CRISPR perturbation methods are limited in their ability to study non-coding elements and genetic interactions. In this study, we developed a system for bidirectional epigenetic editing, called CRISPRai, in which we apply activating (CRISPRa) and repressive (CRISPRi) perturbations to two loci simultaneously in the same cell. We developed CRISPRai Perturb-seq by coupling dual perturbation gRNA detection with single-cell RNA sequencing, enabling study of pooled perturbations in a mixed single-cell population. We applied this platform to study the genetic interaction between two hematopoietic lineage transcription factors, SPI1 and GATA1, and discovered novel characteristics of their co-regulation on downstream target genes, including differences in SPI1 and GATA1 occupancy at genes that are regulated through different modes. We also studied the regulatory landscape of IL2 (interleukin-2) in Jurkat T cells, primary T cells and chimeric antigen receptor (CAR) T cells and elucidated mechanisms of enhancer-mediated IL2 gene regulation. CRISPRai facilitates investigation of context-specific genetic interactions, provides new insights into gene regulation and will enable exploration of non-coding disease-associated variants.

CRISPR 扰动方法研究非编码元件和遗传相互作用的能力有限。在这项研究中，我们开发了一种名为 CRISPRai 的双向表观遗传编辑系统，其中我们同时对同一细胞中的两个基因座应用激活 (CRISPRa) 和抑制 (CRISPRi) 扰动。我们通过将双扰动 gRNA 检测与单细胞 RNA 测序相结合开发了 CRISPRai Perturb-seq，从而能够研究混合单细胞群体中的汇总扰动。我们应用该平台研究了两种造血谱系转录因子SPI1和GATA1之间的遗传相互作用，发现了它们对下游靶基因共同调控的新特征，包括SPI1和GATA1在通过不同模式调控的基因上的占据差异。我们还研究了 Jurkat T 细胞、原代 T 细胞和嵌合抗原受体 (CAR) T 细胞中IL2 （白细胞介素 2）的调控格局，并阐明了增强子介导的IL2基因调控机制。 CRISPRai 有助于研究特定背景的遗传相互作用，提供对基因调控的新见解，并将使得探索非编码疾病相关变异成为可能。