利用CRISPR/Cas9高通量筛选技术研究超级增强子的功能

Super-enhancers (SEs) play important roles in the regulation of essential genes in human and mouse, and have been reported to be involved in various human diseases such as cancer. Although previous studies have functionally characterized a number of SEs, a high-throughput functional analysis method is needed to study the large numbers of SE in mammalian genome. We have fused deactivated Cas9 (dCas9) with the catalytic core of the human histone acetyltransferase p300, which could efficiently and specifically activate mammalian promoters and enhancers by acetylating its targeted genomic regions. We propose to use such dCas9/p300 system and newly designed sgRNA library targeting all of the known and predicted SEs in mouse genome to perform functional screening of the SEs involved in NIH3T3 cell proliferation and transformation as well as the regulation of TNFα expression in bone marrow derived dendritic cells from dCas9 knockin mice. In addition, we have designed capture probes for all mouse SEs which will be used in capture Hi-C to identify target genes of SEs in a whole genome scale. We will also use various methods to validate and study the mechanisms of the candidate super-enhancers identified from the above screening. This proposed study will provide the first high throughput genome wide method to study mammalian super-enhancers, and will give important insights to the functions and mechanisms of super-enhancers in cancer transformation and other physiological processes.

超级增强子在人类及小鼠关键基因的表达调控中起着重要作用，并且与多种疾病的发生和发展有关。但是目前功能已知的超级增强子还很少, 亟需开发高通量研究超级增强子功能的方法。本课题通过构建无内切酶活性的Cas9（dCas9）与组蛋白乙酰化酶p300的融合蛋白，可以实现对目标基因组区域的组蛋白乙酰化从而激活增强子，再利用靶向所有小鼠超级增强子的gRNA文库，在NIH3T3细胞系和dCas9敲入小鼠中筛选参与调控细胞增殖和转化的超级增强子。同时，本课题设计了靶向全基因组超级增强子生物素标记的捕获探针，可以通过Capture Hi-C方法高通量定位超级增强子的靶向调控基因，并将进一步在体内和体外验证和深入研究以上筛选得到的超级增强子的作用机制。这一研究将为深入了解超级增强子在癌症转化及其他生理过程中的功能和作用机制提供新的方法思路和重要结果。

超级增强子在人类及小鼠关键基因的表达调控中起着重要作用，并且与多种疾病的发生和发展有关。但是目前功能已知的超级增强子还很少,亟需开发高通量研究超级增强子功能的方法。本课题利用无内切酶活性的Cas9（dCas9）与组蛋白乙酰化酶p300活性结构域或者Kox1的转录阻遏物结构域（KRAB）结合，可以实现对目标增强子的激活或者抑制，再结合靶向所有小鼠超级增强子的gRNA文库，可以在多种模型中筛选起到调控作用的超级增强子，包括黑色素瘤细胞系B16F10中PDL1表达模型和LPS诱导BMDC表达TNFa模型。这一研究将为深入了解超级增强子在癌症免疫治疗及其他生理过程中的功能和作用机制提供新的方法思路和重要结果。. 建立了靶向小鼠所有超级增强子的gRNA文库并建立了dCas9筛选的方法。利用dCas9-p300激活系统在NIH3T3克隆形成模型中筛选超级增强子，实验结果提示该筛选实验重复性差，尝试更换激活效率更高的SAM激活系统，但是该系统遇到了脱靶率高的问题，所以转向利用B16F10的PDL1表达系统来做筛选，目前该系统已通过SAM激活系统和全基因组基因表达激活文库成功建立起来并且筛选结果与预期一致，该体系可用于超级增强子筛选。. 构建了SunTag-dCas9基因敲入小鼠发现其BMDC细胞可以通过瞬时转染scFv-p65-HSF1和scFv-KRAB实现目的基因的激活和抑制，但是无法得到高滴度的scFv病毒，导致该小鼠暂时无法用来做大规模的筛选实验。构建了dCas9-KRAB基因敲入小鼠，该小鼠只需要转染gRNA表达载体即可实现抑制，目前实验证明该小鼠dCas9-KRAB具有抑制基因表达的活性，其BMDC细胞可用下一步的超级增强子筛选。