Development of compact transcriptional effectors using high-throughput measurements in diverse contexts

HT-recruit and CRISPR HT-recruit processed datasets Abstract: Transcriptional effectors are protein domains known to activate or repress gene expression, however, a systematic understanding of which effector domains regulate transcription robustly 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 containing 114,288 sequences 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 NFZ by combining several domains, which enables efficient CRISPRa with better viral delivery, and inducible control of CAR T-cells. Together, this effector-by-context functional map reveals context-dependence across human effectors and guides effector selection for manipulating transcription.HT-recruit及CRISPR HT-recruit处理后数据集 摘要：转录效应结构域是一类已知可激活或抑制基因表达的蛋白质结构域，但目前学界仍缺乏对哪些效应结构域能够在不同基因组背景、细胞类型以及DNA结合结构域（DNA-binding domain, DBD）背景下稳定调控转录的系统性认知。本研究开发了dCas9介导的高通量募集（HT-recruit）技术，这是一种可在内源靶基因位点定量分析效应蛋白功能的混池筛选方法，并针对涵盖5092个核蛋白Pfam结构域的文库在多种背景下开展了效应蛋白功能测试。此外，本研究利用包含114288条覆盖染色质调控因子与转录因子序列的更大规模文库，对源自未注释蛋白质区域的效应蛋白的背景依赖性进行了图谱绘制。研究发现，诸多效应蛋白的功能依赖于靶位点与DNA结合结构域背景，例如螺旋-环-螺旋（Helix-Loop-Helix, HLH）结构域可同时作为激活子或抑制子发挥功能。为实现高效的基因扰动，我们筛选得到了背景稳定性较强的结构域，其中包括ZNF705 KRAB，该结构域可优化CRISPR干扰（CRISPRi）工具以沉默启动子与增强子区域。本研究通过组合多个结构域工程化构建了一种紧凑型人类激活因子NFZ，该因子可实现高效的CRISPR激活（CRISPRa），并具备更优异的病毒递送能力，同时可实现嵌合抗原受体T（CAR-T）细胞的诱导性调控。综上，这套基于效应蛋白-背景的功能图谱揭示了人类效应蛋白的功能背景依赖性，可为转录调控相关的效应蛋白筛选提供指导。