Tycko Josh, Van Mike V, DelRosso Nicole, Ye Hanrong, Yao David, Valbuena Raeline, Vaughan-Jackson Alun, Xu Xiaoshu, Ludwig Connor, Spees Kaitlyn, Liu Katherine, Gu Mingxin, Khare Venya, Mukund Adi Xiyal, Suzuki Peter H, Arana Sophia, Zhang Catherine, Du Peter P, Ornstein Thea S, Hess Gaelen T, Kamber Roarke A, Qi Lei S, Khalil Ahmad S, Bintu Lacramioara, Bassik Michael C
Department of Genetics, Stanford University, Stanford, CA, USA.
Department of Neurobiology, Harvard Medical School, Boston, MA, USA.
Nat Biotechnol. 2024 Nov 1. doi: 10.1038/s41587-024-02442-6.
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.
转录效应物是已知可激活或抑制基因表达的蛋白质结构域；然而，目前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）背景下调节转录的系统理解。在这里，我们开发了dCas9介导的高通量招募（HT-recruit）方法，这是一种用于在内源靶基因上定量效应物功能的汇集筛选方法，并针对包含5092个核蛋白Pfam结构域的文库在不同背景下测试效应物功能。我们还使用一个更大的覆盖染色质调节因子和转录因子的文库，绘制了从未注释蛋白质区域提取的效应物的背景依赖性。我们发现许多效应物依赖于靶标和DBD背景，例如HLH结构域既可以作为激活剂也可以作为抑制剂。为了实现有效的干扰，我们选择了背景稳健的结构域，包括ZNF705 KRAB，它改进了CRISPRi工具以沉默启动子和增强子。我们通过组合NCOA3、FOXO3和ZNF473结构域设计了一种紧凑的人类激活剂NFZ，它能够实现高效的CRISPRa，具有更好的病毒递送能力，并可诱导控制嵌合抗原受体T细胞。
