MOOC遗传学Chapter 17-3b

chapteroutlinechapter17主讲教师：王亚梅•17.1Creatingtransgenicorganisms•17.2Usesoftransgenicorganisms•17.3Targetedmutagenesis(2)•17.4Humangenetherapy17.3.1GenetargetinginEScellstogenerateknockoutmice17.3.2Knockinsintroducespecificmutationsintospecificgenes17.3.3TALENsgenomeediting17.3Targetedmutagenesis(2)17.3.4CRISPR/Cas9genomeediting17.3.3TALENgenomeeditingtechniqueTranscriptionactivator-likeeffectornucleases(转录激活因子样效应物核酸酶,TALEN)arerestrictionenzymesthatcanbeengineeredtocutspecificsequencesofDNA.TALEDNA-bindingdomainTALEN=识别FokIDNAcleavagedomain+切割TALE(Transcriptionactivator-likeeffector转录激活因子样效应物)n1989植物病原体黄单胞杆菌(Xanthomonasspp.)avrBs3基因被克隆n2007发现avrBs3-TA氨基酸序列能特异结合DNAn2009破译TA氨基酸序列与DNA靶序列的对应关系TALEDNA-bindingdomainnTALEDNA结合结构域由13-29个重复氨基酸单元组成n每个单元含有33-35个串联排列的氨基酸n仅第12和13个氨基酸是可变的（RepeatVariantDi-residue,RVD重复可变双残基)，对应识别一个目标碱基TTALEDNAbindingdomainLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGNGTNIANNG/AHDC识别碱基RVD序列33-35aa模块NNA/T/G/CRVD序列EndonucleaseFokIdimerizationisrequiredforDNAcleavagenThedimerizationofferstwolevelsofcontroltopreventFokIfromcleavingDNAnonspecifically.nFokIisanunusualrestrictionenzymesthatrecognizeaspecificDNAsequenceandcleaveDNAnonspecificallyashortdistanceawayfromthatsequenceandhasbeenusedtocreateartificialenzymeswithnewspecificities.TwowaystorepairdoublestrandbreakcreatedbyTALEN随机修复同源修复模板遵照同源修复模板来修复Dobulestrandbreak(DSB)NHEJ敲除敲入靶序列1靶序列2GeneralstepsofTALENgenomeeditingn确定靶点：n克隆构建TALE靶点识别模块选择目标基因编码区或外显子和内含子的交界处相隔14-20bp的两段16-20bp序列作为靶点n按照DNA序列将相应TALE氨基酸单元串联克隆,即可获得识别某一特定核酸序列的TALE。n选择的特异DNA序列长度一般为16-20bp，作为识别靶点。克隆构建TALE靶点识别模块TALE蛋白模块识别DNA序列靶序列1靶序列2GeneralstepsofTALENgenomeeditingn确定靶点：选择目标基因编码区或外显子和内含子的交界处中相隔14-20bp的两段16-20bp序列作为靶点n克隆构建TALE靶点识别模块n将两个靶点识别模块分别融合到FokI的N-末端，得到TALEN质粒对n将重组真核表达质粒转入细胞n筛选突变体TheapplicationofTALENgenomeeditingtechniquen靶向基因敲除-无同源修复模板NHEJn靶向基因敲入-有同源修复模板HRTALEproteinstructuraldomainsn调控基因表达主讲教师：王亚梅