Evolution of gene regulatory networks in primates inferred by perturbations

了解基因调控如何进化对理解生物多样性和人类生物学至关重要。灵长类动物之间的比较具有特别的相关性，不仅因为它们为人类特有的进化提供了信息，而且因为它们弥合了人类与小鼠（我们最重要的模式生物）之间的差距。虽然跨物种表达水平的比较已经进行了充分的研究，但为了充分利用灵长类动物分子进化中编码的独特信息，有必要扩大研究范围，以研究遗传可及和可比较的灵长类动物细胞系统中的基因调控。随着基于crispr的扰动和单细胞转录组读出的结合，最近出现了一种改变游戏规则的技术来研究基因调控。这种单细胞CRISPR筛选的一个突出策略是使用单个引导RNA （sgRNA）将催化无活性Cas9 （dCas9）和抑制结构域（如KRAB）的融合蛋白靶向到启动子上。这显著降低了靶基因的表达，单细胞RNA-seq对转录组产生了相应的影响。这样的筛选可能会大大有助于我们对基因调控网络（grn）的理解。然而，目前尚不清楚这种微扰推断的grn是如何演变的。在这里，我们建议通过比较灵长类动物神经祖细胞（npc）中微扰推断的grn来缩小这一知识差距。我们的目标是通过使用我们已经建立的来自人类、大猩猩、猩猩和猕猴的诱导多能干细胞（iPSCs），在特定的基因组位置从每个物种的两个个体中产生含有可诱导dCas9-KRAB的iPSCs来实现这一目标。这些细胞系将分化为神经祖细胞（NPCs），并用sgrna靶向NPCs中表达的100个转录因子进行转导。dCas9-KRAB诱导9天后，收集细胞用于生成scRNA-seq文库。我们的目标是为每个物种和目标TF生成500个细胞的crispr - scrna -seq数据，这有望为分析这些TF网络的保守性提供足够的统计能力。将这种保守性与受干扰的tf及其目标的特性联系起来，将使我们能够收集到灵长类grn进化的独特见解。最后，使用一种新颖的结合表观遗传和表达读出，我们将跟踪18个进化上有趣的tf子集。这将揭示反式效应相互作用的详细信息，即TF的下调，以及靶基因的顺式调控格局，为证实调控进化的推断提供真正可比的功能数据。

Understanding how gene regulation evolves is crucial to understand biological diversity and human biology. Comparisons across primates are of particular relevance not only because they inform human-specific evolution but also because they bridge the gap between human and mouse, our most important model organism. While cross-species comparisons of expression levels have been amply conducted, broadening the scope to study gene regulation in genetically accessible and comparable primate cell systems is necessary to fully leverage the unique information encoded in the molecular evolution of primates. With the combination of CRISPR-based perturbations and single-cell transcriptomic read-outs, a game-changing technology to study gene regulation has recently emerged. One prominent strategy for such single-cell CRISPR screens is to target a fusion protein of catalytically inactive Cas9 (dCas9) and a repressor domain such as KRAB to a promoter using a single guide RNA (sgRNA). This significantly reduces the expression of the targeted gene and single-cell RNA-seq informs on the corresponding effect on the transcriptome. Such screens are likely to contribute substantially to our understanding of gene regulatory networks (GRNs). However, it is currently unknown how such perturbation-inferred GRNs evolve.Here, we propose to start closing this knowledge gap by comparing perturbation-inferred GRNs in neural progenitor cells (NPCs) of primates. We aim to achieve this by using our established induced pluripotent stem cells (iPSCs) from human, gorilla, orangutan and cynomolgus macaque to generate iPSCs from two individuals per species containing an inducible dCas9-KRAB at a defined genomic location. These lines will be differentiated to neural progenitor cells (NPCs) and transduced with sgRNAs targeting 100 transcription factors expressed in NPCs. After nine days of dCas9-KRAB induction, pooled cells will be harvested for scRNA-seq library generation. We aim to generate CRISPRi-scRNA-seq data for 500 cells per species and targeted TF, which is expected to provide sufficient statistical power to analyse the conservation of these TF networks. Correlating this conservation with the properties of the perturbed TFs and the properties of their targets will enable us to gather unique insights into the evolution of primate GRNs. Finally, using a novel combined epigenetic and expression read-out, we will follow up on a subset of 18 evolutionarily interesting TFs. This will reveal detailed information about the interplay of trans-effects, i.e. the downregulation of a TF, and the target gene's cis-regulatory landscape providing truly comparable functional data to corroborate inferences about regulatory evolution.