Wellcome Sanger Institute, Hinxton, Cambridge CB10 1SA, UK.
Wellcome Sanger Institute, Hinxton, Cambridge CB10 1SA, UK; Institute of Animal Science and Veterinary Medicine, Hubei Academy of Agricultural Sciences, Wuhan 430064, China.
Mol Cell. 2023 Apr 6;83(7):1125-1139.e8. doi: 10.1016/j.molcel.2023.02.011. Epub 2023 Mar 13.
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.
CRISPR 激活（CRISPRa）是一种重要的转录调控工具，但在不同的靶基因中其效果存在差异。我们利用数千个随机整合的带有条形码报告基因的人多能干细胞来评估影响 CRISPRa 效果的表观遗传特征。基础表达水平受基因组背景影响，并在向神经元分化过程中发生显著变化。dCas9-VPR 对大多数基因组背景中的基因激活都是成功的，包括发育抑制区域，并且激活水平与基础基因表达呈负相关，而 dCas9-p300 在干细胞中无效。某些染色质状态，如双价染色质，对 dCas9-VPR 特别敏感，而组成型异染色质的反应性较低。我们在内源性基因上验证了这些规则，并表明某些基因的激活会引起干细胞转录组的变化，有时表现出分化细胞的特征。我们的数据提供了预测 CRISPRa 结果的规则，并强调了其用于筛选驱动干细胞分化的因素的实用性。
