Transcriptional effectors are protein domains known to activate or repress gene expression; however, a systematic understanding of which effector domains regulate transcription across genomic, cell type and DNA-binding domain (DBD) contexts is lacking. Here we develop dCas9-mediated high-throughput recruitment (HT-recruit), a pooled screening method for quantifying effector function at endogenous target genes and test effector function for a library containing 5,092 nuclear protein Pfam domains across varied contexts. We also map context dependencies of effectors drawn from unannotated protein regions using a larger library tiling chromatin regulators and transcription factors. We find that many effectors depend on target and DBD contexts, such as HLH domains that can act as either activators or repressors. To enable efficient perturbations, we select context-robust domains, including ZNF705 KRAB, that improve CRISPRi tools to silence promoters and enhancers. We engineer a compact human activator called NFZ, by combining NCOA3, FOXO3 and ZNF473 domains, which enables efficient CRISPRa with better viral delivery and inducible control of chimeric antigen receptor T cells.

转录效应子是已知可激活或抑制基因表达的蛋白质结构域;然而，缺乏对哪些效应结构域在基因组、细胞类型和 DNA 结合结构域 （DBD） 环境中调节转录的系统理解。在这里，我们开发了 dCas9 介导的高通量募集 （HT-recruit），这是一种用于量化内源性靶基因效应器功能的混合筛选方法，并测试在不同环境中包含 5,092 个核蛋白 Pfam 结构域的文库的效应器功能。我们还使用更大的文库平铺染色质调节因子和转录因子来绘制从未注释的蛋白质区域提取的效应子的上下文依赖性。我们发现许多效应子依赖于靶标和 DBD 环境，例如可以充当激活因子或抑制因子的 HLH 结构域。为了实现有效的扰动，我们选择了上下文稳健的结构域，包括 ZNF705 KRAB，它们改进了 CRISPRi 工具以沉默启动子和增强子。我们通过结合 NCOA3、FOXO3 和 ZNF473 结构域，设计了一种名为 NFZ 的紧凑型人类激活剂，可实现高效的 CRISPRa，具有更好的病毒递送和嵌合抗原受体 T 细胞的诱导控制。