Wellcome Sanger Institute, Hinxton, Cambridge CB10 1SA, UK
:
Wellcome Sanger Institute, Hinxton, Cambridge CB10 1SA, UK
Metabolic Research Laboratories, Addenbrooke's Treatment Center, Institute of Metabolic Science, Addenbrooke's Hospital, University of Cambridge, Cambridge, UK
Centro de Investigación Príncipe Felipe, 46012 Valencia, Spain
Kotter MR:
Department of Clinical Neurosciences, University of Cambridge, Cambridge CB2 0QQ, UK
Bassett A:
Wellcome Sanger Institute, Hinxton, Cambridge CB10 1SA, UK
Green Published, Green Accepted, hybrid
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