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CRISPR Activator Approaches Assayed in Human Stem Cells, Neurons

For a paper in Molecular Cell, a team from the Wellcome Sanger Institute and other centers in the UK and China share findings from a barcoded reporter-based analysis of CRISPR activation (CRISPRa) activity in human induced pluripotent stem cells (iPSCs) or differentiated neurons. Starting with iPSCs or neurons containing thousands of barcoded receptor sequences peppered randomly across the genome, the researchers used targeted amplicon sequencing to assess a CRISPRa efficacy in the presence of "dead" Cas9 enzymes paired with a transcriptionally activating VPR domain or chromatin-modifying p300 protein sequence, bringing in RNA sequencing-based expression profiles and chromatin immunoprecipitation sequencing profiles focused on half a dozen chromatin modifications. The analyses pointed to molecular influencing dCas9-VPR or dCas9-p300 activity in each cell type, particularly chromatin-dependent activity, that was assessed further with single-cell experiments and single-cell RNA-seq. "Our data demonstrate, for the first time, that dCas9-VPR-mediated transcriptional activation is generally applicable across chromatin states and cell types, but that basal expression level and chromatin state can impact both the degree of activation and its variability" the authors report. "These features will be important in the design and analysis of CRISPRa screens and the use of these systems for disease modeling or therapeutic intervention."