TALE的靶向基因操作.pptx

,Genecopoeia Inc.,广州复能基因有限公司,4006 020 200,李华平,Precision genome engineering based on TALE,基于,TALE,的靶向基因,修,饰,技术,1,报,告提纲,基,因,组操作的现状,运用,TALE,进行基因组操作,的特点和优势,TALE,的构建体系,TAL-TFs Validation,TALENs Validation,In vitro digestion of TALE Site,Mammalian cell based TALEN Assay,Surveyor Nuclease Assay,Knock-out and Knock-in based on TALENs,2,基因功能的确证,当该基因表达时，呈现某种功能,当该基因被敲除时，该功能消失或下降,当该基因回复时，该功能得到恢复,Gene trap,:,化学诱变、转座子,-,随机性的海量筛选,自,杀质粒，同源重,组,：,效率低（,1 HR/10,6,cells,），操作难,RNAi,：,不完全敲除、临时性,表达克隆瞬时转,染,：,操作简便、临,时性,病毒介导的整,合,：,整合位点不特异、潜在风险高,ZFN,：,难以完全找到匹配的,3,连子锌,指、易,Off-target,TALEN,：,识别任意目,标,序,列、效率高,-,基因操作技术的新里程碑,常用的基因操作技术,TALE,（,transcription,activator,like effector,）,1989,年，植物病原体黄单胞菌属（,Xanthomonas spp.,）,avrBs3,基因被克,隆,2007,年，,发现其序列特异性核酸结合特性，,avrBs3-,TA,2009,年，,TAL,effector,氨基酸序列与核酸靶序列的密码被破译,2011,年，,Nature Biotechonlogy,同时发表,4,篇,TALEN,基因敲除文章及一篇综述,AvrBs3/Bs3,UPA10,UPA12,UPA14,UPA19,UPA20,UPA21,UPA23,UPA25,AvrBs3Drep16/Bs3-E,AvrBs3Drep109/Bs3,AvrHah1/Bs3,AvrXa27/Xa27,PthXo1/Xa13,PthXo6/OsTFX1,and PthXo7/OsTFIIAg1,Boch J,et al.Breaking the code of DNA binding specificity of TAL-type III effectors.Science.2009;326:1509,RVD,Optimized Sequence(,H.sapiens,),Target Base,NI,CTGACCCCCGACCAGGTGGTGGCCATCGCCAGC,AACATC,GGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCCGTGCTGTGCCAGGACCACGGC,A,HD,CTGACCCCCGACCAGGTGGTGGCCATCGCCAGC,CACGAC,GGAGGCAAGCAGGCCCTGGAGACAGTGCAGAGGCTGCTGCCCGTGCTGTGCCAGGACCACGGC,C,NN,CTGACCCCCGACCAGGTGGTGGCCATCGCCAGC,AACAAC,GGCGGCAAGCAGGCCCTGGAGACAGTGCAGAGGCTGCTGCCCGTGCTGTGCCAGGACCACGGC,G/A,NG,CTGACCCCCGACCAGGTGGTGGCCATCGCCAGC,AACGGC,GGAGGCAAGCAGGCCCTGGAGACAGTGCAGAGGCTGCTGCCCGTGCTGTGCCAGGACCACGGC,T,Genecopoeia,的,TALE,识别靶点的构建体系,激,活基,因的转录,-TALE-TF,特,定序列进行切割,-TALEN,抑,制基因表达,-TALE-TR,启动子附近的甲基化,-TALEM,特定序列的重组酶,-TALER,。,Activators,Nucleases,Repressors,Mythylases,Recombinase,将识别特,DNA,序列的,TALE,与不同的蛋,白融,合，实现不同的靶向基因操作的应,用,TALE,的应用,TAL-TFs,激活任意目标基因的转录激活因子,TALE,与转录因子激活区域,VP64(VP64 Activation Domain),融,合,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,PolII,结合，从而激活基因的转,录,无,需构建表达克隆，启动基因转录表达,8,TAL-TFs,激,活转录示例,TGAGCGCGGAGCCATCTGGC,NTF3,TSS,TATA,+1,-46,5,3,12p13,Target site,Human NTF3 genomic context,NTF3 helps to support the survival and differentiation of existing neurons,and encourages the growth and differentiation of new neurons and synapses.NTF3 was the third neurotrophic factor to be characterized.NTF3,encodes a secreted nerve growth,factor that has therapeutic potential for neurodegenerative diseases.,HEK 293T cells transfected with the NTF3 TALE-TF(6 well plate,1 g plasmid per well)exhibited a 17-fold increase in the amount of NTF3 mRNA compared with empty vector-transfected cells.Measurements were performed in triplicate.,TALENs,识别任意特定核酸靶序列的重组核酸酶,构建,TALE,与,核酸内切酶的融合蛋,白,FokI,需形成二聚,体才具有活性，因,此在目标,DNA,中选择两,处靶序列，形成,TALEN,对,TALE,是识别靶基因，而,FokI,是起了切割,DNA,的作,用,特,异位点打断目标基因组,DNA,序列，从而可在该位点进行,DNA,编辑修饰操,作,DSB,NHEJ,HR,通,过,NHEJ,（,Non-homologous End Joining,）修复,DNA,，缺,乏修复模,板，在,此修过程中或多或少地删除或插入了一定数目的碱基，造成移码，形成目标基因敲除突变,体,如,果有同源修复模板，细胞可通过同源重组,HR(Homologous Recombination),方式修复,DNA,，如果在细胞中转入的质粒含有修复模板，就可以对目标,DNA,做修饰，如点突变、碱基替换、碱基磷酸化、加入标记,细胞内基因组,DNA,被,TALEN,剪切后，诱发,DNA,损伤修复机制,TALENs,靶向基因修饰的原理,enhancer,t,ranscription factor,promoter,exon1,exon2,exon3,UTR,intron,intron,intron,Add/delete transcription factor binding sites,i,ntegrate constitutive,inducible promoter,gene,knock-out,integrate,point mutations,Elongation,trunction,fuse,reporter gene,add/delete,miRNA regulate element,基于,TALEN,的基因,修饰,gene,knock-out,gene,knock-in,gene tagging,promoter swapping,nucleotide,substitution,protein truncation,reading-frame,disruption,m,odification of regulation by miRNA,运用,TALEN,进,行基,因修饰的,特点和优,势,无,基因序列、细胞、物种限,制,TAL,的核酸识别单元与,A,、,G,、,C,、,T,有恒定的对应关,系,，,整,个实验简单准,确,实,验周期,短,，,成,本低,毒性低、脱靶情况,少,，,成,功率可达,90%,以,上,靶,序列识别模块不受上下游影响，特异识别并打断基因组中的任,意序列,成,对的,TAL,识别模块保证了基因敲除靶点的准确,性,克,服了常规的,ZFN,方法不能识别任意目标基因序列，以及识别序列经常受上下游序列影响等问题，而具有,ZFN,相等或更好的活,性,成,功应用到了植物、细胞、酵母、斑马鱼及大、小鼠,等,TALEN,技术需要注意的问题,TALEN,的效率差异很大：,1%50%,染色体结构；,表观修饰（甲基化修饰）,SNP,位点,CNV,不,同细胞的效率有变化,需要有,TALEN,效率的检验和确证方法,Genecopoeia,多层次的,TALEN,功能验证,Western-blot,of TALENs expression in 293T cell,Digestion,in,vitro,Episomal cleavage,validation,Surrogate reporter assay(,HDR,Assay),Surveyor,nuclease,a,ssay,15,250,150,100,70,55,kDa,1 2 3,TALEN Expression Validation:,eGFP TALENs expression validation:80%confluence HEK293T cells were transfected with 0.8 g plasmid per well in a 6-well plate.The cells were harvested 48 hrs post-transfection.1/20,th,of the cell lysate per well was analyzed for western blot using anti-Flag antibody in a SDS-PAGE(8%resolve gel),with the un-transfected cell lysate as the negative control.,Expected size:110 kDa,1:Left TALEN,2:Right TALEN,3:Cells alone,NLS,N-terminus,3 Flag,18-mer TAL,0.5 TAL,C-terminus,Fok I C,TGCACCACCGGCAAGCTGCC,cgtgccctggcccacc,CTCGTGACCACCCTGACCTA,Left TALEN Target site,Right TALEN Target site,Target site(half-sites in CAPS,spacer in lowercase):,TALEN Structure,Western-blot detect the expression of TALEN,16,In vitro,target DNA cleavage by EGFP-TALENs.(A)The TALEN target plasmid(6110 bp)contains an unique EcoRI site and an eGFP TALEN target site.The two sites are1066 bp apart.(B)1 g of the plasmid was incubated with the indicated enzymes for 30 min at 37C.0.5 volume of the digestion reaction was analyzed by the agarose gel electrophoresis.*The indicated fragment was checked by PCR,data not shown,.,In vitro target DNA cleavage with Purified TALEN,s,Recombinant Proteins:,A pair iof TALEN gene were cloned into pET28a and expressed in BL21.,The His6-tagged TALENs were purified with Nickel column,17,EX-EGFP-Lv105(200 ng)+Control Plasmid(800 ng),EX-EGFP-Lv105(200 ng)+EGFP TALENs(800 ng),a,b,TALENs knockdown eGFP expression,TALENs knockdown eGFP expression:HEK293T cells in a 6-well plate were co-transfected with EX-EGFP-Lv105 and TALEN plasmid or control plasmid.EGFP expression was checked under microscope(Nikon Eclipse Ti,exposure time:600ms)48hrs post-transfection.,Episomal cleavage,validation,24h A,eGFP cut sample;,24h B,eGFP non-cut sample;,24h C,P53 non-cut sample;,24h D,P53 cut sample;,1 2 3 4,kDa,62,49,38,28,light,The,surrogated reporter plasmid was constructed by disrupting a CMV-driven Gaussia luciferase(GLuc)with a in-frame stop codon followed by eGFP TALEN target sequences.,The,donor plasmid contains a promoter-less wild type GLuc,which can replace the interrupted GLuc in the surrogate reporter plasmid and restore the GLuc expression through homologous recombination,which is enhanced by TALEN cleavage.,Validation of TALENs via HDR Assay,Validation of TALENs via HDR Assay,Restore disrupted,Gaussia,luciferase function via TALEN-mediated homologous recombination.,HEK293T,cells in a 6-well plate were co-transfected with the eGFP-TALEN pair(1 g),the surrogate reporter plasmid(0.5 g)and the donor plasmid(0.5 g).48hours post-transfection,the restored Gluc activity was determined to evaluate the TALEN function.Internal control SEAP activity was used for normalization.,A,:,shows,a pair of TALENs designed to,target.,B:,shows,NHEJ reaction after TALEN cleavage;,C:,schematic is,designed to detect TALE-Nuclease mediated cutting events at a defined genomic position.,The,PCR products were denatured and re-hybridized then treated with,T7 Endo,nuclease.The resulting cleavage products were separated on a 2%agarose gel and,imaged,D,.,The,efficiency was,2.5%,and,3.6%,at the,24,48-hour,and,72-hour,time-points,respectively.,Surveyor Nuclease Assay,A,B,C,D,通过,TALEN,介导的基因敲除,(knock-out),，该基因功能消失或下降,TALEN mediated Knock-out,TALEN mediated Knock-in,通过,TALEN,介,导基因,敲,入,(,Knock-in,）,，该基因功能得到恢复,应用,TALEN,技术进行基因功能的研究和确证,Safe Harbor,是指,基,因组上转录活跃的安全区域,，该,区域能有效地转录导入的外源基,因,同,时打断该区域也不会影响细胞的表,型,，,克,服,了随,机整,合中,常见的转基因沉默、癌变和插入突变等问题,。,AAVS1,是确证的、广泛使用,的,、,位,于人类,19,号染色体上的,Safe,Harbor,TALEN-mediated knock-in,at the safe-harbor,site,of,genome,Human genome safe harbor AAVS1 gene targeting,(B),AAVS1 donor control plasmid,pAAVS1D-RFP-copGFPpuro(800 ng)was co-transfected with AAVS1 TALEN pair(600 ng for each)or control TALEN pair into HEK293T cells in a 6-well pate.,(A),48 hr post-transfection,the cells were splited 1:10 into a new 6-well pate and selected,against 1.0 g/ml of puromycin.The images were taken after two weeks of selection.The cells with the transfection of pAAVS1D-RFP-copGFPpuro +control TALEN were completely killed by puromycin selection(data no shown).,(C)(D),PCR primers designed to detect the HR junction were used to validate the successful integration.,5 Primer Set(GCI),AAVS1,TALENs,Control,TALENs,1500,1250,1000,750,500,bp,TALEN safe-harbor Control,(A),(,B,),(C),(D),Components,Description,Qty,AAVS1,TALEN pair-,AAVS1 left TALEN and AAVS1 right TALEN,Specifically cleave AAVS1,target site to create DSBs,1 pair,AAVS1 donor cloning,vector,or,AAVS1 donor clone*,For cloning,GOI or containing GOI -Two AAVS1 flanking arms for HR,-Inducible or non-inducible promoter,-GFP/Puro marker for selection,-Over18,000 sequence-verified human full length ORFs,1,AAVS1,positive control donor clone,RFP,instead of GOI for positive control purpose,1,Knock-in,d,etection Kit,PCR mixture,and,primer pairs for 5 and 3 HR,j,unctions to verify successful integration,1,Genome-TALER,TM,Human AAVS1 safe harbor gene targeting kits,One of the biggest roadblocks to the application of programmable nucleases is,the lack of systems to enrich or select,gene-modified,cells.,nature,methods,2011.,VOL.8,941,How to selection and enrichment,Selection,and,enrichment of,TALEN modified cells,Single cell culture and PCR analysis to identify modified cells,Sorting or/and puromycin selection,HR,DSBs,TALEN pair,Chr,Chr,Chr,GFP,2A,EF1,Puro,L,R,Donor clone,Mutated,GFP,2A,EF1,Puro,Mutated,Sorting,and,puromycin selection,Loxp,Mutated,Cre,TALEN-mediated gene mutation,Sorting,and PCR analysis,28,HR,DSBs,TALEN pair,Chr,Chr,Chr,GFP,2A,EF1,Puro,R,Donor clone,Gene tag,GFP,2A,EF1,Puro,Sorting,and,puromycin selection,Loxp,Cre,TALEN-mediated gene tagging,L,Gene tag,Gene tag,Sorting,and PCR analysis,29,Genecopoeia,提供的,TALENs,和,TAL-TF,技术,服务,Customer,Tech support,TAL target site,Generate Cat No.by TALE interface,Target Gene,Design sequence,Enginee service,Value service,Premium service,Project service,engineering&sequencing,in vitro cleavage,surrogate reporter assay,Chromosomal-level functional validation,cloning and sequence analyze,expression and purification,construct cleavage substrate,cleavage assay,Design and construct assay substrate,surrogate assay,Design and validation primers,Nuclease survor assay,Generate Cat No.by TALE interface,TALEN case,engineering&sequencing,engineering&sequencing,in vitro cleavage,surrogate reporter assay,gene mutangesis,gene tagging,reading-frame disruption,promoter swapping,30,谢 谢,GeneCopoeia Inc.,广州复能基因有限公,司,电话：,4006 020 200,传真：,+86,（,20,）,3205 2877,地址：广州市科学城国际企业孵化器,D,区,8,楼,510663,订购：,sales,客服：,support,或,[phone],网址：,英,文：,32,表达谱分析,细胞分析,功能验证,杂交芯片,测,序,qPCR,