Long range regulation of transcription scales with genomic distance in a gene specific manner. Long range regulation of transcription scales with genomic distance in a gene specific manner

While critical for tuning the timing and level of transcription, enhancer communication with distal promoters is not well understood. Here we aim to bypass the need for sequence-specific transcription factors and recruit activators directly using catalytically dead Cas9 fused to the synthetic activator VPR. We perform a CRISPRa tiling screen in mouse ESCs that express doxycycline-inducible dCas9-VPR and were engineered with an endogenous T2A-EGFP reporter at the end of the Fgf5 gene. We used a library of over 13,000 guides spanning a 500kb window centered at the Prdm8-Fgf5 TAD. We demonstrate that this approach is unable to achieve detectable dCas9-VPR binding to arbitrary genomic sites making it impossible to address whether any genomic site to which activators are recruited can function as an enhancer. To overcome this limitation, we turned to CARGO-VPR, an approach for targeting dCas9-VPR using a multiplexed array of RNA guides. We show that this approach achieves effective activator recruitment to arbitrary genomic sites, even those inaccessible when targeted with a single guide. We utilize CARGO-VPR across the Prdm8-Fgf5 locus in mESCs, where neither gene is expressed. We demonstrate that while activator recruitment to any tested region results in transcriptional induction of at least one gene, the expression level strongly depends on the genomic distance between the promoter and activator recruitment site. However, the expression-distance relationship for each gene scales distinctly in a manner not attributable to differences in 3D contact frequency, promoter DNA sequence or presence of the repressive chromatin marks at the locus. Overall design: R1 mESCs expressing doxycycline-inducible dCas9-VPR and an endogenous T2A-EGFP reporter were transfected with an sgRNA library. Cells expressing high EGFP were isolated along with a roughly equal number of unsorted cells. Four paired replicates of EGFP positive and unsorted populations were sequenced to identify sgRNAs in each population.

尽管增强子与远端启动子的通讯对于调控转录的时序与强度至关重要，但目前学界对其机制仍缺乏深入了解。本研究拟绕过序列特异性转录因子的依赖需求，通过与合成激活因子VPR融合的催化失活Cas9（catalytically dead Cas9，dCas9）直接招募激活蛋白。
我们在表达四环素诱导型dCas9-VPR、且在Fgf5基因末端整合了内源性T2A-EGFP报告基因的小鼠胚胎干细胞（mouse ESCs，mESCs）中开展了CRISPR激活（CRISPRa）平铺筛选。我们使用了覆盖以Prdm8-Fgf5拓扑关联结构域（Topologically Associating Domain，TAD）为中心的500kb区间的13000余条sgRNA向导序列文库。
我们发现，该策略无法实现dCas9-VPR对任意基因组位点的可检测结合，因此无法验证被招募激活蛋白的基因组位点是否能够充当增强子。
为克服这一局限，我们转而采用CARGO-VPR策略——该策略通过多路RNA向导阵列实现dCas9-VPR的靶向递送。
我们证实，该策略可实现激活蛋白对任意基因组位点的有效招募，即便采用单向导RNA（single guide RNA，sgRNA）无法靶向的位点亦能完成靶向。
我们在mESCs中的Prdm8-Fgf5基因座开展了CARGO-VPR应用，该位点的两个基因均未发生表达。
我们发现，尽管向任意测试区域招募激活蛋白均可诱导至少一个基因的转录，但基因表达水平强烈依赖于启动子与激活蛋白招募位点之间的基因组距离。
然而，每个基因的表达-距离关系呈现出显著差异，且该差异无法通过三维基因组接触频率、启动子DNA序列或该位点抑制性染色质修饰的有无来解释。
实验整体设计：将sgRNA文库转染至表达四环素诱导型dCas9-VPR及内源性T2A-EGFP报告基因的R1型mESCs。随后分离高EGFP表达细胞与大致等量的未分选细胞，对4对EGFP阳性细胞群与未分选细胞群的平行重复样本进行测序，以鉴定各群体中的sgRNA。