TALEN和CRISPER基因靶向技术.pptx

2013,11,13; Outline;我们研究一个作物的性状，最终都需要做一件事：改变基因，从必要性和充分性两方面阐明基因功能。 1）Loss function：降低基因或删除基因 Give function: 提高基因的表达 ;生物学家的梦想：随心所欲地操作基因 ;TALEN技术形成的关键研究; TALE domain: DNA 识别域 TALE，植物病原体黄单胞菌 Xantomonas – 用于调控宿主基因 由34 个氨基酸长度的重复单位构成 第12，13位点氨基酸（ repeat-variable di-residue，RVD）不同 RVD决定识别位点不同 Nuclease domain: DNA剪切域 FokI，发现于海床黄杆菌 Flavobacterium okeanokoites 采取其非特异性DNA剪切结构域 形成二聚体时发生剪切;FastTALETM连接TALEN;亚克隆到植物表达载体上;We targeted the rice bacterial blight susceptibility gene?Os11N3?(also called?OsSWEET14) for TALEN-based disruption. This rice gene encodes a member of the SWEET sucrose-efflux transporter family and is hijacked by?X. oryzae?pv.?oryzae, using its endogenous TAL effectors AvrXa7 or PthXo3, to activate the gene and thus divert sugars from the plant cell so as to satisfy the pathogen‘s nutritional needs and enhance its persistence. The?Os11N3?promoter contains an effector-binding element (EBE) for AvrXa7, overlapping with another EBE for PthXo3 and with the TATA box. ;24bp;T2代表型;All mutant plants were morphologically normal compared to wild-type plants, indicating that the developmental function of Os11N3 was not disrupted.;Removal of T-DNA sequences containing TALEN genes from TALEN-modified rice plants using genetic crossing.;将识别特异DNA序列的TALE与转录激活因子VP64 融合，表达的融合蛋白结合启动子附近的特异DNA序列， 并通过VP64激活区域与Pol II 结合，激活基因的转录，提高了内源目标基因的表达。;TALE应用展望-TALEX;TALE蛋白新功能;CRISPR/Cas9 System;CRISPR-Cas主要由两部分组成：;CRISPR-Cas系统赋予原核细胞针对外源DNA特异性免疫，而这种特异性是由间隔序列（spacer）决定的。这种免疫功能的有无是由CRISPR 间隔序列的动态性变化决定的，即通过增加或删除间隔序列（spacer）来实现的。;42nt;The choice of the target sites also affects the level of transcrip -tion repression. By using fluorescent reporter genes in E. coli, we have observed that the repression is inversely correlated with the distance of the target site from the transcription start site. Thus, to achieve better repression in bacteria, target sites within the 5 ′ end of the gene should be selected. In human cells, we recom -mend selecting multiple target sites withi