CN116887853A - 用于定点诱变的组合物和方法 - Google Patents
用于定点诱变的组合物和方法 Download PDFInfo
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- CN116887853A CN116887853A CN202180093810.4A CN202180093810A CN116887853A CN 116887853 A CN116887853 A CN 116887853A CN 202180093810 A CN202180093810 A CN 202180093810A CN 116887853 A CN116887853 A CN 116887853A
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Abstract
本公开提供了用于编辑双链DNA靶位点的改进的基因组编辑组合物和方法。本公开进一步提供了通过所描述的组合物和方法产生的基因组编辑的细胞。
Description
相关申请交叉引用
本申请根据35U.S.C.§119(e)要求于2020年12月21日提交的美国临时申请第63/128,391号的权益,所述美国临时申请通过引用以其整体并入本文。
关于序列表的声明
与本申请相关的序列表以文本形式提供以代替纸质副本,并且特此通过引用并入本说明书中。含有序列表的文本文件的名称为BLUE-132PC_ST25.txt。所述文本文件为541KB,创建于2021年12月15日并且通过EFS-Web以电子方式提交。
技术领域
本公开涉及改进的基因组编辑组合物。更具体地,本公开涉及包括DNA结合结构域和与核酸外切酶连接的归巢核酸内切酶变体的融合多肽、组合物以及使用其在细胞内对dsDNA进行定点诱变的方法。
背景技术
基因组编辑技术的相对较新的发展开辟了在几乎所有真核细胞和哺乳动物中直接靶向和修饰基因组序列的可能性。此类技术包含但不限于转录激活因子样效应物核酸酶(TALEN)、锌指核酸酶(ZFN)、成簇的规律间隔的短回文重复序列(CRISPR)-Cas相关核酸酶和归巢核酸内切酶(HE)。所有这些编辑技术的共同点是它们在靶核苷酸序列中产生断点,而天然细胞修复机制通过非同源末端连接(NHEJ)或同源定向修复(HDR)重新连接核苷酸序列。然而,修复并不总是完美的。因此,最终产物是包括多种基因损害中的任一种基因损害的核苷酸序列。
应用基因编辑核酸酶最常观察到的基因损害是插入和缺失(习惯上称为‘插入缺失(indel)’)。当双链DNA断裂(DSB)被NHEJ DNA修复机制加工和重新密封时,插入缺失出现。在不存在递送的过量同源DNA序列的情况下,NHEJ插入缺失通常占主导地位,所述递送的过量同源DNA序列可以将DSB转向各种同源重组结果。
每种基因损害的具体性质会导致不同的表型结果。例如,任何给定的基因损害都可能导致广泛的表型结果,从基因的完全敲除跨越到功能的获得或丧失,到完全没有表型影响。因此,越来越需要进一步理解在基因编辑时导致不同突变的机制,并且开发组合物和方法来实现治疗上有意义的基因编辑。
发明内容
本公开总体上部分地涉及融合多肽和使用其的方法,所述融合多肽包括DNA结合结构域、切割人基因中的靶位点的归巢核酸内切酶变体、连接子结构域和核酸外切酶。
一方面,提供了一种融合多肽,其包括DNA结合结构域和归巢核酸内切酶(HE)变体,所述HE变体结合并且切割细胞中的选定双链DNA(dsDNA)靶位点;连接子结构域;以及核酸外切酶或其生物活性片段。
在特定实施例中,所述核酸外切酶是Trex2、ExoI或ExoX或其生物活性片段。在一些实施例中,所述核酸外切酶是ExoX或其生物活性片段。在一些实施例中,所述核酸外切酶是ExoI或其生物活性片段。在一些实施例中,所述核酸外切酶是Trex2或其生物活性片段。
在各个实施例中,所述归巢核酸内切酶是工程化的归巢核酸内切酶。
在各个实施例中,所选择的dsDNA靶位点是非天然归巢核酸内切酶靶位点。
在各个实施例中,所述DNA结合结构域结合所述核酸内切酶dsDNA靶位点上游的dsDNA靶位点。在一些实施例中,所述DNA结合结构域包括TALE DNA结合结构域。在一些实施例中,所述TALE DNA结构域包括约9.5个TALE重复单元到约15.5个TALE重复单元。在一些实施例中,所述TALE DNA结构域包括11.5个TALE重复单元或12.5个TALE重复单元。在一些实施例中,所述DNA结合结构域包括锌指DNA结合结构域。在一些实施例中,所述锌指DNA结合结构域包括2个、3个、4个、5个、6个、7个或8个锌指基序。
在各个实施例中,所述连接子结构域是肽连接子。在一些实施例中,所述肽连接子是自切割肽连接子。在一些实施例中,所述肽连接子包括约4个至约30个氨基酸。在一些实施例中,所述肽连接子包括约10个至约16个氨基酸。在一些实施例中,所述肽连接子包括约12个氨基酸。在一些实施例中,所述肽连接子是(GGGGS)1-4连接子(SEQ ID No:117、150-152)。在一些实施例中,所述肽连接子包括(GGGGS)2连接子(SEQ ID NO:150)。
在各个实施例中,所述HE变体是LAGLIDADG归巢核酸内切酶(LHE)变体。在一些实施例中,与对应的野生型HE相比,所述HE变体缺少1个、2个、3个、4个、5个、6个、7个或8个N末端氨基酸。在一些实施例中,与对应的野生型HE相比,所述HE变体缺少4个N末端氨基酸。在一些实施例中,与对应的野生型HE相比,所述HE变体缺少8个N末端氨基酸。在一些实施例中,与对应的野生型HE相比,所述HE变体缺少1个、2个、3个、4个或5个C末端氨基酸。在一些实施例中,与对应的野生型HE相比,所述HE变体缺少C末端氨基酸。在一些实施例中,与对应的野生型HE相比,所述HE变体缺少2个C末端氨基酸。
在特定实施例中,所述HE变体是选自由以下组成的组的LHE的变体:I-AabMI、I-AaeMI、I-AniI、I-ApaMI、I-CapIII、I-CapIV、I-CkaMI、I-CpaMI、I-CpaMII、I-CpaMIII、I-CpaMIV、I-CpaMV、I-CpaV、I-CraMI、I-EjeMI、I-GpeMI、I-GpiI、I-GzeMI、I-GzeMII、I-GzeMIII、I-HjeMI、I-LtrII、I-LtrI、I-LtrWI、I-MpeMI、I-MveMI、I-NcrII、I-Ncrl、I-NcrMI、I-OheMI、I-OnuI、I-OsoMI、I-OsoMII、I-OsoMIII、I-OsoMIV、I-PanMI、I-PanMII、I-PanMIII、I-PnoMI、I-SceI、I-ScuMI、I-SmaMI、I-SscMI和I-Vdi141I。在一些实施例中,所述HE变体是选自由以下组成的组的LHE的变体:I-CpaMI、I-HjeMI、I-OnuI、I-PanMI和I-SmaMI。在优选的实施例中,所述HE变体是I-OnuI LHE变体。
在各个实施例中,所述HE靶位点位于免疫系统检查点基因、珠蛋白基因、编码有助于抑制γ-珠蛋白基因表达和HbF的多肽的基因或免疫抑制信号传导基因内。在一些实施例中,所述HE靶位点位于选自由以下组成的组的基因内:程序性细胞死亡蛋白1(PD-1;PDCD1)、淋巴细胞激活基因3蛋白(LAG-3)、T细胞免疫球蛋白结构域和粘蛋白结构域蛋白3(TIM-3)、细胞毒性T淋巴细胞抗原-4(CTLA-4)、带T淋巴细胞弱化因子(BTLA)、T细胞免疫球蛋白和基于免疫受体酪氨酸的抑制基序结构域(TIGIT)、T细胞激活的V结构域Ig抑制因子(VISTA)和杀伤细胞免疫球蛋白样受体(KIR)、CCR5、TRAC(TCRα)、TCRβ、IL10Rα、IL10Rβ、TGFBR1、TGFBR2、CBL-B、PCSK9、AHR、BTK、α-珠蛋白、β-珠蛋白、γ-珠蛋白、BCL11A、KLF1、SOX6、GATA1、LSD1、α叶酸受体(FRα)、αvβ6整合素、B细胞成熟抗原(BCMA)、B7-H3(CD276)、B7-H6、碳酸酐酶IX(CAIX)、CD16、CD19、CD20、CD22、CD30、CD33、CD37、CD38、CD44、CD44v6、CD44v7/8、CD70、CD79a、CD79b、CD123、CD133、CD138、CD171、癌胚抗原(CEA)、C型凝集素样分子-1(CLL-1)、CD2子集1(CS-1)、硫酸软骨素蛋白聚糖4(CSPG4)、皮肤T细胞淋巴瘤相关抗原1(CTAGE1)、表皮生长因子受体(EGFR)、表皮生长因子受体变体III(EGFRvIII)、上皮糖蛋白2(EGP2)、上皮糖蛋白40(EGP40)、上皮细胞粘附分子(EPCAM)、肝配蛋白A型受体2(EPHA2)、成纤维细胞激活蛋白(FAP)、Fc受体样5(FCRL5)、胎儿乙酰胆碱酯酶受体(AchR)、神经节苷脂G2(GD2)、神经节苷脂G3(GD3)、磷脂酰肌醇蛋白聚糖-3(GPC3)、包含ErbB2(HER2)的EGFR家族、IL-11Rα、IL-13Rα2、κ、癌/睾丸抗原2(LAGE-1A)、λ、路易斯-Y(Lewis-Y,LeY)、L1细胞粘附分子(L1-CAM)、黑色素瘤抗原基因(MAGE)-A1、MAGE-A3、MAGE-A4、MAGE-A6、MAGEA10、T细胞识别的黑色素瘤抗原1(MelanA或MART1)、间皮素(MSLN)、MUC1、MUC16、MHC I类链相关蛋白A(MICA)、MHC I类链相关蛋白B(MICB)、神经细胞粘附分子(NCAM)、癌/睾丸抗原1(NY-ESO-1)、聚唾液酸;胎盘特异性1(PLAC1)、黑色素瘤中优先表达的抗原(PRAME)、前列腺干细胞抗原(PSCA)、前列腺特异性膜抗原(PSMA)、受体酪氨酸激酶样孤儿受体1(ROR1)、滑膜肉瘤、X断点2(SSX2)、存活蛋白、肿瘤相关糖蛋白72(TAG72)、肿瘤内皮标志物1(TEM1/CD248)、肿瘤内皮标志物7相关(TEM7R)、TEM5、TEM8、滋养层糖蛋白(TPBG)、UL16结合蛋白(ULBP)1、ULBP2、ULBP3、ULBP4、ULBP5、ULBP6、血管内皮生长因子受体2(VEGFR2)和威尔姆斯瘤1(WT-1)基因。在一些实施例中,所述HE靶位点位于选自由以下组成的组的基因内:程序性细胞死亡蛋白1(PD-1;PDCD1)、淋巴细胞激活基因3蛋白(LAG-3)、T细胞免疫球蛋白结构域和粘蛋白结构域蛋白3(TIM-3)、细胞毒性T淋巴细胞抗原-4(CTLA-4)、带T淋巴细胞弱化因子(BTLA)、T细胞免疫球蛋白和基于免疫受体酪氨酸的抑制基序结构域(TIGIT)、T细胞激活的V结构域Ig抑制因子(VISTA)和杀伤细胞免疫球蛋白样受体(KIR)、CCR5、TRAC(TCRα)、IL10Rα、TGFBR2、CBL-B、PCSK9、AHR、BTK、α-珠蛋白、β-珠蛋白、γ-珠蛋白和BCL11A基因。在一些实施例中,所述HE靶位点位于TRAC(TCRα)基因、CBL-B基因或PDCD1(PD-1)基因内。在特定实施例中,所述TCRα基因靶位点包括SEQ ID NO:1中所示的氨基酸序列。在特定实施例中,所述CBL-B基因靶位点包括SEQ ID NO:2中所示的氨基酸序列。在特定实施例中,所述PD-1基因靶位点包括SEQ ID NO:3中所示的氨基酸序列。
在各个实施例中,所述DNA结合结构域包括具有如SEQ ID NO:4中所示的靶位点的TALE DNA结合结构域。
在各个实施例中,所述ExoX或其生物活性片段包括与如SEQ ID NO:109中所示的氨基酸序列具有至少85%、90%、95%、96%、97%、98%或99%同一性的氨基酸序列。在特定实施例中,所述ExoX或其生物活性片段包括如SEQ ID NO:109中所示的氨基酸氨基酸序列。
在各个实施例中,所述融合多肽包括与如SEQ ID NO:46、64、73和82中的任一个中所示的氨基酸序列具有至少85%、90%、95%、96%、97%、98%或99%同一性的氨基酸序列。在特定实施例中,所述融合多肽包括如SEQ ID NO:46、64、73和82中的任一个中所示的氨基酸序列。
在各个实施例中,所述ExoI或其生物活性片段包括与如SEQ ID NO:112中所示的氨基酸序列具有至少85%、90%、95%、96%、97%、98%或99%同一性的氨基酸氨基酸序列。在特定实施例中,所述ExoI或其生物活性片段包括如SEQ ID NO:112中所示的氨基酸氨基酸序列。
在各个实施例中,所述融合多肽包括与如SEQ ID NO:43中所示的氨基酸序列具有至少85%、90%、95%、96%、97%、98%或99%同一性的氨基酸序列。在一些实施例中,所述融合多肽包括如SEQ ID NO:43中所示的氨基酸序列。
在各个实施例中,提供了一种对本文所设想的融合多肽进行编码的多核苷酸。在一些实施例中,所述多核苷酸包括与如SEQ ID NO:44、62、71和80中的任一个中所示的多核苷酸序列具有至少85%、90%、95%、96%、97%、98%或99%同一性的核苷酸序列。在特定实施例中,所述多核苷酸包括如SEQ ID NO:44、62、71和80中的任一个中所示的多核苷酸序列。
在各个实施例中,提供了一种编码本文所设想的融合多肽的mRNA。在一些实施例中,所述mRNA包括与如SEQ ID NO:45、63、72和81中的任一个中所示的RNA序列具有至少85%、90%、95%、96%、97%、98%或99%同一性的RNA序列。在特定实施例中,所述mRNA包括如SEQ ID NO:45、63、72和81中的任一个中所示的RNA序列。在一些实施例中,所述mRNA包括与如SEQ ID NO:42中所示的RNA序列具有至少85%、90%、95%、96%、97%、98%或99%同一性的RNA序列。在特定实施例中,所述mRNA包括如SEQ ID NO:42中的任一个所示的RNA序列。
在各个实施例中,提供了一种编码本文所设想的融合多肽的载体。在一些实施例中,所述载体包括编码本文所设想的融合多肽的多核苷酸。
在各个实施例中,提供了一种细胞,所述细胞包括编码本文所设想的融合多肽的融合多肽。在一些实施例中,所述细胞包括编码本文所设想的融合多肽的多核苷酸。在一些实施例中,包括编码本文所设想的融合多肽的mRNA。在一些实施例中,所述细胞包括本文所设想的载体。在一些实施例中,所述细胞包括一个或多个基因组修饰。
在各个实施例中,所述细胞是造血细胞。在一些实施例中,所述细胞是造血干细胞或祖细胞。在一些实施例中,所述细胞是CD34+细胞。在一些实施例中,所述细胞是CD133+细胞。
在各个实施例中,所述细胞是免疫效应细胞。在一些实施例中,所述免疫效应细胞是细胞毒性T淋巴细胞(CTL)、肿瘤浸润淋巴细胞(TIL)或辅助T细胞。在一些实施例中,所述免疫效应细胞是T细胞。在一些实施例中,所述免疫效应细胞是αβT细胞、γδT细胞、自然杀伤(NK)细胞或自然杀伤T(NKT)细胞。
在各个实施例中,提供了一种本文所设想的细胞群体。
在各个实施例中,提供了一种组合物,其包括本文所设想的融合多肽。在一些实施例中,提供了一种组合物,其包括本文所设想的多核苷酸。在一些实施例中,提供了一种组合物,其包括本文所设想的mRNA。在一些实施例中,提供了一种组合物,其包括本文所设想的载体。在一些实施例中,提供了一种组合物,其包括本文所设想的细胞。在一些实施例中,提供了一种组合物,其包括本文所设想的细胞群体。在特定实施例中,所述组合物包括药学上可接受的载体。
另一方面,提供了一种定点诱变方法,所述方法包括:(a)选择双链DNA(dsDNA)靶位点;以及(b)向细胞中引入本文所设想的融合多肽、多核苷酸、mRNA或载体;其中融合肽产生定向偏向的缺失,所述定向偏向的缺失的缺失中心位置靠近所述细胞中的选定dsDNA靶切割位点。
在各个实施例中,大于50%、大于51%、大于52%、大于53%、大于54%、大于55%、大于56%、大于57%、大于58%、大于59%、大于60%、大于65%、大于70%、大于75%或大于80%的所述定向偏向的缺失的缺失中心位置位于HE靶位点中心位置的一侧上。
在各个实施例中,相对于所述HE靶位点中心位置,所述缺失中心位置与所述DNA结合结构域靶位点位于同一侧上。在特定实施例中,所述缺失中心位置位于HE靶位点中心位置的5'端。
在各个实施例中,至少50%、至少51%、至少52%、至少53%、至少54%、至少55%、至少56%、至少57%、至少58%、至少59%、至少60%、至少65%、至少70%、至少75%或至少80%的缺失的缺失中心距所述HE靶位点中心位置大于4个核苷酸。
在各个实施例中,至少10%、至少11%、至少12%、至少13%、至少14%、至少15%、至少16%、至少17%、至少18%、至少19%、至少20%、至少25%、至少30%或至少35%的缺失的缺失中心距所述HE靶位点中心位置大于8个核苷酸。
在各个实施例中,至少50%、至少51%、至少52%、至少53%、至少54%、至少55%、至少56%、至少57%、至少58%、至少50%、至少59%、至少60%、至少65%、至少70%、至少75%或至少80%的缺失的长度为6bp或更长。
在各个实施例中,至少30%、至少31%、至少32%、至少33%、至少34%、至少35%、至少36%、至少37%、至少38%、至少39%、至少40%、至少45%、至少50%、至少55%或至少60%的缺失的长度为12bp或更长。
在各个实施例中,所述定向偏向的缺失的长度包括约10个、约11个、约12个、约13个、约14个、约15个、约16个、约17个、约18个、约19个或约20个核苷酸。
在各个实施例中,所述缺失延伸到DNA结合结构域靶位点中。在一些实施例中,所述缺失中心位置位于所述DNA结合结构域靶位点内。
在各个实施例中,所述方法进一步包括向所述细胞中引入末端加工酶或其生物活性片段。在一些实施例中,末端加工酶或其生物活性片段选自由以下组成的组:Trex2、Trex1、不含跨膜结构域的Trex1、Apollo、Artemis、DNA2、ExoI、ExoT、ExoIII、ExoX、Fen1、Fan1、MreII、Rad2、Rad9、TdT(末端脱氧核苷酸转移酶)、PNKP、RecE、RecJ、RecQ、λ核酸外切酶、Sox、牛痘DNA聚合酶、核酸外切酶I、核酸外切酶III、核酸外切酶VII、NDK1、NDK5、NDK7、NDK8、WRN、T7-核酸外切酶基因6、禽类成髓细胞瘤病毒整合蛋白(IN)、Bloom、热敏磷酸酶(Antarctic Phosphatase)、碱性磷酸酶、多核苷酸激酶(PNK)、ApeI、绿豆核酸酶(MungBean nuclease)、Hex1、TTRAP(TDP2)、Sgs1、Sae2、CUP、Polμ、Polλ、MUS81、EME1、EME2、SLX1、SLX4以及UL-12。在一些实施例中,所述末端加工酶是核酸外切酶或其生物活性片段。在特定实施例中,核酸外切酶是Trex2或其生物活性片段。
在各个实施例中,所述方法为体外方法。在各个实施例中,所述方法为离体方法。在各个实施例中,所述方法为体内方法。
另一方面,提供了一种治疗、预防或改善疾病或与其相关的病状的至少一种症状的方法,所述方法包括:从受试者采集细胞群体;根据本文所设想的方法编辑所述细胞群体;以及向所述受试者施用经编辑的细胞群体。
另一方面,提供了一种本文所设想的细胞用于治疗、预防或改善疾病或与其相关的病状的至少一种症状的用途。
另一方面,提供了一种本文所设想的群体用于治疗、预防或改善疾病或与其相关的病状的至少一种症状的用途。
另一方面,提供了一种本文所设想的组合物用于治疗、预防或改善疾病或与其相关的病状的至少一种症状的用途。
在各个实施例中,所述疾病或病状是免疫病症或癌症。
附图说明
图1A示出了TALE DNA结合结构域与归巢核酸内切酶连接的草图,所述核酸内切酶被重新编程以靶向TCRα核苷酸序列(TCRαmegaTAL)。
图1B示出了TCRαmegaTAL的下一代测序(NGS)的插入缺失活性。含有基因编辑事件的插入缺失特性的NGS数据读段根据其长度制成表格。
图2示出了如通过NGS评估的低活性TCRαmegaTAL和高活性TCRαmegaTAL两者在两个不同供体中的插入缺失位置分布(“指纹”)。NGS数据根据其长度和其相对于megaTAL引起的断点中心的纵向位置两者制成表格。此分析排除了插入。
图3A-3C示出了通过NGS进行的对TCRαmegaTAL(图3A)、TCRαmegaTAL-Trex2融合体(图3B)和TCRαmegaTAL+Trex2共表达(图3C)的插入缺失活性。含有基因编辑事件的插入缺失特性的NGS数据读段根据其长度制成表格。
图4示出了如通过NGS评估的针对TCRαmegaTAL、TCRαmegaTAL-Trex2融合体以及TCRαmegaTAL+Trex2共表达的插入缺失位置分布(“指纹”)。NGS数据根据其长度和其相对于megaTAL引起的断点中心的纵向位置两者制成表格。此分析排除了插入。
图5示出了与各种Trex2同源物融合的TCRαmegaTAL的编辑效率,如通过对细胞表面上与TCRα和β链组合的CD3组分进行染色,然后进行流式细胞仪分析来评估的。
图6示出了通过NGS进行的针对与Trex2同源物融合的TCRαmegaTAL的插入缺失活性。含有基因编辑事件的插入缺失特性的NGS数据读段根据其长度制成表格。
图7示出了如通过NGS评估的与Trex2鸭嘴兽、负鼠、人类和小鼠同源物融合的TCRαmegaTAL的插入缺失位置分布(“指纹”)。NGS数据根据其长度和其相对于megaTAL引起的断点中心的纵向位置两者制成表格。此分析排除了插入。
图8示出了在有或没有小鼠Trex2的共表达的情况下,与核酸外切酶RAD1、RAD9A、ExoI、ExoX、T5FEN、lanbdaExo和RecJ融合的TCRαmegaTAL的编辑效率。Trex2同源物,如通过CD3表达进行染色,然后进行流式细胞术分析来评估的。
图9示出了在有或没有Trex2共表达的情况下,如通过NGS评估的针对仅TCRαmegaTAL以及与核酸外切酶ExoI或ExoX融合的TCRαmegaTAL的插入缺失位置分布(“指纹”)。NGS数据根据其长度和其相对于megaTAL引起的断点中心的纵向位置两者制成表格。此分析排除了插入。
图10示出了如通过NGS评估的单独的或与Trex2或ExoX融合的高活性CBL-BmegaTAL的插入缺失位置分布(“指纹”)。NGS数据根据其长度和其相对于megaTAL引起的断点中心的纵向位置两者制成表格。此分析排除了插入。
图11示出了如通过NGS评估的单独的或与Trex2或ExoX融合的低活性CBL-BmegaTAL的插入缺失位置分布(“指纹”)。NGS数据根据其长度和其相对于megaTAL引起的断点中心的纵向位置两者制成表格。此分析排除了插入。
图12示出了如通过NGS评估的单独的或与Trex2或ExoX融合的PD-1megaTAL的插入缺失位置分布(“指纹”)。NGS数据根据其长度和其相对于megaTAL引起的断点中心的纵向位置两者制成表格。此分析排除了插入。
图13示出了PDCD1基因、22个碱基对PD-1megaTAL靶位点和13个碱基TALE阵列结合位点(SEQ ID NO:154和159),以及来自图12所示实验的4个代表性缺失物种(SEQ ID NO:155-158和160-162)。
图14示出了如通过NGS评估的单独的或与Trex2或ExoX融合的PD-1megaTAL的插入缺失位置分布(“指纹”)。缺失物质被编码以指示其所得阅读框类别。NGS数据根据其长度和其相对于megaTAL引起的断点中心的纵向位置两者制成表格。此分析排除了插入。
图15示出了定量图14所示的四种缺失物种类别中的每种类别的归一化的分数的堆积直方图。
图16示出了用聚腺苷酸化mRNA电穿孔的激活的原代人T细胞的流式细胞术分析,所述mRNA编码以下中的任一者或两者:用于追踪转染效率的青色荧光蛋白(CFP),以及在三个可能的阅读框中的每个阅读框中的野生型或模拟物编辑的PD-1等位基因。
图17示出了通过NGS评估的低编辑TCRαmegaTAL、高编辑TCRαmegaTAL(TCRα2.2)以及各自与Trex2或ExoX的直接融合体的插入缺失位置分布(“指纹”)。NGS数据根据其长度和其相对于megaTAL引起的断点中心的纵向位置两者制成表格。此分析排除了插入。
图18示出了在有和没有与Trex2或ExoX直接融合的情况下,低编辑TCRαmegaTAL在已知KAT2B脱靶位点处的插入缺失位置分布(“指纹”)。NGS数据根据其长度和其相对于megaTAL引起的断点中心的纵向位置两者制成表格。此分析排除了插入。
图19示出了在有和没有与ExoX直接融合的情况下,高编辑TCRαmegaTAL(TCRα2.2)在已知AC016700.3脱靶位点(SEQ ID NO:163和164)处的插入缺失位置分布(“指纹”)。NGS数据根据其长度和其相对于megaTAL引起的断点中心的纵向位置两者制成表格。此分析排除了插入。
序列标识符简要说明
SEQ ID NO:1-3是说明性归巢核酸内切酶靶位点。
SEQ ID NO:4是PD-1TALE阵列靶位点。
SEQ ID NO:5-7是低活性TCRαmegaTAL DNA、RNA和蛋白质序列。
SEQ ID NO:8-10是高活性TCRαmegaTAL DNA、RNA和蛋白质序列。
SEQ ID NO:11-13是低活性TCRαmegaTAL-Trex2 DNA、RNA和蛋白质序列。
SEQ ID NO:14-16是Trex2核酸外切酶DNA、RNA和蛋白质序列。
SEQ ID NO:17-55是与Trex2同源物融合的低活性TCRαmegaTAL的DNA、RNA和蛋白质序列。
SEQ ID NO:56-64是与Trex2或ExoX融合或不融合的高活性CBL-BmegaTAL的DNA、RNA和蛋白质序列。
SEQ ID NO:65-73是与Trex2或ExoX融合或不融合的低活性CBL-BmegaTAL的DNA、RNA和蛋白质序列。
SEQ ID NO:74-82是与Trex2或ExoX融合或不融合的PD-1megaTAL的DNA、RNA和蛋白质序列。
SEQ ID NO:83是编码PD-1的mRNA序列。
SEQ ID NO:84是编码模拟物编辑的PD-1开放阅读框(ORF)的mRNA序列,其在密码子3处包括1bp缺失。
SEQ ID NO:85是编码模拟物编辑的PD-1开放阅读框(ORF)的mRNA序列,其在密码子3处包括2bp缺失。
SEQ ID NO:86是编码模拟物编辑的PD-1开放阅读框(ORF)的mRNA序列,其在密码子3处包括3bp缺失。
SEQ ID NO:87-101是TCRα、CBL-B和PD-1归巢核酸内切酶的DNA、RNA和蛋白质序列。
SEQ ID NO:102-106是野生型I-OnuI核酸内切酶和其各部分。
SEQ ID NO:107-109是ExoX核酸外切酶DNA、RNA和蛋白质序列。
SEQ ID NO:110-112是ExoI核酸外切酶DNA、RNA和蛋白质序列。
SEQ ID NO:113-123示出了各个连接子的氨基酸序列。
SEQ ID NO:124-148示出了蛋白酶切割位点和自切割多肽切割位点的氨基酸序列。
在前述序列中,X(如果存在的话)是指任何氨基酸或不存在氨基酸。
具体实施方式
A.概述
本公开总体上部分涉及经改进的基因组编辑组合物及其使用方法。不希望受任何特定理论的束缚,本文所设想的基因组编辑组合物用于1)将由基因编辑诱导的缺失的大小增加到特定的目标大小,和2)使缺失中心位置偏向。在特定实施例中,相对于原始断点中心或核酸内切酶靶位点中心,缺失中心位置主要与DNA结合结构域靶位点位于相同的一侧上。在特定实施例中,缺失中心位置偏向原始断点中心或核酸内切酶靶位点中心的5'侧。进一步设想了通过控制断点处或其附近的基因损害的大小和位置,可以更精确地控制基因组编辑的表型结果,例如,调节性DNA序列和/或靶基因表达的破坏。
应用基因编辑核酸酶最常观察到的基因损害是NHEJ插入缺失。关于NHEJ机制如何达到被重新密封并对进一步的DNA切割具有抗性的给定的插入缺失,从而成为基因编辑的等位基因库中的永久基因型,知之甚少。然而,已知插入缺失几乎只在核酸酶产生的断裂点出现的DNA序列处观察到,并且直接接近所述序列。先前对各种核酸酶平台(ZFN、TALEN和CRISPR)的研究表明,基因编辑的等位基因在观察到的插入缺失的定性性质方面显示出一定量度的一致性。这意味着有决定性的生物物理和/或生物化学过程控制着基因编辑结果。然而,尽管在不同DNA靶位点的不同基因编辑核酸酶产生的编辑的等位基因的观察谱中存在一些细微差异,但影响插入缺失性质的能力仍然难以捉摸。
定义给定插入缺失的定性性质是:(i)其长度,以插入或缺失的碱基数量表示;(ii)其沿染色体的纵向位置,通常相对于核酸酶的靶位点或断点而言;以及(iii)对于插入,插入的序列的长度和组成。缺失是最显著的结果,通常占观察到的事件的90-95%。它们最常报道的大小特性趋于变小(即,长度为1-20个碱基对,频率偏向所述范围的低端),并且已经发现它们的位置分布是均匀分布的,覆盖了DNA断裂点并且在没有明显偏向的情况下向两个方向向外发散。这些性质的例外经常被假设为由定位于DNA断裂点两侧的微同源物(长度为大约3-6个碱基对的小的重复片段)驱动。在基因组编辑工具的应用期间,很少有关于插入性质的报道。另外地,使每个插入缺失物种与表型相关的基因型特征(例如,它如何影响开放阅读框或它是否破坏转录因子结合基序)对于每个给定的应用来说是潜在巨大的和特异的。
本文设想了具有不同编辑模式(例如,缺失)的独特融合多肽,所述融合多肽可用于精确控制基因组编辑的表型结果。在各个实施例中,融合多肽包括DNA结合结构域、归巢核酸内切酶变体和末端加工酶(例如,核酸外切酶)。在某些实施例中,所述核酸外切酶是ExoX、ExoI或其生物活性片段。在特定实施例中,融合多肽诱导延长的遗传缺失,所述缺失在与DNA结合结构域靶位点相同的一侧或原始编辑/断点的5'端具有定向偏向的缺失中心。在特定实施例中,融合多肽产生由DNA结合结构域靶位点包围的缺失中心位置。在特定实施例中,融合多肽产生延伸到DNA结合结构域靶位点中(或被其包围)的缺失。
在特定实施例中,融合多肽包括DNA结合结构域和结合并切割细胞中的选定双链DNA(dsDNA)靶位点的归巢核酸内切酶(HE)变体、多肽连接子和核酸外切酶(例如,ExoX或ExoI)或其生物活性片段。
在各个实施例中,设想了编码融合多肽的载体、多核苷酸、mRNA或cDNA。在各个实施例中,设想了经基因组编辑的细胞。在各个实施例中,设想了包括融合多肽、载体、多核苷酸、mRNA、cDNA或细胞的组合物。
在各个实施例中,设想了基因组编辑、定点诱变、增加定点诱变缺失的长度、使缺失的位置偏向以及治疗有需要的受试者的方法。
因此,与现有的基因编辑策略相比,本文所设想的组合物和方法表现出显著的改进,因为它允许对期望的诱变结果进行策略控制和选择。
用于重组(即,经工程化的)DNA、肽和寡核苷酸合成、免疫测定、组织培养、转化(例如,电穿孔、脂质转染)、酶促反应、纯化的技术以及相关技术和程序总体上可以如本说明书通篇引用和讨论的微生物学、分子生物学、生物化学、分子遗传学、细胞生物学、病毒学和免疫学中的各种通用且更具体的参考文献中所描述的来执行。参见例如,Sambrook等人,《分子克隆:实验室手册(Molecular Cloning:ALaboratory Manual)》,第3版,纽约冷泉港冷泉港实验室出版社(Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.);《当代分子生物学实验指南(Current Protocols in Molecular Biology)》(约翰·威利父子出版公司(Wiley and Sons),2008年7月更新);《精编分子生物学实验指南:当代分子生物学实验指南的方法概要(Short Protocols in Molecular Biology:A Compendium ofMethods from Current Protocols in Molecular Biology)》,格林出版协会和威利跨学科出版社(Greene Pub.Associates and Wiley-Interscience);Glover,《DNA克隆:实用方法(DNA Cloning:A Practical Approach)》,第I卷和第II卷(美国牛津大学出版社IRL出版社(IRL Press,Oxford Univ.Press USA),1985);《当代免疫学手册(Current Protocolsin Immunology)》,编辑:John E.Coligan、Ada M.Kruisbeek、David H.Margulies、EthanM.Shevach、Warren Strober 2001纽约州纽约市约翰威立父子公司(John Wiley&Sons,NY,NY);《实时PCR:当前技术和应用(Real-Time PCR:Current Technology andApplications)》,编辑:Julie Logan、Kirstin Edwards和Nick Saunders,2009,英国诺福克凯斯特学术出版社(Caister Academic Press,Norfolk,UK);Anand,《复杂基因组分析技术(Techniques for the Analysis of Complex Genomes)》,(纽约学术出版社公司(Academic Press),1992);Guthrie和Fink,《酵母遗传学和分子生物学指南(Guide toYeast Genetics and Molecular Biology)》,(纽约学术出版社公司,1991);《寡核苷酸合成(Oligonucleotide Synthesis)》(N.Gait编辑,1984);《核酸:杂交(Nucleic Acid TheHybridization)》(B.Hames和S.Higgins编辑,1985);《转录和转译(Transcription andTranslation)》(B.Hames和S.Higgins编辑,1984);《动物细胞培养(Animal CellCulture)》(R.Freshney编辑,1986);Perbal,《分子克隆实用指南(A Practical Guide toMolecular Cloning)》(1984);《下一代基因组测序(Next-Generation GenomeSequencing)》(Janitz,2008Wiley-VCH出版社(Wiley-VCH));《PCR方案(分子生物学方法)(PCR Protocols(Methods in Molecular Biology))》(Park编辑,第3版,2010,胡马纳出版社(Humana Press));《固定化细胞和酶(Immobilized Cells And Enzymes)》(IRL出版社,1986);论文《酶学方法(Methods In Enzymology)》(纽约学术出版社公司);《哺乳动物细胞的基因转移载体(Gene Transfer Vectors For Mammalian Cells)》(J.H.Miller和M.P.Calos编辑,1987,冷泉港实验室出版社);Harlow和Lane,《抗体(Antibodies)》,(纽约冷泉港冷泉港实验室出版社,1998);《细胞和分子生物学中的免疫化学方法(Immunochemical Methods In Cell And Molecular Biology)》(Mayer和Walker编辑,伦敦学术出版社公司(Academic Press,London),1987);《实验免疫学手册(Handbook OfExperimental Immunology)》,第I-IV卷(D.M.Weir和CC Blackwell编辑,1986);Roitt,《基础免疫学(Essential Immunology)》,第6版,(牛津布莱克韦尔科学出版社(BlackwellScientific Publications,Oxford),1988);《当代免疫学方案》(Q.E.Coligan、A.M.Kruisbeek、D.H.Margulies、E.M.Shevach和W.Strober编辑,1991);《免疫学年度评论(Annual Review of Immunology)》;以及如《免疫学进展(Advances in Immunology)》等期刊上的专著。
B.定义
在更加详细地阐述本公开之前,提供本文要使用的某些术语的定义可能有助于理解本公开。
除非另外定义,否则本文所用的所有技术和科学术语都具有与本发明所属领域的一般技术人员通常所了解相同的含义。尽管可以使用与本文所描述的方法和材料类似或相当的任何方法和材料来实践或测试特定实施例,但本文中描述组合物、方法和材料的优选实施例。出于本公开的目的,下文定义以下术语。贯穿本公开阐述了另外的定义。
本文使用的冠词“一(a/an)”是指一个(种)或超过一个(种)(即,至少一个(种)或一个(种)或多个(种))该冠词的语法对象。举例来说,“一要素”意指一个要素或一个或多个要素。
替代方案(例如,“或”)的使用应理解为意指替代方案中的任一个、两个或其任何组合。
术语“和/或”应理解为意指替代方案中的一种或两种。
如本文所用,术语“约”或“大约”是指相对于参考数量、水平、值、数目、频率、百分比、尺寸、大小、量、重量或长度变化高达15%、10%、9%、8%、7%、6%、5%、4%、3%、2%或1%的数量、水平、值、数目、频率、百分比、尺寸、大小、量、重量或长度。在一个实施例中,术语“约”或“大约”是指参考数量、水平、值、数目、频率、百分比、尺寸、大小、量、重量或长度的±15%、±10%、±9%、±8%、±7%、±6%、±5%、±4%、±3%、±2%或±1%的数量、水平、值、数目、频率、百分比、尺寸、大小、量、重量或长度。
在一个实施例中,范围,例如,1至5、约1至5或约1至约5指代所述范围所涵盖的每个数值。例如,在一个非限制性且仅说明性实施例中,范围“1至5”相当于表达1、2、3、4、5;或1.0、1.5、2.0、2.5、3.0、3.5、4.0、4.5或5.0;或1.0、1.1、1.2、1.3、1.4、1.5、1.6、1.7、1.8、1.9、2.0、2.1、2.2、2.3、2.4、2.5、2.6、2.7、2.8、2.9、3.0、3.1、3.2、3.3、3.4、3.5、3.6、3.7、3.8、3.9、4.0、4.1、4.2、4.3、4.4、4.5、4.6、4.7、4.8、4.9或5.0。
如本文所用,术语“基本上”是指数量、水平、值、数目、频率、百分比、尺寸、大小、量、重量或长度是参考数量、水平、值、数目、频率、百分比、尺寸、大小、量、重量或长度的80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或更高百分比。在一个实施例中,“基本上相同”是指数量、水平、值、数目、频率、百分比、尺寸、大小、量、重量或长度产生的作用,例如,生理作用与参考数量、水平、值、数目、频率、百分比、尺寸、大小、量、重量或长度大约相同。
在本说明书通篇,除非上下文另外要求,否则“包含(comprise/comprises/comprising)”一词应理解为暗指包括所述步骤或要素或一组步骤或要素,但不排除任何其它步骤或要素或任何其它组步骤或要素。“由…组成”意指包含且限于短语“由…组成”之后的事物。因此,短语“由…组成”指示所列要素是必不可少或必需的,并且不能存在其它要素。“基本上由…组成”意指包含在所述短语之后列出的任何元件,并且限于不干扰或促进本公开中对所列元件指定的活动或动作的其它元件。因此,短语“基本上由……组成”指示所列要素是必需的或强制性的,但不存在实质上影响所列要素的活动或动作的其它要素。
贯穿本说明书中对“一个实施例”、“实施例”、“特定实施例”、“相关实施例”、“某个实施例”、“另外的实施例”或“进一步的实施例”或其组合的引用意味着结合所述实施例所描述的特定特征、结构或特性包含在至少一个实施例中。因此,上述短语在贯穿本说明书的各个地方的出现不一定全部是指同一个实施例。此外,特定特征、结构或特性可以通过任何适合方式组合于一个或多个实施例中。还应理解的是,在一个实施例中对特征的肯定叙述充当在特定实施例中排除所述特征的基础。
术语“离体(ex vivo)”通常是指在生物体外发生的活动,如在生物体外的人工环境中的活组织中或上进行的实验或测量,优选地具有最小的自然条件改变。在特定实施例中,“离体”程序涉及从生物体取得并在实验室设备中通常在无菌条件下并且通常在几小时或高达约24小时但包含高达48小时或72小时(视情况而定)内培养或调节的活细胞或活组织。在某些实施例中,可以收集和冷冻这种组织或细胞,并且随后将其解冻用于离体治疗。使用活细胞或组织持续时间长于几天的组织培养实验或程序通常被视为“体外”,但在某些实施例中,这个术语可以与离体互换使用。
术语“体内”一般是指在生物体内部进行的活动。在一个实施例中,细胞基因组在体内被工程化、编辑或修饰。
“增强(enhance)”或“促进(promote)”或“增加(increase)”或“扩展(expand)”或“加强(potentiate)”通常是指与由媒剂或对照物引起的应答相比,本文所设想的产生、引发或引起更大应答(即,生理应答)的融合多肽、核酸酶变体、基因组编辑组合物或经基因组编辑的细胞的能力。可测量的应答可以包含编辑事件(例如,插入缺失)、缺失、插入、缺失长度和/或靶基因表达的增加,以及从本领域的理解和本文的描述中显而易见的其它增加。“增加的”或“增强的”量通常是“统计上显著”的量并且可以包含是由媒剂或对照组合物产生的应答的1.1倍、1.2倍、1.5倍、2倍、3倍、4倍、5倍、6倍、7倍、8倍、9倍、10倍、15倍、20倍、30倍或更多倍(例如,500倍、1000倍)(包括其间且在1以上的所有整数和小数点,例如,1.5、1.6、1.7、1.8等)于由媒剂或对照物产生的应答的增加。
“减少(decrease或lower或lessen或reduce)”或“减轻(abate)”或“消除(ablate)”或“抑制(inhibit或dampen)”通常是指与由媒剂或对照物引起的应答相比,本文所设想的融合多肽、核酸酶变体、基因组编辑组合物或经基因组编辑的细胞产生、引发或引起更小的应答(即,生理应答)的能力。可测量的应答可以包含编辑事件(例如,插入缺失)、缺失、插入、缺失长度、靶基因表达和/或一种或多种与疾病相关的症状的减少。“减小的”或“减少的”量通常是“统计上显著”的量并且可以包含是由媒剂、对照组合物或特定细胞谱系中的应答产生的应答(参考应答)的1.1倍、1.2倍、1.5倍、2倍、3倍、4倍、5倍、6倍、7倍、8倍、9倍、10倍、15倍、20倍、30倍或更多倍(例如,500倍、1000倍)(包含其间且在1以上的所有整数和小数点,例如1.5、1.6、1.7、1.8等)于由媒剂或对照物产生的应答(参考应答)的减少。
“维持(maintain)”或“保持(preserve)”或“维持(maintenance)”或“无变化(nochange)”或“无实质性变化(no substantial change)”或“无实质性减少(no substantialdecrease)”通常是指与由媒剂或对照物引起的应答相比,本文所设想的融合多肽、核酸酶变体、基因组编辑组合物或经基因组编辑的细胞产生、引发或引起基本相似或相当的生理应答(即,下游效应)的能力。相当的应答是与参考应答没有显著差异或可测量的差异的应答。
如本文所用,术语“特异性结合亲和力(specific binding affinity)”或“特异性结合(specifically binds)”或“特异性结合(specifically bound)”或“特异性结合(specific binding)”或“特异性地靶向(specifically targets)”描述了与背景结合相比,一个分子以更大的结合亲和力与另一个分子结合,例如多肽的DNA结合结构域与DNA结合。如果结合结构域以例如大于或等于约105M-1的亲和力或Ka(即,特定结合相互作用的以1/M为单位的平衡缔合常数)与靶位点结合或相关联,则所述结合被称为与靶位点“特异性结合”。在某些实施例中,结合结构域与靶位点以大于或等于约106M-1、107M-1、108M-1、109M-1、1010M-1、1011M-1、1012M-1或1013M-1的Ka结合。“高亲和力”结合结构域是指Ka为至少107M-1、至少108M-1、至少109M-1、至少1010M-1、至少1011M-1、至少1012M-1、至少1013M-1或更大的那些结合结构域。
可替代地,亲和力可以被定义为以M为单位的特定结合相互作用的平衡解离常数(Kd)(例如,10-5M至10-13M或更低)。包括用于特定实施例中所设想的DNA靶位点的一个或多个DNA结合结构域的核酸酶变体的亲和力可以使用常规技术例如酵母细胞表面展示、或通过结合缔合、或使用标记配体的置换分析法容易地确定。
在一个实施例中,特异性结合亲和力为背景结合的约2倍、为背景结合的约5倍、为背景结合的约10倍、为背景结合的约20倍、为背景结合的约50倍、为背景结合的约100倍或为背景结合的约1000倍或更多倍。
术语“选择性结合(selectively binds)”或“选择性结合(selectively bound)”或“选择性结合(selectively binding)”或“选择性地靶向(selectively targets)”描述了在存在多个脱靶分子的情况下一个分子与靶分子的优先结合(中靶结合)。在特定的实施例中,HE或megaTAL选择性地结合中靶DNA结合位点比HE或megaTAL结合脱靶DNA结合位点更频繁约5倍、10倍、15倍、20倍、25倍、50倍、100倍或1000倍。
“中靶(on-target)”是指靶位点序列。
“脱靶(off-target)”是指与靶位点序列相似但不相同的序列。
“靶位点(target site)”或“靶序列(target sequence)”是染色体或染色体外核酸序列,其限定核酸的结合分子将与其结合和/或将切割的一部分,条件是存在充分的结合和/或切割条件。当参考多核苷酸序列或参考仅引用靶位点或靶序列的一条链的SEQ ID NO时,将应理解,由核酸酶变体结合和/或切割的靶位点或靶序列是双链的并且包括参考序列及其互补序列。在各个实施例中,所述靶位点位于免疫系统检查点基因、珠蛋白基因、编码有助于抑制γ-珠蛋白基因表达和/或HbF的多肽的基因或免疫抑制信号传导基因中。在某些实施例中,所述靶位点是人TRAC基因、CBL-B基因或PDCD1基因中的序列。
“重组(recombination)”是指在两个多核苷酸之间交换遗传信息的过程,其包含但不限于通过非同源末端接合(NHEJ)和同源重组进行的供体捕获。出于本公开的目的,“同源重组(HR)”是指在例如通过同源定向修复(HDR)机制修复细胞中的双链断裂期间发生的这种交换的专门形式。此过程需要核苷酸序列同源性,使用“供体”分子作为模板以修复“靶”分子(即,经历双链断裂的分子)并且以各种方式被称为“非交换基因转换(non-crossover gene conversion)”或“短链基因转换(short tract gene conversion)”,因为其导致遗传信息从供体转移到靶。不希望受任何特定理论的束缚,这种转移可以涉及在断裂的靶与供体之间形成的异源双链DNA的失配校正和/或“合成依赖性链退火(synthesis-dependent strand annealing)”,其中供体用于重新合成将成为靶的一部分的遗传信息和/或相关过程。这种专门的HR通常导致靶分子序列的改变,使得供体多核苷酸的序列的一部分或全部并入靶多核苷酸中。
“切割(cleavage)”是指DNA分子的共价骨架的断裂。切割可以通过多种方法引发,包含但不限于磷酸二酯键的酶促水解或化学水解。单链切割和双链切割两者都是可能的。由于两个不同的单链切割事件,可能发生双链切割。DNA切割可以导致平末端或交错末端的产生。在某些实施例中,本文所设想的多肽和核酸酶变体例如归巢核酸内切酶变体、megaTAL和相关的融合多肽用于靶双链DNA切割。核酸内切酶切割识别位点可能位于任一DNA链上。
“定向偏向的”或“定向偏向的缺失”是指由细胞的内源性修复机制响应于由核酸内切酶诱导的双链DNA断裂而产生的缺失的位置。如果缺失是定向偏向的,它们将主要出现在相对于靶切割位点或双链DNA断裂(断点或断点中心)的5'端或3'端。也就是说,相对于断点中心或靶位点中心位置,一侧比另一侧发生基本上更多的缺失。在特定实施例中,融合多肽诱导基因缺失,相对于断点中心或HE靶位点中心位置,所述基因缺失具有发生在与DNA结合结构域靶位点相同的一侧的定向偏向的缺失。在另外的实施例中,融合多肽诱导基因缺失,所述基因缺失具有相对于原始断点中心或HE靶位点中心位置定向偏向的缺失中心5'端。在优选的实施例中,定向偏向的缺失也被拉长。此外,由本文所设想的融合蛋白诱导的定向偏向的缺失或延伸的量可以与由相同核酸外切酶和包括相同DNA结合结构域和相同归巢核酸内切酶的融合多肽的共表达产生的缺失分布或缺失长度进行比较。
“外源”分子是通常不存在于细胞中但通过一种或多种遗传方法、生物化学方法或其它方法引入到细胞中的分子。示例性外源分子包含但不限于小型有机分子、蛋白质、核酸、碳水化合物、脂质、糖蛋白、脂蛋白、多糖、上述分子的任何经过修饰的衍生物或包括上述分子中的一个或多个的任何复合物。用于将外源分子引入到细胞中的方法是本领域技术人员已知的,并且包含但不限于脂质介导的转移(即,脂质体,包含中性和阳离子脂质)、电穿孔、直接注射、细胞融合、粒子轰击、生物聚合物纳米颗粒、磷酸钙共沉淀法、DEAE-葡聚糖介导的转移和病毒载体介导的转移。
“内源”分子是通常在特定环境条件下在特定发育阶段存在于特定细胞中的分子。其它内源分子可以包含蛋白质。
“基因”是指编码基因产物的DNA区以及调控基因产物的产生的所有DNA区,无论这种调控序列是否与编码序列和/或转录序列相邻。基因包含但不限于启动子序列、增强子、沉默子、绝缘子、边界元件、终止子、多腺苷酸化序列、转录后应答元件、转译调控序列如核糖体结合位点和内部核糖体进入位点、复制起点、基质附着位点以及基因座控制区。
“基因表达”是指将基因中含有的信息转化为基因产物。基因产物可以是基因的直接转录产物(例如,mRNA、tRNA、rRNA、反义RNA、核酶、结构RNA或任何其它类型的RNA)。基因产物还包含通过如封端、多腺苷酸化、甲基化和编辑等过程修饰的RNA,以及通过例如甲基化、乙酰化、磷酸化、泛素化、ADP-核糖基化、十四烷基化和糖基化修饰的蛋白质。
如本文所用,术语“基因工程化的(genetically engineered)”或“基因修饰的(genetically modified)”是指以DNA或RNA的形式将额外的遗传材料的染色体或染色体外添加到细胞中的总基因材料中。基因修饰可以靶向或不靶向细胞的基因组中的特定位点。在一个实施例中,基因修饰是位点特异性的。在一个实施例中,基因修饰不是位点特异性的。
如本文所用,术语“基因组编辑”是指在细胞的基因组中的靶位点处对遗传材料进行的取代、缺失和/或引入,其恢复、校正、破坏和/或修饰基因或基因产物的表达。在特定实施例中所设想的基因组编辑包括将一个或多个核酸酶变体引入到细胞中以在细胞的基因组中的靶位点处或附近产生DNA病变,任选地在存在供体修复模板的情况下。
如本文所用,术语“基因疗法”是指将额外的基因材料引入到细胞中的总基因材料中,所述额外的基因材料恢复、校正或修饰基因或基因产物的表达或用于表达治疗性多肽。在特定实施例中,通过基因组编辑将基因材料引入到细胞的基因组中被认为是基因疗法,所述基因材料恢复、校正、破坏或修饰基因或基因产物的表达或用于表达治疗性多肽。
C.融合多肽
本文中的特定实施例中设想的适于编辑靶位点的融合多肽包括DNA结合结构域、归巢核酸内切酶和末端加工结构域(例如,核酸外切酶)。在各个实施例中,融合多肽包括DNA结合结构域和归巢核酸内切酶变体、多肽连接子和核酸外切酶(例如,ExoX)或其生物活性片段。在特定实施例中,融合多肽以N末端到C末端的顺序包括DNA结合结构域、第一连接子结构域、归巢核酸内切酶变体、第二连接子结构域和核酸外切酶(例如,ExoX)或其生物活性片段。
在各个实施例中,DNA结合结构域是TALE DNA结合结构域或锌指DNA结合结构域。
在各个实施例中,归巢核酸内切酶变体是工程化核酸酶。术语“重编程的核酸酶”、“工程化的核酸酶”或“核酸酶变体”可互换使用,并且是指包括一个或多个DNA结合结构域以及一个或多个DNA切割结构域(例如,核酸酶或归巢核酸内切酶)的核酸酶,其中所述核酸酶已经被从亲代或天然存在的核酸酶中设计和/或修饰,以结合并切割双链DNA靶序列或位点。可以从天然存在的核酸酶或先前的核酸酶变体中设计和/或修饰核酸酶变体。在一些实施例中,核酸酶变体被设计为结合并切割非天然靶序列或位点。
结合并切割靶序列的融合多肽的说明性实例包含但不限于通过连接子结构域(例如,多肽连接子)与核酸外切酶(例如,ExoX核酸外切酶)连接的megaTAL,或其生物活性片段。
在优选的实施例中,与由相似的非融合/连接的多肽,例如单独的megaTAL和核酸外切酶被一起引入到细胞中时诱导的缺失相比,融合多肽可用于产生位置偏向的和/或延长的缺失。在特定实施例中,与由相似的非融合/连接的多肽诱导的缺失相比,缺失更具方向性偏向。在更具体的实施例中,本文所设想的融合多肽在断点的一侧比另一侧产生基本上更多的缺失。例如,所描述的融合多肽诱导基本上更多的诱变缺失,其在断点或核酸酶靶位点中心位置的5'端具有缺失中心。
1.归巢核酸内切酶(大范围核酸酶)变体
在各个实施例中,归巢核酸内切酶或大范围核酸酶被重编程以在靶基因内的靶位点处引入双链断裂(DSB)。“归巢核酸内切酶”和“大范围核酸酶”可互换使用,并且是指识别12-45个碱基对切割位点(例如,靶位点)并且通常基于序列和结构基序分为五个家族的天然存在的核酸酶:LAGLIDADG、GIY-YIG、HNH、His-Cys盒子以及PD-(D/E)XK。
“参考归巢核酸内切酶”或“参考大范围核酸酶”是指在自然界中发现的野生型归巢核酸内切酶或归巢核酸内切酶。在一个实施例中,“参考归巢核酸内切酶”是指经过修饰以增加基础活性的野生型归巢核酸内切酶。
“工程化归巢核酸内切酶”、“重编程的归巢核酸内切酶”、“归巢核酸内切酶变体”、“工程化大范围核酸酶”、“重编程的大范围核酸酶”或“大范围核酸酶变体”是指包括一个或多个DNA结合结构域和一个或多个DNA切割结构域的归巢核酸内切酶,其中归巢核酸内切酶已经从亲本或天然存在的归巢核酸内切酶设计和/或修饰以结合和切割DNA靶序列或位点。可以从天然存在的归巢核酸内切酶或另一种归巢核酸内切酶变体中设计和/或修饰归巢核酸内切酶变体。
归巢核酸内切酶(HE)变体在自然界中不存在,并且可以通过重组DNA技术或通过随机诱变获得。HE变体可以通过在天然存在的HE或HE变体中进行一个或多个氨基酸改变例如突变、替代、添加或缺失一个或多个氨基酸来获得。在特定实施例中,HE变体包括DNA识别界面的一个或多个氨基酸改变。
特定实施例中所设想的HE变体可能进一步包括一个或多个连接子和/或另外的功能结构域,例如,展现出5′-3′核酸外切酶、5′-3′碱性核酸外切酶、3′-5′核酸外切酶(例如,Trex2、ExoI或ExoX)、5′瓣状核酸内切酶、解旋酶、模板依赖性DNA聚合酶或模板非依赖性DNA聚合酶活性的末端加工酶的末端加工酶结构域。在各个实施例中,提供了包括通过连接子结构域(例如,多肽连接子)与末端加工酶连接的HE变体的多肽。在各个实施例中,末端加工酶表现出3′-5′核酸外切酶活性。在特定实施例中,末端加工酶是Trex2、ExoI或ExoX。HE变体和末端加工酶可以单独地引入例如在不同的载体或单独的mRNA中或一起引入例如作为融合蛋白或引入在由病毒自切割肽或IRES元件分离的多顺反子构建体中。
“DNA识别界面”是指与核酸靶碱基相互作用的HE氨基酸残基以及相邻的那些残基。对于每个HE,DNA识别界面包括侧链到侧链和侧链到DNA接触的广泛网络,所述接触中的大多数必定对识别特定核酸靶序列来说是唯一的。因此,DNA识别界面的对应于特定核酸序列的氨基酸序列显著变化,并且是任何天然的或HE变体的特征。通过非限制性实例,可以通过构建HE变体库来导出特定实施例中所设想的HE变体,在所述库中,定位在天然HE(或先前产生的HE变体)的DNA识别界面中一个或多个氨基酸残基是变化的。可以使用切割分析法从所述文库中筛选针对每个预测的BTK靶位点的靶切割活性(参见例如,Jarjour等人,2009,《核酸研究(Nuc.Acids Res.)》37(20):6871-6880)。
LAGLIDADG归巢核酸内切酶(LHE)是最充分研究的归巢核酸内切酶家族,其主要编码于古细菌以及绿藻和真菌中的细胞器DNA中并显示出最高的整体DNA识别特异性。LHE包括每个蛋白质链一个或两个LAGLIDADG催化基序,并分别起同源二聚体或单链单体的作用。对LAGLIDADG蛋白的结构研究识别了高度保守的核心结构(Stoddard 2005),其特征在于αββαββα折叠,其中LAGLIDADG基序属于此折叠的第一个螺旋。LHE的高效和特异性切割表示用于衍生新颖的高度特异性的核酸内切酶的蛋白质支架。然而,将LHE工程化为结合并切割非天然或非规范靶位点需要选择适当的LHE支架、检查靶基因座、选择推定靶位点以及广泛改变LHE以改变其DNA接触点和靶位点中多达三分之二的碱基对位置处的切割特异性。
在一个实施例中,可以由其设计出重编程的LHE或LHE变体的LHE包含但不限于I-CreI和I-SceI。
可以由其设计出重编程的LHE或LHE变体的LHE的说明性实例包含但不限于I-AabMI、I-AaeMI、I-AniI、I-ApaMI、I-CapIII、I-CapIV、I-CkaMI、I-CpaMI、I-CpaMII、I-CpaMIII、I-CpaMIV、I-CpaMV、I-CpaV、I-CraMI、I-EjeMI、I-GpeMI、I-GpiI、I-GzeMI、I-GzeMII、I-GzeMIII、I-HjeMI、I-LtrII、I-LtrI、I-LtrWI、I-MpeMI、I-MveMI、I-NcrII、I-Ncrl、I-NcrMI、I-OheMI、I-OnuI、I-OsoMI、I-OsoMII、I-OsoMIII、I-OsoMIV、I-PanMI、I-PanMII、I-PanMIII、I-PnoMI、I-ScuMI、I-SmaMI、I-SscMI以及I-Vdi141I。
在一个实施例中,重编程的LHE或LHE变体选自由以下组成的组:I-CpaMI变体、I-HjeMI变体、I-OnuI变体、I-PanMI变体以及I-SmaMI变体。
在一个实施例中,靶向靶基因的重编程的I-OnuI LHE或I-OnuI变体可以由天然的I-OnuI或其生物活性片段产生(SEQ ID NO:102-106)。
在各个实施例中,所述靶基因是免疫系统检查点基因、珠蛋白基因、编码有助于抑制γ-珠蛋白基因表达和/或HbF的多肽的基因或免疫抑制信号传导基因。
在一个实施例中,所述靶基因选自由以下组成的组:程序性细胞死亡蛋白1(PD-1;PDCD1)、淋巴细胞激活基因3蛋白(LAG-3)、T细胞免疫球蛋白结构域和粘蛋白结构域蛋白3(TIM-3)、细胞毒性T淋巴细胞抗原-4(CTLA-4)、带T淋巴细胞弱化因子(BTLA)、T细胞免疫球蛋白和基于免疫受体酪氨酸的抑制基序结构域(TIGIT)、T细胞激活的V结构域Ig抑制因子(VISTA)和杀伤细胞免疫球蛋白样受体(KIR)、CCR5、TRAC(TCRα)、TCRβ、IL10Rα、IL10Rβ、TGFBR1、TGFBR2、CBL-B、PCSK9、AHR、BTK、α-珠蛋白、β-珠蛋白、γ-珠蛋白、BCL11A、KLF1、SOX6、GATA1、LSD、α叶酸受体(FRα)、αvβ6整合素、B细胞成熟抗原(BCMA)、B7-H3(CD276)、B7-H6、碳酸酐酶IX(CAIX)、CD16、CD19、CD20、CD22、CD30、CD33、CD37、CD38、CD44、CD44v6、CD44v7/8、CD70、CD79a、CD79b、CD123、CD133、CD138、CD171、癌胚抗原(CEA)、C型凝集素样分子-1(CLL-1)、CD2子集1(CS-1)、硫酸软骨素蛋白聚糖4(CSPG4)、皮肤T细胞淋巴瘤相关抗原1(CTAGE1)、表皮生长因子受体(EGFR)、表皮生长因子受体变体III(EGFRvIII)、上皮糖蛋白2(EGP2)、上皮糖蛋白40(EGP40)、上皮细胞粘附分子(EPCAM)、肝配蛋白A型受体2(EPHA2)、成纤维细胞激活蛋白(FAP)、Fc受体样5(FCRL5)、胎儿乙酰胆碱酯酶受体(AchR)、神经节苷脂G2(GD2)、神经节苷脂G3(GD3)、磷脂酰肌醇蛋白聚糖-3(GPC3)、包含ErbB2(HER2)的EGFR家族、IL-11Rα、IL-13Rα2、κ、癌/睾丸抗原2(LAGE-1A)、λ、路易斯-Y(Lewis-Y,LeY)、L1细胞粘附分子(L1-CAM)、黑色素瘤抗原基因(MAGE)-A1、MAGE-A3、MAGE-A4、MAGE-A6、MAGEA10、T细胞识别的黑色素瘤抗原1(MelanA或MART1)、间皮素(MSLN)、MUC1、MUC16、MHC I类链相关蛋白A(MICA)、MHC I类链相关蛋白B(MICB)、神经细胞粘附分子(NCAM)、癌/睾丸抗原1(NY-ESO-1)、聚唾液酸;胎盘特异性1(PLAC1)、黑色素瘤中优先表达的抗原(PRAME)、前列腺干细胞抗原(PSCA)、前列腺特异性膜抗原(PSMA)、受体酪氨酸激酶样孤儿受体1(ROR1)、滑膜肉瘤、X断点2(SSX2)、存活蛋白、肿瘤相关糖蛋白72(TAG72)、肿瘤内皮标志物1(TEM1/CD248)、肿瘤内皮标志物7相关(TEM7R)、TEM5、TEM8、滋养层糖蛋白(TPBG)、UL16结合蛋白(ULBP)1、ULBP2、ULBP3、ULBP4、ULBP5、ULBP6、血管内皮生长因子受体2(VEGFR2)和威尔姆斯瘤1(WT-1)基因。
在某些实施例中,所述靶基因选自由以下组成的组:程序性细胞死亡蛋白1(PD-1;PDCD1)、淋巴细胞激活基因3蛋白(LAG-3)、T细胞免疫球蛋白结构域和粘蛋白结构域蛋白3(TIM-3)、细胞毒性T淋巴细胞抗原-4(CTLA-4)、带T淋巴细胞弱化因子(BTLA)、T细胞免疫球蛋白和基于免疫受体酪氨酸的抑制基序结构域(TIGIT)、T细胞激活的V结构域Ig抑制因子(VISTA)和杀伤细胞免疫球蛋白样受体(KIR)、CCR5、TRAC(TCRα)、IL10Rα、TGFBR2、CBL-B、PCSK9、AHR、BTK、α-珠蛋白、β-珠蛋白、γ-珠蛋白和BCL11A基因。
在某些实施例中,所述靶基因是TRAC(TCRα)、CBL-B或PDCD1(PD-1)基因。
在一个实施例中,所述靶基因是TRAC基因、CBL-B基因或PD-1基因。在特定实施例中,所述靶基因/位点包括SED ID NO:1、2或3中所示的核苷酸序列。
在一个实施例中,从现有的I-OnuI变体中产生靶向人TRAC(TCRα)基因的重编程的I-OnuI LHE或I-OnuI变体。在一些实施例中,针对SEQ ID NO:1中所示的人TRAC基因靶位点产生重编程的I-OnuI LHE。
在另一个实施例中,从现有的I-OnuI变体中产生靶向人PDCD1(PD-1)基因的重编程的I-OnuI LHE或I-OnuI变体。在一些实施例中,针对SEQ ID NO:2中所示的人PD-1基因靶位点产生重编程的I-OnuI LHE。
在另一个实施例中,从现有的I-OnuI变体中产生靶向人CBL-B基因的重编程的I-OnuI LHE或I-OnuI变体。在一些实施例中,针对SEQ ID NO:3中所示的人CBL-B基因靶位点产生重编程的I-OnuI LHE。
在特定实施例中,结合并切割人靶基因的I-OnuI LHE变体包括如SEQ ID NO:89、92、95、98、101或102-106中任一个中所示的I-OnuI的DNA识别界面中的一个或多个氨基酸取代或修饰、其生物活性片段和/或其另外的变体。
在特定实施例中,结合并切割人靶基因的重编程的I-OnuI LHE或I-OnuI变体包括DNA识别界面中的一个或多个氨基酸取代。在特定实施例中,结合并切割人靶基因的I-OnuILHE包括与I-OnuI(Taekuchi等人,2011,《美国国家科学院院刊(Proc Natl Acad SciU.S.A)》,2011年8月9日;108(32):13077-13082)或与如SEQ ID NO:89、92、95、98、101或102-106中的任一个中所示的I-OnuI LHE变体、其生物活性片段和/或其另外的变体的DNA识别界面的DNA识别界面至少70%、至少71%、至少72%、至少73%、至少74%、至少75%、至少76%、至少77%、至少78%、至少79%、至少80%、至少81%、至少82%、至少83%、至少84%、至少85%、至少86%、至少87%、至少88%、至少89%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%或至少99%的序列同一性。
在一个实施例中,结合并切割靶基因的I-OnuI LHE包括与I-OnuI(Taekuchi等人,2011,《美国国家科学院院刊》,2011年8月9日;108(32):13077-13082)或与如SEQ ID NO:89、92、95、98、101或102-106中的任一个中所示的I-OnuI LHE变体、其生物活性片段和/或其另外的变体的DNA识别界面的至少70%、更优选地至少80%、更优选地至少85%、更优选地至少90%、更优选地至少95%、更优选地至少97%、更优选地至少99%序列同一性。
在特定实施例中,结合并切割靶基因的I-OnuI LHE变体包括DNA识别界面中的一个或多个氨基酸取代或修饰,特别是在位于I-OnuI(SEQ ID NO:102-106)或如SEQ ID NO:89、92、95、98和101中任一个中所示的I-OnuI变体、其生物活性片段和/或其另外的变体的位置24-50、68-82、180-203和223-240中的子结构域中。
在特定实施例中,结合并切割靶基因的I-OnuI LHE变体包括DNA识别界面中在选自由以下组成的组的氨基酸位置处的一个或多个氨基酸取代或修饰:I-OnuI(SEQ ID NO:103-107)或如SEQ ID NO:89、92、95、98和101中所示的I-OnuI变体、其生物活性片段和/或其另外的变体的24、26、28、30、32、34、35、36、37、38、40、42、44、46、48、68、70、72、75、76、78、80、82、180、182、184、186、188、189、190、191、192、193、195、197、199、201、203、223、225、227、229、231、232、234、236、238和240。
在一个实施例中,结合并切割靶基因的I-OnuI LHE变体包括在位于整个I-OnuI序列内任何位置的额外位置处的一个或多个氨基酸取代或修饰。可以被取代和/或修饰的残基包含但不限于与核酸靶接触或直接地或通过水分子与核酸骨架或与核苷酸碱基相互作用的氨基酸。在一个非限制性实例中,本文所设想的结合并切割靶基因的I-OnuI LHE变体包括在选自由以下位置组成的位置组的至少一个位置中的一个或多个取代和/或修饰,优选地至少5个、优选地至少10个、优选地至少15个、优选地至少20个、更优选地至少25个、更优选地至少30个、甚至更优选地至少35个、或甚至更优选地至少40个取代和/或修饰:I-OnuI(SEQ ID NO:102-106)或如SEQ ID NO:89、92、95、98和101中所示的I-OnuI变体、其生物活性片段和/或其另外的变体的24、26、28、30、32、34、35、36、37、38、40、42、44、46、48、61、68、70、72、75、76、78、80、82、85、116、135、138、143、147、159、164、168、178、180、182、184、186、188、189、190、191、192、193、195、197、199、201、203、210、223、225、227、229、231、232、234、236、238、240和246。
在特定实施例中,结合并切割靶基因的I-OnuI LHE变体包括在选自由以下组成的组的氨基酸位置处的至少5个、至少15个、优选地至少25个、更优选地至少35个或甚至更优选地至少40个或更多个氨基酸取代:I-OnuI(SEQ ID NO:102-106)或如SEQ ID NO:89、92、95、98和101中所示的I-OnuI变体、其生物活性片段和/或其另外的变体的24、26、28、30、32、34、35、36、37、38、40、42、44、46、48、61、68、70、72、75、76、78、80、82、85、116、135、138、143、147、159、164、168、178、180、182、184、186、188、189、190、191、192、193、195、197、199、201、203、210、223、225、227、229、231、232、234、236、238、240和246。
在特定实施例中,结合并切割靶基因的I-OnuI LHE变体包括与SEQ ID NO:89、92、95、98和101中的任一个中所示的氨基酸序列至少80%、优选地至少85%、更优选地至少90%、或甚至更优选地至少95%相同的氨基酸序列,或其生物活性片段。
在特定实施例中,I-OnuI LHE变体包括SEQ ID NO:89、92、95、98和101中的任一个中所示的氨基酸序列或其生物活性片段。
在特定实施例中,I-OnuI LHE变体包括SEQ ID NO:89中所示的氨基酸序列或其生物活性片段。
在特定实施例中,I-OnuI LHE变体包括SEQ ID NO:92中所示的氨基酸序列或其生物活性片段。
在特定实施例中,I-OnuI LHE变体包括SEQ ID NO:95中所示的氨基酸序列或其生物活性片段。
在特定实施例中,I-OnuI LHE变体包括SEQ ID NO:98中所示的氨基酸序列或其生物活性片段。
在特定实施例中,I-OnuI LHE变体包括SEQ ID NO:101中所示的氨基酸序列或其生物活性片段。
2.DNA结合结构域
在各个实施例中,本文所设想的融合多肽包括DNA结合结构域。在某些实施例中,DNA结合结构域定位于归巢核酸内切酶的N末端。在特定实施例中,融合多肽包括定位于归巢核酸内切酶的N末端的DNA结合结构域,所述归巢核酸内切酶位于末端加工结构域(例如,核酸外切酶)的N末端。换言之,归巢核酸内切酶夹在DNA结合结构域与核酸外切酶之间。因此,按照N末端到C末端的顺序,本文所设想的说明性融合多肽包括DNA结合结构域、第一多肽连接子、归巢核酸内切酶、第二多肽连接子和核酸外切酶。
一方面,所述DNA结合结构域包括TALE DNA结合结构域。在特定实施例中,融合多肽包括megaTAL。“megaTAL”是指包括TALE DNA结合结构域和结合并切割靶基因中的DNA靶序列的归巢核酸内切酶变体的多肽,并且进一步包括一个或多个连接子和/或另外的功能结构域,例如展现出5′-3′核酸外切酶、5′-3′碱性核酸外切酶、3′-5′核酸外切酶(例如,Trex2、ExoI或ExoX)、5′瓣状核酸内切酶、解旋酶或模板非依赖性DNA聚合酶活性的末端加工酶的末端加工酶结构域。
在各个实施例中,包括归巢核酸内切酶变体的megaTAL被重编程以在靶基因中引入双链断裂(DSB)。在一些实施例中,包括归巢核酸内切酶变体的megaTAL被重新编程以在免疫系统检查点基因、珠蛋白基因、编码有助于抑制γ-珠蛋白基因表达和/或HbF的多肽的基因或免疫抑制信号传导基因的靶序列中引入双链断裂。在一些实施例中,包括归巢核酸内切酶变体的megaTAL被重新编程以在人TRAC基因、PD-1基因或CBL-B基因(例如,分别为SEQ ID NOs:1-3)的靶序列中引入双链断裂。
“TALE DNA结合结构域”是转录激活子样效应子(TALE或TAL效应子)的DNA结合部分,其模拟植物转录激活子以操纵植物转录组(参见例如,Kay等人,2007,《科学》318:648-651)。在特定实施例中设想的TALE DNA结合结构域是从头工程化的或来自天然存在的TALE,例如来自野油菜黄单胞菌叶斑病致病变种(Xanthomonas campestrispv.vesicatoria)、加德纳黄单胞菌(Xanthomonas gardneri)、雀麦褐条病菌(Xanthomonastranslucens)、地毯草黄单胞菌(Xanthomonas axonopodis)、番茄黄单胞菌(Xanthomonasperforans)、苜蓿黄单胞杆菌(Xanthomonas alfalfa)、柑桔溃疡病菌(Xanthomonascitri)、番茄细菌性斑点致病黄单胞菌(Xanthomonas euvesicatoria)和白叶枯病菌(Xanthomonas oryzae)的AvrBs3以及来自青枯雷尔氏菌(Ralstonia solanacearum)的brg11和hpx17。在美国专利号9,017,967和其中引用的参考文献中公开了用于衍生和设计DNA结合结构域的TALE蛋白的说明性实例,所有这些文献都通过引用整体并入本文。
在特定实施例中,TALE DNA结合结构域包括参与TALE DNA结合结构域与其对应的靶DNA序列结合的一个或多个重复单元。单个“重复单元”(也称为“重复”)的长度通常为33个到35个氨基酸。每个TALE DNA结合结构域重复单元包含构成重复可变二残基(RVD)的1个或2个DNA结合残基,通常在重复的位置12和/或位置13处。已经确定了用于DNA识别这些TALE DNA结合结构域的天然(规范)代码,使得位置12和位置13处的HD序列导致与胞嘧啶(C)结合、NG与T结合、NI与A结合、NN与G或A结合并且NG与T结合。在某些实施例中,设想了非规范(非典型)RVD。
适用于特定实施例中所设想的特定megaTAL的非规范RVD的说明性实例包含但不限于:用于识别鸟嘌呤(G)的HH、KH、NH、NK、NQ、RH、RN、SS、NN、SN、KN;用于识别腺嘌呤(A)的NI、KI、RI、HI、SI;用于识别胸腺嘧啶(T)的NG、HG、KG、RG;用于识别胞嘧啶(C)的RD、SD、HD、ND、KD、YG;用于识别A或G的NV、HN;以及用于识别A或T或G或C的H*、HA、KA、N*、NA、NC、NS、RA、S*,其中(*)意指在位置13处的氨基酸不存在。适用于特定实施例中所设想的特定megaTAL的RVD的另外的说明性实例进一步包含美国专利号8,614,092中公开的那些内容,所述美国专利通过引用整体并入本文。
在特定实施例中,本文所设想的融合多肽或megaTAL包括TALE DNA结合结构域,其包括3个到30个重复单元。在某些实施例中,TALE DNA结合结构域包括3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、17个、18个、19个、20个、21个、22个、23个、24个、25个、26个、27个、28个、29个或者30个TALE DNA结合结构域重复单元。在优选的实施例中,TALE DNA结合结构域包括5个到15个重复单元、更优选地7个到15个重复单元、更优选地9个到15个重复单元、并且更优选地9个、10个、11个、12个、13个、14或15个重复单元。
在特定实施例中,本文所设想的融合多肽或megaTAL包括TALE DNA结合结构域,其包括3个到30个重复单元和另外的单个截短的TALE重复单元,所述单个截短的TALE重复单元包括位于一组TALE重复单元的C末端处的20个氨基酸,即另外的C末端半TALE DNA结合结构域重复单元(本文下文其它地方公开的C-帽的氨基酸-20到氨基酸-1)。因此,在特定实施例中,本文所设想的融合多肽或megaTAL包括TALE DNA结合结构域,其包括3.5个到30.5个重复单元。在某些实施例中,融合多肽或megaTAL包括3.5个、4.5个、5.5个、6.5个、7.5个、8.5个、9.5个、10.5个、11.5个、12.5个、13.5个、14.5个、15.5个、16.5个、17.5个、18.5个、19.5个、20.5个、21.5个、22.5个、23.5个、24.5个、25.5个、26.5个、27.5个、28.5个、29.5个或30.5个TALE DNA结合结构域重复单元。在优选的实施例中,本文所设想的融合多肽或megaTAL包括TALE DNA结合结构域,其包括5.5个到15.5个重复单元、更优选地7.5个到15.5个重复单元、更优选地9.5个到15.5个重复单元、并且更优选地9.5个、10.5个、11.5个、12.5个、13.5个、14.5或15.5个重复单元。
在特定实施例中,本文所设想的融合多肽或megaTAL包括TAL效应子架构,其包括“N末端结构域(NTD)”多肽、一个或多个TALE重复结构域/单元、“C末端结构域(CTD)”多肽以及归巢核酸内切酶变体。在一些实施例中,NTD、TALE重复和/或CTD结构域来自相同物种。在其它实施例中,NTD、TALE重复和/或CTD结构域中的一个或多个来自不同物种。
如本文所用,术语“N末端结构域(NTD)”多肽是指天然存在的TALE DNA结合结构域的N末端部分或片段两侧的序列。NTD序列(如果存在的话)可以具有任何长度,只要TALEDNA结合结构域重复单元保留结合DNA的能力即可。在特定实施例中,NTD多肽在TALE DNA结合结构域的N末端处包括至少120个到至少140个或更多个氨基酸(0是最N末端重复单元的氨基酸1)。在特定实施例中,NTD多肽在TALE DNA结合结构域的N末端处包括至少约120个、121个、122个、123个、124个、125个、126个、127个、128个、129个、130个、131个、132个、133个、134个、135个、136个、137个、138个、139个或至少140个氨基酸。在一个实施例中,本文所设想的融合多肽或megaTAL包括黄单胞菌属TALE蛋白的至少约氨基酸+1到氨基酸+122到至少约氨基酸+1到氨基酸+137的NTD多肽(0是最N末端重复单元的氨基酸1)。在特定实施例中,NTD多肽在黄单胞菌属TALE蛋白的TALE DNA结合结构域的N末端处包括至少约122个、123个、124个、125个、126个、127个、128个、129个、130个、131个、132个、133个、134个、135个、136个或137个氨基酸。在一个实施例中,本文所设想的融合多肽或megaTAL包括雷氏菌属TALE蛋白的至少氨基酸+1到氨基酸+121的NTD多肽(0是最N末端重复单元的氨基酸1)。在特定实施例中,NTD多肽在雷氏菌属TALE蛋白的TALE DNA结合结构域的N末端处包括至少约121个、122个、123个、124个、125个、126个、127个、128个、129个、130个、131个、132个、133个、134个、135个、136个或137个氨基酸。
如本文所用,术语“C末端结构域(CTD)”多肽是指天然存在的TALE DNA结合结构域的C末端部分或片段两侧的序列。CTD序列(如果存在的话)可以具有任何长度,只要TALEDNA结合结构域重复单元保留结合DNA的能力即可。在特定实施例中,CTD多肽在TALE DNA结合结构域的最后一个完整重复的C末端处包括至少20个到至少85个或更多个氨基酸(前20个氨基酸是最后一个C末端完整重复单元的C末端处的半重复单元)。在特定实施例中,CTD多肽在TALE DNA结合结构域的最后一个完整重复的C末端处包括至少约20个、21个、22个、23个、24个、25个、26个、27个、28个、29个、30个、31个、32个、33个、34个、35个、36个、37个、38个、39个、40个、41个、42个、443个、44个、45个、46个、47个、48个、49个、50个、51个、52个、53个、54个、55个、56个、57个、58个、59个、60个、61个、62个、63个、64个、65个、66个、67个、68个、69个、70个、71个、72个、73个、74个、75个、76个、77个、78个、79个、80个、81个、82个、83个、84个或至少85个氨基酸。在一个实施例中,本文所设想的融合多肽或megaTAL包括黄单胞菌属TALE蛋白的至少约氨基酸-20到氨基酸-1的CTD多肽(-20是最后一个C末端完整重复单元C末端处的半重复单元的氨基酸1)。在特定实施例中,CTD多肽在黄单胞菌属TALE蛋白的TALE DNA结合结构域的最后一个完整重复的C末端处包括至少约20个、19个、18个、17个、16个、15个、14个、13个、12个、11个、10个、9个、8个、7个、6个、5个、4个、3个、2个或1个氨基酸。在一个实施例中,本文设想的融合多肽或megaTAL包括雷氏菌属TALE蛋白的至少约氨基酸-20到氨基酸-1的CTD多肽(-20是最后一个C末端完整重复单元C末端处的半重复单元的氨基酸1)。在特定实施例中,CTD多肽在雷氏菌属TALE蛋白的TALE DNA结合结构域的最后一个完整重复的C末端处包括至少约20个、19个、18个、17个、16个、15个、14个、13个、12个、11个、10个、9个、8个、7个、6个、5个、4个、3个、2个或1个氨基酸。
在特定实施例中,本文所设想的融合多肽或megaTAL包括被工程化成结合靶序列的TALE DNA结合结构域、被重编程成结合并切割靶序列/位点的归巢核酸内切酶以及任选地NTD和/或CTD多肽,其任选地通过本文其它地方设想的一个或多个连接子多肽彼此连接。进一步设想了包括TALE DNA结合结构域以及任选地NTD和/或CTD多肽的融合多肽或megaTAL可以与连接子多肽融合,所述连接子多肽进一步与归巢核酸内切酶变体融合。因此,TALE DNA结合结构域结合DNA靶序列,所述DNA靶序列位于距离与归巢核酸内切酶变体的DNA结合结构域结合的靶序列约1个、2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个或15个核苷酸内。以这种方式,本文所设想的融合多肽或megaTAL增加了基因组编辑的特异性和效率。
在一个实施例中,融合多肽或megaTAL包括归巢核酸内切酶变体以及TALE DNA结合结构域,所述TALE DNA结合结构域结合在重编程的归巢核酸内切酶的结合/靶位点上游约4个、5个或6个核苷酸、优选地6个核苷酸内的核苷酸/靶序列/位点。
在特定实施例中,本文所设想的融合多肽或megaTAL包括一个或多个TALE DNA结合重复单元以及由选自由以下组成的组的LHE设计或重编程的LHE变体:I-AabMI、I-AaeMI、I-AniI、I-ApaMI、I-CapIII、I-CapIV、I-CkaMI、I-CpaMI、I-CpaMII、I-CpaMIII、I-CpaMIV、I-CpaMV、I-CpaV、I-CraMI、I-EjeMI、I-GpeMI、I-GpiI、I-GzeMI、I-GzeMII、I-GzeMIII、I-HjeMI、I-LtrII、I-LtrI、I-LtrWI、I-MpeMI、I-MveMI、I-NcrII、I-Ncrl、I-NcrMI、I-OheMI、I-OnuI、I-OsoMI、I-OsoMII、I-OsoMIII、I-OsoMIV、I-PanMI、I-PanMII、I-PanMIII、I-PnoMI、I-ScuMI、I-SmaMI、I-SscMI、I-Vdi141I及其变体、或优选地I-CpaMI、I-HjeMI、I-OnuI、I-PanMI、SmaMI及其变体、或更优选地I-OnuI及其变体。
在特定实施例中,本文所设想的融合多肽或megaTAL包括NTD、一个或多个TALEDNA结合重复单元、CTD以及选自由以下组成的组的LHE变体:I-AabMI、I-AaeMI、I-AniI、I-ApaMI、I-CapIII、I-CapIV、I-CkaMI、I-CpaMI、I-CpaMII、I-CpaMIII、I-CpaMIV、I-CpaMV、I-CpaV、I-CraMI、I-EjeMI、I-GpeMI、I-GpiI、I-GzeMI、I-GzeMII、I-GzeMIII、I-HjeMI、I-LtrII、I-LtrI、I-LtrWI、I-MpeMI、I-MveMI、I-NcrII、I-Ncrl、I-NcrMI、I-OheMI、I-OnuI、I-OsoMI、I-OsoMII、I-OsoMIII、I-OsoMIV、I-PanMI、I-PanMII、I-PanMIII、I-PnoMI、I-ScuMI、I-SmaMI、I-SscMI、I-Vdi141I及其变体、或优选地I-CpaMI、I-HjeMI、I-OnuI、I-PanMI、SmaMI及其变体、或更优选地I-OnuI及其变体。
在特定实施例中,本文所设想的融合多肽或megaTAL包括NTD、约9.5个到约15.5个TALE DNA结合重复单元以及选自由以下组成的组的LHE变体:I-AabMI、I-AaeMI、I-AniI、I-ApaMI、I-CapIII、I-CapIV、I-CkaMI、I-CpaMI、I-CpaMII、I-CpaMIII、I-CpaMIV、I-CpaMV、I-CpaV、I-CraMI、I-EjeMI、I-GpeMI、I-GpiI、I-GzeMI、I-GzeMII、I-GzeMIII、I-HjeMI、I-LtrII、I-LtrI、I-LtrWI、I-MpeMI、I-MveMI、I-NcrII、I-Ncrl、I-NcrMI、I-OheMI、I-OnuI、I-OsoMI、I-OsoMII、I-OsoMIII、I-OsoMIV、I-PanMI、I-PanMII、I-PanMIII、I-PnoMI、I-ScuMI、I-SmaMI、I-SscMI、I-Vdi141I及其变体、或优选地I-CpaMI、I-HjeMI、I-OnuI、I-PanMI、SmaMI及其变体、或更优选地I-OnuI及其变体。
在特定实施例中,本文所设想的融合多肽或megaTAL包括:约122个氨基酸到137个氨基酸的NTD;约9.5个、约10.5个、约11.5个、约12.5个、约13.5个、约14.5个或约15.5个结合重复单元;约20个氨基酸到约85个氨基酸的CTD;以及I-OnuI LHE变体。在特定实施例中,NTD、DNA结合结构域和CTD中的任何一个、任何两个或全部可以由相同的物种或不同的物种以任何合适的组合设计而成。
在某些实施例中,本文所设想的融合多肽或megaTAL包括:包括约9.5、约10.5、约11.5、约12.5、约13.5、约14.5或约15.5个结合重复单元的TALE DNA结合结构域,本文其它地方所设想的归巢核酸内切酶变体和本文其它地方所设想的末端加工结构域(例如,核酸外切酶),或其生物活性片段。
在特定实施例中,本文所设想的融合多肽以N末端至C末端的顺序包括:包括约9.5、约10.5、约11.5、约12.5、约13.5、约14.5或约15.5个结合重复单元的TALE DNA结合结构域、第一连接子结构域、I-OnuI LHE变体、第二连接子结构域和末端加工酶(例如,3'至5'核酸外切酶)或其生物活性片段。在特定实施例中,TALE结合重复序列中的任何一个、任何两个或全部可以由相同的物种或不同的物种以任何合适的组合设计而成。在优选的实施例中,核酸外切酶是ExoX核酸外切酶或其生物活性片段。
在特定实施例中,本文所设想的融合多肽或megaTAL包括与如SEQ ID NO:7、10、58、67或76中的任一个中所示的氨基酸序列具有至少85%、90%、95%、96%、97%、98%或99%同一性的氨基酸序列。在特定实施例中,本文所设想的融合多肽或megaTAL包括SEQ IDNO:7、10、58、67或76中的任一个中所示的氨基酸序列。在特定实施例中,本文所设想的融合多肽或megaTAL-Trex2融合蛋白包括与如SEQ ID NO:13、61、70或79中的任一个中所示的氨基酸序列具有至少85%、90%、95%、96%、97%、98%或99%同一性的氨基酸序列。在特定实施例中,本文所设想的融合多肽或megaTAL-Trex2融合蛋白包括SEQ ID NO:13、61、70或79中所示的氨基酸序列。在特定实施例中,本文所设想的融合多肽或megaTAL-ExoX融合蛋白包括与如SEQ ID NO:46、64、73或82中的任一个中所示的氨基酸序列具有至少85%、90%、95%、96%、97%、98%或99%同一性的氨基酸序列。在特定实施例中,本文所设想的融合多肽或megaTAL-ExoX融合蛋白包括SEQ ID NO:46、64、73或82中的任一个中所示的氨基酸序列。在特定实施例中,本文所设想的融合多肽或megaTAL-ExoI融合蛋白包括与如SEQID NO:43中所示的氨基酸序列具有至少85%、90%、95%、96%、97%、98%或99%同一性的氨基酸序列。在特定实施例中,本文所设想的融合多肽或megaTAL-ExoI融合蛋白包括SEQID NO:43中所示的氨基酸序列。
在某些实施例中,本文所设想的融合多肽或megaTAL由mRNA序列编码,所述序列与如SEQ ID NO:6、9、57、66或75中的任一个中所示的mRNA序列具有至少85%、90%、95%、96%、97%、98%或99%同一性。在某些实施例中,本文所设想的融合多肽或megaTAL由SEQID NO:6、9、57、66或75中的任一个中所示的mRNA序列编码。在某些实施例中,本文所设想的融合多肽或megaTAL-Trex2由与如SEQ ID NO:12、60、69或78中的任一个中所示的mRNA序列具有至少85%、90%、95%、96%、97%、98%或99%同一性的mRNA序列编码。在某些实施例中,本文所设想的融合多肽或megaTAL-Trex2由SEQ ID NO:12、60、69或78中的任一个中所示的mRNA序列编码。在某些实施例中,本文所设想的融合多肽或megaTAL-ExoX由与如SEQID NO:45、63、72或81中的任一个中所示的mRNA序列具有至少85%、90%、95%、96%、97%、98%或99%同一性的mRNA序列编码。在某些实施例中,本文所设想的融合多肽或megaTAL-ExoX由与SEQ ID NO:45、63、72或81中的任一个中所示的mRNA序列编码。在某些实施例中,本文所设想的融合多肽或megaTAL-ExoI由与如SEQ ID NO:42中所示的mRNA序列具有至少85%、90%、95%、96%、97%、98%或99%同一性的mRNA序列编码。在某些实施例中,本文所设想的融合多肽或megaTAL-ExoI由SEQ ID NO:42中所示的mRNA序列编码。
在某些实施例中,本文所设想的融合多肽或megaTAL由与如SEQ ID NO:5、8、56、65或74中的任一个中所示的DNA序列具有至少85%、90%、95%、96%、97%、98%或99%同一性的DNA序列编码。在某些实施例中,本文所设想的融合多肽或megaTAL由SEQ ID NO:5、8、56、65或74中的任一个中所示的DNA序列编码。在某些实施例中,本文所设想的融合多肽或megaTAL-Trex2由与如SEQ ID NO:11、59、68或77中的任一个中所示的DNA序列具有至少85%、90%、95%、96%、97%、98%或99%同一性的DNA序列编码。在某些实施例中,本文所设想的融合多肽或megaTAL-Trex2由SEQ ID NO:11、59、68或77中的任一个中所示的DNA序列编码。在某些实施例中,本文所设想的融合多肽或megaTAL-ExoX由与如SEQ ID NO:44、62、71或80中的任一个中所示的DNA序列具有至少85%、90%、95%、96%、97%、98%或99%同一性的DNA序列编码。在某些实施例中,本文所设想的融合多肽或megaTAL-ExoX由SEQ IDNO:44、62、71或80中的任一个中所示的DNA序列编码。在某些实施例中,本文所设想的融合多肽或megaTAL-ExoI由与如SEQ ID NO:41中所示的DNA序列具有至少85%、90%、95%、96%、97%、98%或99%同一性的DNA序列编码。在某些实施例中,本文所设想的融合多肽或megaTAL-ExoI由SEQ ID NO:41中所示的DNA序列编码。
在某些实施例中,megaTAL包括TALE DNA结合结构域和I-OnuI LHE变体,所述变体结合并切割SEQ ID NO:1-3中的任一个中所示的核苷酸序列。
另一方面,所述DNA结合结构域包括锌指DNA结合结构域。在特定实施例中,本文所设想的融合多肽包括锌指DNA结合结构域、本文其它地方所设想的归巢核酸内切酶结构域和本文其它地方所设想的末端加工结构域(例如,核酸外切酶)。
在具体实施例中,锌指DNA结合结构域具有一个、两个、三个、四个、五个、六个、七个或八个或更多个锌指基序。典型地,单个锌指基序的长度为约30个氨基酸。锌指基序包含规范的C2H2锌指和如例如C3H锌指和C4锌指等非规范的锌指。
锌指DNA结合结构域可以被工程化成结合任何DNA序列。已经识别了给定3bp DNA靶序列的候选锌指DNA结合结构域,并且已经设计了用于将多个结构域连接成靶向对应复合DNA靶序列的多指肽的模块化装配策略。本领域已知的其它适合的方法还可以用于设计和构建对锌指DNA结合结构域进行编码的核酸,例如噬菌体显示、随机诱变、组合库、计算机/合理设计、亲和力选择、PCR、从cDNA或基因组库克隆、合成构造等。(参见例如,美国专利第5,786,538号;Wu等人,《美国国家科学院院刊》92:344-348(1995);Jamieson等人,《生物化学》33:5689-5695(1994);Rebar和Pabo,《科学》263:671-673(1994);Choo和Klug,《美国国家科学院院刊》91:11163-11167(1994);Choo和Klug,《美国国家科学院院刊》9 1:11168-1 1172(1994);Desjarlais和Berg,《美国国家科学院院刊》90:2256-2260(1993);Desjarlais和Berg,《美国国家科学院院刊》89:7345-7349(1992);Pomerantz等人,《科学》267:93-96(1995);Pomerantz等人,《美国国家科学院院刊》92:9752-9756(1995);Liu等人,《美国国家科学院院刊》94:5525-5530(1997);Griesman和Pabo,《科学》275:657-661(1997);Desjarlais和Berg,《美国国家科学院院刊》91:1 1-99-1 1103(1994))。
单个锌指基序结合到三个或四个核苷酸序列。锌指结合结构域被工程化成与其结合的序列(例如,靶序列)的长度将决定工程化的锌指DNA结合结构域中的锌指基序的数目。例如,当锌指基序不与重叠子位点结合时,六核苷酸靶序列由双指DNA结合结构域结合;九核苷酸靶序列由三指结合结构域等结合。在具体实施例中,靶位点中的单独锌指基序的DNA结合位点不需要是连续的,而是可以被一个或几个核苷酸分开,这取决于多指结合结构域中的锌指基序之间的连接子序列的长度和性质。
在某些实施例中,本文所设想的融合多肽包括包含一个或多个锌指基序的锌指DNA结合结构域、连接子、归巢核酸内切酶变体、连接子和末端加工结构域(例如,核酸外切酶)。在特定实施例中,所述归巢核酸内切酶是从选自由以下组成的组的LHE设计或重编程的LHE变体LHE变体:I-AabMI、I-AaeMI、I-AniI、I-ApaMI、I-CapIII、I-CapIV、I-CkaMI、I-CpaMI、I-CpaMII、I-CpaMIII、I-CpaMIV、I-CpaMV、I-CpaV、I-CraMI、I-EjeMI、I-GpeMI、I-GpiI、I-GzeMI、I-GzeMII、I-GzeMIII、I-HjeMI、I-LtrII、I-LtrI、I-LtrWI、I-MpeMI、I-MveMI、I-NcrII、I-Ncrl、I-NcrMI、I-OheMI、I-OnuI、I-OsoMI、I-OsoMII、I-OsoMIII、I-OsoMIV、I-PanMI、I-PanMII、I-PanMIII、I-PnoMI、I-ScuMI、I-SmaMI、I-SscMI、I-Vdi141I及其变体、或优选地I-CpaMI、I-HjeMI、I-OnuI、I-PanMI、SmaMI及其变体、或更优选地I-OnuI及其变体。
在某些实施例中,本文所设想的融合多肽包括:包括2个、3个、4个、5个、6个、7个或8个或更多个锌指基序的锌指DNA结合结构域,本文其它地方所设想的归巢核酸内切酶变体,以及本文其它地方所设想的末端加工结构域(例如,核酸外切酶)或其生物活性片段。
在特定实施例中,本文所设想的融合多肽以N末端至C末端的顺序包括:包括约2个、3个、4个、5个、6个、7个或8个或更多个锌指基序的锌指DNA结合结构域、第一连接子结构域、I-OnuI LHE变体、第二连接子结构域和末端加工结构域(例如,核酸外切酶)或其生物活性片段。在特定实施例中,锌指基序中的任何一个、任何两个或全部可以由相同的物种或不同的物种以任何合适的组合设计而成。在优选的实施例中,核酸外切酶是ExoX核酸外切酶或其生物活性片段。
3.末端加工酶
特定实施例中所设想的基因组编辑组合物(例如,融合多肽)和方法包括使用DNA结合结构域、归巢核酸内切酶变体和末端加工酶来编辑细胞基因组。在特定实施例中,融合多肽编码DNA结合结构域、归巢核酸内切酶变体和一种或多种末端加工酶(例如,核酸外切酶),每种酶都被连接子结构域(例如,肽连接子)分开。
术语“末端加工酶”是指修饰多核苷酸链的暴露末端的酶。多核苷酸可以是双链DNA(dsDNA)、单链DNA(ssDNA)、RNA、DNA和RNA的双链混杂体、以及合成DNA(例如,含有除A、C、G和T以外的碱基)。末端加工酶可以通过添加一个或多个核苷酸、移除一个或多个核苷酸、移除或修饰磷酸基和/或移除或修饰羟基来修饰暴露的多核苷酸链末端。末端加工酶可以修饰核酸内切酶切割位点处或通过其它化学或机械手段产生的末端处的末端,如剪切(例如通过穿过细针、加热、声波处理、迷你珠子翻滚和雾化)、电离辐射、紫外线辐射、氧自由基、化学水解和化学治疗剂。
在特定实施例中,特定实施例中所设想的基因组编辑组合物和方法包括使用融合多肽编辑细胞基因组,所述融合多肽包括DNA结合结构域、归巢核酸内切酶变体和DNA末端加工酶。
术语“DNA末端加工酶”是指修饰DNA暴露末端的酶。DNA末端加工酶可以修饰平末端或交错末端(具有5′突出端或3′突出端的末端)。DNA末端加工酶可以修饰单链DNA或双链DNA。DNA末端加工酶可以修饰核酸内切酶切割位点处或通过其它化学或机械手段产生的末端处的末端,如剪切(例如通过穿过细针、加热、声波处理、迷你珠子翻滚和雾化)、电离辐射、紫外线辐射、氧自由基、化学水解和化学治疗剂。DNA末端加工酶可以通过添加一个或多个核苷酸、移除一个或多个核苷酸、移除或修饰磷酸基和/或移除或修饰羟基来修饰暴露的DNA末端。
适用于本文所设想的特定实施例的DNA末端加工酶的说明性实例包含但不限于:5′-3′核酸外切酶、5′-3′碱性核酸外切酶、3′-5′核酸外切酶、5′瓣状核酸内切酶、解旋酶、磷酸酶、水解酶和模板非依赖性DNA聚合酶。
适用于本文所设想的特定实施例的DNA末端加工酶的另外的说明性实例包含但不限于:Trex2、Trex1、不含跨膜结构域的Trex1、Apollo、Artemis、DNA2、ExoI、ExoT、ExoIII、ExoX、Fen1、Fan1、MreII、Rad2、Rad9、TdT(末端脱氧核苷酸转移酶)、PNKP、RecE、RecJ、RecQ、λ核酸外切酶、Sox、牛痘DNA聚合酶、核酸外切酶I、核酸外切酶III、核酸外切酶VII、NDK1、NDK5、NDK7、NDK8、WRN、T7-核酸外切酶基因6、禽类成髓细胞瘤病毒整合蛋白(IN)、Bloom、热敏磷酸酶、碱性磷酸酶、多核苷酸激酶(PNK)、ApeI、绿豆核酸酶、Hex1、TTRAP(TDP2)、Sgs1、Sae2、CUP、Polμ、Polλ、MUS81、EME1、EME2、SLX1、SLX4以及UL-12。
在特定实施例中,用于编辑本文所设想的细胞基因组的基因组编辑组合物和方法包括融合多肽,所述融合多肽包括DNA结合结构域、归巢核酸内切酶变体和核酸外切酶。术语“核酸外切酶”是指通过在3′末端或5′末端处破坏磷酸二酯键的水解反应在多核苷酸链末端切割磷酸二酯键的酶。在特定实施例中,核酸外切酶是3′-5′核酸外切酶。在一些实施例中,核酸外切酶是ExoX核酸外切酶或其生物活性片段。在一些实施例中,核酸外切酶是ExoI核酸外切酶或其生物活性片段。在一些实施例中,核酸外切酶是Trex2核酸外切酶或其生物活性片段。
ExoX是来自大肠杆菌(大肠杆菌(E.coli))的3′-5′分布核酸外切酶,并且是DnaQ超家族的成员。ExoX也被称为脱氧核糖核酸外切酶10、脱氧核糖核酸外切酶X、核酸外切酶X和Exo X。特定实施例中使用的示例性ExoX参考序列号包含但不限于:NP_416358.1;NC_000913.3;WP_000944256.1;NZ_STEB01000009.1AAC74914。
在本文所设想的优选实施例中,融合多肽包括DNA结合结构域、归巢核酸内切酶变体和ExoX核酸外切酶或其生物活性片段。在各个实施例中,融合多肽包括DNA结合结构域和通过连接子结构域(例如,多肽连接子)与ExoX核酸外切酶连接的归巢核酸内切酶变体,或其生物活性片段。
在各个实施例中,ExoX包括与如SEQ ID NO:109中所示的氨基酸序列具有至少85%、90%、95%、96%、97%、98%或99%同一性的氨基酸序列。在一些实施例中,ExoX包括与如SEQ ID NO:109中所示的氨基酸序列具有至少85%同一性的氨基酸序列。在一些实施例中,ExoX包括与如SEQ ID NO:109中所示的氨基酸序列具有至少90%同一性的氨基酸序列。在一些实施例中,ExoX包括与如SEQ ID NO:109中所示的氨基酸序列具有至少95%同一性的氨基酸序列。在一些实施例中,ExoX包括与如SEQ ID NO:109中所示的氨基酸序列具有至少96%同一性的氨基酸序列。在一些实施例中,ExoX包括与如SEQ ID NO:109中所示的氨基酸序列具有至少97%同一性的氨基酸序列。在一些实施例中,ExoX包括与如SEQ ID NO:109中所示的氨基酸序列具有至少98%同一性的氨基酸序列。在一些实施例中,ExoX包括与如SEQ ID NO:109中所示的氨基酸序列具有至少99%同一性的氨基酸序列。
在特定实施例中,ExoX或其生物活性片段包括如SEQ ID NO:109中所示的氨基酸氨基酸序列。
ExoI是来自大肠杆菌(大肠杆菌)的3′-5′进行性核酸外切酶,并且是DnaQ超家族的成员。ExoI也被称为脱氧核糖核酸外切酶I、核酸外切酶I、DNA脱氧核糖磷酸二酯酶和dRP酶。特定实施例中使用的示例性ExoI参考序列号包含但不限于:NP_416515.1、NC_000913.3、WP_000980589.1、NZ_LN832404.1。
在本文所设想的优选实施例中,融合多肽包括DNA结合结构域、归巢核酸内切酶变体和ExoI核酸外切酶或其生物活性片段。在各个实施例中,融合多肽包括DNA结合结构域和通过连接子结构域(例如,多肽连接子)与ExoI核酸外切酶连接的归巢核酸内切酶变体,或其生物活性片段。
在各个实施例中,ExoI包括与如SEQ ID NO:112中所示的氨基酸序列具有至少85%、90%、95%、96%、97%、98%或99%同一性的氨基酸序列。在一些实施例中,ExoI包括与如SEQ ID NO:112中所示的氨基酸序列具有至少85%同一性的氨基酸序列。在一些实施例中,ExoI包括与如SEQ ID NO:112中所示的氨基酸序列具有至少90%同一性的氨基酸序列。在一些实施例中,ExoI包括与如SEQ ID NO:112中所示的氨基酸序列具有至少95%同一性的氨基酸序列。在一些实施例中,ExoI包括与如SEQ ID NO:112中所示的氨基酸序列具有至少96%同一性的氨基酸序列。在一些实施例中,ExoI包括与如SEQ ID NO:112中所示的氨基酸序列具有至少97%同一性的氨基酸序列。在一些实施例中,ExoI包括与如SEQ ID NO:112中所示的氨基酸序列具有至少98%同一性的氨基酸序列。在一些实施例中,ExoI包括与如SEQ ID NO:112中所示的氨基酸序列具有至少99%同一性的氨基酸序列。
在特定实施例中,ExoI或其生物活性片段包括如SEQ ID NO:112中所示的氨基酸氨基酸序列。
D.靶位点
与天然存在的核酸酶相比,特定实施例中所设想的归巢核酸内切酶变体可以被设计成与任何合适的靶序列(例如,人基因组中的序列)结合并且可以具有新颖的结合特异性。在特定实施例中,靶位点是基因的调控区,所述调控区包含但不限于启动子、增强子、阻遏子元件等。在特定实施例中,靶位点是基因或剪接位点的编码区。在特定实施例中,核酸酶变体和供体修复模板可以被设计成插入治疗性多核苷酸。在特定实施例中,核酸酶变体和供体修复模板可以被设计成在内源性基因调节元件或表达控制序列的控制下插入治疗性多核苷酸。在各个实施例中,核酸酶变体结合并切割免疫系统检查点基因、珠蛋白基因、编码有助于抑制γ-珠蛋白基因表达和/或HbF的多肽的基因或免疫抑制信号传导基因中的靶序列。
免疫系统检查点基因的说明性实例包含但不限于:PD-1、LAG-3、TIM-3、CTLA-4、BTLA、TIGIT、VISTA和KIR。
编码免疫抑制信号传导组分的基因的说明性实例包含但不限于:IL-10Rα、TGFβR1、TGFβR2、AHR、SGK1、TSC2、VHL、A2AR和CBL-B。
抑制γ-珠蛋白基因表达和HbF的多肽的说明性实例包含但不限于:BCL11A、KLF1、SOX6、GATA1和LSD1。
在各个实施例中,核酸酶变体结合并切割选自由以下组成的组的基因中的靶序列:程序性细胞死亡蛋白1(PD-1;PDCD1)、淋巴细胞激活基因3蛋白(LAG-3)、T细胞免疫球蛋白结构域和粘蛋白结构域蛋白3(TIM-3)、细胞毒性T淋巴细胞抗原-4(CTLA-4)、带T淋巴细胞弱化因子(BTLA)、T细胞免疫球蛋白和基于免疫受体酪氨酸的抑制基序结构域(TIGIT)、T细胞激活的V结构域Ig抑制因子(VISTA)和杀伤细胞免疫球蛋白样受体(KIR)、CCR5、TRAC(TCRα)、TCRβ、IL10Rα、IL10Rβ、TGFBR1、TGFBR2、CBL-B、PCSK9、AHR、BTK、α-珠蛋白、β-珠蛋白、γ-珠蛋白、BCL11A、KLF1、SOX6、GATA1、LSD、α叶酸受体(FRα)、αvβ6整合素、B细胞成熟抗原(BCMA)、B7-H3(CD276)、B7-H6、碳酸酐酶IX(CAIX)、CD16、CD19、CD20、CD22、CD30、CD33、CD37、CD38、CD44、CD44v6、CD44v7/8、CD70、CD79a、CD79b、CD123、CD133、CD138、CD171、癌胚抗原(CEA)、C型凝集素样分子-1(CLL-1)、CD2子集1(CS-1)、硫酸软骨素蛋白聚糖4(CSPG4)、皮肤T细胞淋巴瘤相关抗原1(CTAGE1)、表皮生长因子受体(EGFR)、表皮生长因子受体变体III(EGFRvIII)、上皮糖蛋白2(EGP2)、上皮糖蛋白40(EGP40)、上皮细胞粘附分子(EPCAM)、肝配蛋白A型受体2(EPHA2)、成纤维细胞激活蛋白(FAP)、Fc受体样5(FCRL5)、胎儿乙酰胆碱酯酶受体(AchR)、神经节苷脂G2(GD2)、神经节苷脂G3(GD3)、磷脂酰肌醇蛋白聚糖-3(GPC3)、包含ErbB2(HER2)的EGFR家族、IL-11Rα、IL-13Rα2、κ、癌/睾丸抗原2(LAGE-1A)、λ、路易斯-Y(Lewis-Y,LeY)、L1细胞粘附分子(L1-CAM)、黑色素瘤抗原基因(MAGE)-A1、MAGE-A3、MAGE-A4、MAGE-A6、MAGEA10、T细胞识别的黑色素瘤抗原1(MelanA或MART1)、间皮素(MSLN)、MUC1、MUC16、MHC I类链相关蛋白A(MICA)、MHC I类链相关蛋白B(MICB)、神经细胞粘附分子(NCAM)、癌/睾丸抗原1(NY-ESO-1)、聚唾液酸;胎盘特异性1(PLAC1)、黑色素瘤中优先表达的抗原(PRAME)、前列腺干细胞抗原(PSCA)、前列腺特异性膜抗原(PSMA)、受体酪氨酸激酶样孤儿受体1(ROR1)、滑膜肉瘤、X断点2(SSX2)、存活蛋白、肿瘤相关糖蛋白72(TAG72)、肿瘤内皮标志物1(TEM1/CD248)、肿瘤内皮标志物7相关(TEM7R)、TEM5、TEM8、滋养层糖蛋白(TPBG)、UL16结合蛋白(ULBP)1、ULBP2、ULBP3、ULBP4、ULBP5、ULBP6、血管内皮生长因子受体2(VEGFR2)和威尔姆斯瘤1(WT-1)基因。
在某些实施例中,所述靶基因选自由以下组成的组:程序性细胞死亡蛋白1(PD-1)、淋巴细胞激活基因3蛋白(LAG-3)、T细胞免疫球蛋白结构域和粘蛋白结构域蛋白3(TIM-3)、细胞毒性T淋巴细胞抗原-4(CTLA-4)、带T淋巴细胞弱化因子(BTLA)、T细胞免疫球蛋白和基于免疫受体酪氨酸的抑制基序结构域(TIGIT)、T细胞激活的V结构域Ig抑制因子(VISTA)和杀伤细胞免疫球蛋白样受体(KIR)、CCR5、TRAC(TCRα)、IL10Rα、TGFBR2、CBL-B、PCSK9、AHR、BTK、α-珠蛋白、β-珠蛋白、γ-珠蛋白和BCL11A基因。
在某些实施例中,所述靶基因是TRAC(TCRα)、CBL-B或PDCD1(PD-1)基因。
在各个实施例中,核酸酶变体结合并切割TRAC基因中的靶序列。T细胞受体α(TRAC)基因座是人类中由TRA基因编码的蛋白质。TRAC也被称为TRA、IMD7、TCRA、TRA@、TRAC、T细胞受体α基因座、TCRD、T细胞受体α基因座、TCRα。它将α链贡献给更大的TCR蛋白(T细胞受体)。α-βT细胞受体是免疫应答所必需的抗原特异性受体,并且存在于T淋巴细胞的细胞表面上。它们识别由抗原呈递细胞(APC)展示的肽-主要组织相容性(MH)(pMH)复合物,这是针对病原体的高效T细胞适应性免疫的先决条件。
在特定实施例中,核酸酶变体在人TCRα基因的恒定区的外显子1中引入DSB,优选地在人TCRα基因的恒定区的外显子1中的SEQ ID NO:1处引入,更优选地在人TCRα基因的恒定区的外显子1中的SEQ ID NO:1中的序列“ATTC”处引入。在优选的实施例中,TCRα基因是人TCRα基因。
在各个实施例中,归巢核酸内切酶变体结合并切割程序性死亡受体1(PD-1)基因中的靶序列。PD-1也被称为程序性细胞死亡1(PDCD1)、系统性红斑狼疮易感性2(SLEB2)、CD279、HPD1、PD1、HPD-L和HSLE1。PD-1是B7/CD28共刺激受体家族的成员。PD-1分子由细胞外配体结合IgV结构域、跨膜结构域和细胞内结构域组成,所述细胞内结构域具有潜在的磷酸化位点,所述磷酸化位点定位于基于免疫酪氨酸的抑制性基序(ITIM)和基于免疫受体抑制性酪氨酸的开关基序(ITSM)上。PD-1是在T细胞、Tregs、衰竭T细胞、B细胞、活性单核细胞、树突状细胞(DC)、自然杀伤(NK)细胞和自然杀伤T(NKT)细胞上表达的抑制性共受体。PD-1通过与其配体,程序性死亡配体1(PD-L1)和程序性死亡配体2(PD-L2)的结合负调节T细胞激活。PD-1结合抑制T细胞增殖、干扰素-γ(IFN-γ)、肿瘤坏死因子-α和IL-2产生,并且降低T细胞存活率。PD-1表达是经历高水平刺激的“耗尽”T细胞的标志。这种衰竭状态发生在慢性感染和癌症期间,其特征在于T细胞功能障碍,导致对感染和肿瘤的次优控制。
在特定实施例中,归巢核酸内切酶变体在PD-1基因的外显子1中引入双链断裂(DSB),优选地在PD-1基因的外显子1中的SEQ ID NO:2处引入,更优选地在PD-1基因的外显子1中的SEQ ID NO:2中的序列“ATCC”处引入。在优选的实施例中,归巢核酸内切酶变体或megaTAL切割双链DNA并将DSB引入到SEQ ID NO:2中所示的多核苷酸序列中。在优选的实施例中,PD-1基因是人PD-1基因。
在各个实施例中,归巢核酸内切酶变体结合并切割人casitas B谱系(Cbl)淋巴瘤原癌基因B(CBLB)基因中的靶序列。CBL也被称为CBL2;伴或不伴幼年型粒单核细胞白血病的努南综合征样病症(NSLL);C-CBL;环指蛋白55(RNF55);脆性位点,叶酸型罕见fra(11)(q23.3)(FRA11B);E3泛素-蛋白连接酶CBL;Cas-Br-M(鼠)亲生态逆转录病毒转化序列;Cbl原癌基因,E3泛素蛋白连接酶;环型E3泛素转移酶CBL;casitas B谱系淋巴瘤原癌基因;原癌基因CBL2;原致癌基因c-Cbl;以及信号转导蛋白CBL。
此基因是编码环指E3泛素连接酶的原致癌基因。经编码的蛋白质是蛋白酶体降解靶向底物所需的酶之一。此蛋白质调节泛素从泛素结合酶(E2)转移到特定底物。此蛋白质也含有N末端磷酸酪氨酸结合结构域,使其能够与许多酪氨酸磷酸化底物相互作用并靶向所述底物进行蛋白酶体降解。CBLB充当许多信号转导通路的负性调节因子,包含T细胞激活和持续。
在特定实施例中,归巢核酸内切酶变体在CBLB基因的靶位点中引入双链断裂(DSB)。在特定实施例中,归巢核酸内切酶变体或megaTAL在CBLB基因的外显子6中,优选在CBLB基因外显子6的SEQ ID NO:3处,更优选地在CBLB基因外显子6的SEQ ID NO:3的序列“ATTC”处引入DSB。在特定实施例中,归巢核酸内切酶变体切割双链DNA并将DSB引入到SEQID NO:3中所示的多核苷酸序列中。在优选的实施例中,CBLB基因是人CBLB基因。
E.供体修复模板
融合多肽,包含归巢核酸内切酶变体,可以用于在靶序列中引入DSB;可以在存在一个或多个供体修复模板的情况下通过同源定向修复(HDR)机制修复DSB。在特定实施例中,供体修复模板用于将序列插入到基因组中。在特别优选的实施例中,供体修复模板用于插入编码治疗性多肽的多核苷酸序列。在特别优选的实施例中,供体修复模板用于插入编码治疗性多肽的多核苷酸序列,使得多肽的表达在内源性启动子和/或增强子的控制下。
在各个实施例中,通过用腺相关病毒(AAV)、逆转录病毒例如慢病毒、IDLV等、单纯疱疹病毒、腺病毒、或包括供体修复模板的痘苗病毒载体转导细胞来将供体修复模板引入到造血细胞例如造血干细胞或祖细胞,或CD34+细胞中。
在特定实施例中,供体修复模板包括位于DSB位点侧翼的一个或多个同源臂。
如本文所用,术语“同源臂”是指供体修复模板中与通过核酸酶在靶位点处引入的DNA断裂侧翼的DNA序列完全相同或几乎完全相同的核酸序列。在一个实施例中,供体修复模板包括5′同源臂,其包括与DNA断裂位点的DNA序列5′完全相同或几乎完全相同的核酸序列。在一个实施例中,供体修复模板包括3′同源臂,其包括与DNA断裂位点的DNA序列3′完全相同或几乎完全相同的核酸序列。在优选的实施例中,供体修复模板包括5′同源臂和3′同源臂。供体修复模板可以包括与DSB位点紧邻的基因组序列的同源性或与来自DSB位点的任何数目的碱基对内的基因组序列的同源性。在一个实施例中,供体修复模板包括与基因组序列同源约5bp、约10bp、约25bp、约50bp、约100bp、约250bp、约500bp、约1000bp、约2500bp、约5000bp、约10000bp或更多的核酸序列,包含任何中间长度的同源序列。
特定实施例中所设想的适合长度的同源臂的说明性实例可以独立地选择并且包含但不限于:约100bp、约200bp、约300bp、约400bp、约500bp、约600bp、约700bp、约800bp、约900bp、约1000bp、约1100bp、约1200bp、约1300bp、约1400bp、约1500bp、约1600bp、约1700bp、约1800bp、约1900bp、约2000bp、约2100bp、约2200bp、约2300bp、约2400bp、约2500bp、约2600bp、约2700bp、约2800bp、约2900bp、或约3000bp、或更长的同源臂,包含所有中间长度的同源臂。
合适的同源臂长度的另外的说明性实例包含但不限于:约100bp到约3000bp、约200bp到约3000bp、约300bp到约3000bp、约400bp到约3000bp、约500bp到约3000bp、约500bp到约2500bp,约500bp到约2000bp、约750bp到约2000bp、约750bp到约1500bp、或约1000bp到约1500bp,包含所有中间长度的同源臂。
在特定具体实施例中,5′同源臂和3′同源臂的长度独立地选自约500bp至约1500bp。在一个实施例中,5′同源臂长度为约1500bp,并且3′同源臂长度为约1000bp。在一个实施例中,5′同源臂为约200bp至约600bp,并且3′同源臂为约200bp至约600bp。在一个实施例中,5′同源臂长度为约200bp,并且3′同源臂长度为约200bp。在一个实施例中,5′同源臂长度为约300bp,并且3′同源臂长度为约300bp。在一个实施例中,5′同源臂长度为约400bp,并且3′同源臂长度为约400bp。在一个实施例中,5′同源臂长度为约500bp,并且3′同源臂长度为约500bp。在一个实施例中,5′同源臂长度为约600bp,并且3′同源臂长度为约600bp。
F.多肽
本文设想了各种多肽,包含但不限于融合多肽,包含归巢核酸内切酶变体和/或megaTAL。在优选实施例中,多肽包括SEQ ID NO:7、10、13、19、22、25、28、31、34、37、40、43、46、49、52、55、58、61、64、67、70、73、76、79、82、89、92、95、98和101中的任一个中所示的氨基酸序列。除非相反地规定,否则“多肽”、“多肽片段”、“肽”和“蛋白质”可互换使用,并且根据常规含义使用,即,作为氨基酸序列。在一个实施例中,“多肽”包含融合多肽和其它变体。可以使用各种熟知的重组和/或合成技术中的任何技术来制备多肽。多肽不限于具体长度,例如,多肽可以包括全长蛋白序列、全长蛋白质的片段或融合蛋白,并且可以包含多肽的转译后修饰,例如糖基化、乙酰化、磷酸化等以及本领域已知的天然存在的和非天然存在的其它修饰。
如本文所用,“分离的蛋白质”、“分离的肽”或“分离的多肽”等是指从细胞环境中以及从与细胞的其它组分的缔合中体外合成、分离和/或纯化的肽或多肽分子,即,所述肽或多肽分子未与体内物质显著缔合。
在特定实施例中设想的多肽的说明性实例包含但不限于归巢核酸内切酶变体、megaTAL、末端加工核酸酶、核酸外切酶、融合多肽及其变体。
多肽包含“多肽变体”。多肽变体与天然存在的多肽的不同之处可以在于一个或多个氨基酸取代、缺失、添加和/或插入。此类变体可以是天然存在的或可以通过合成产生,例如通过修饰上述多肽序列的一个或多个氨基酸。例如,在特定实施例中,可能期望通过将一个或多个取代、缺失、添加和/或插入引入到多肽中来改善结合并切割靶位点的融合多肽、归巢核酸内切酶、megaTAL等的生物学特性。在特定实施例中,多肽包含与本文所设想的任何参考序列具有至少约65%、70%、71%、72%、73%、74%、75%、76%、77%、78%、79%、80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%氨基酸同一性的多肽,通常其中变体维持参考序列的至少一种生物活性。
多肽变体包含具有生物活性的“多肽片段”。生物活性多肽片段的说明性实例包含DNA结合结构域、核酸酶结构域、内切加工结构域(例如,核酸外切酶)等。如本文所用,术语“生物活性片段”或“最小生物活性片段”是指保留天然存在的多肽活性的至少100%、至少90%、至少80%、至少70%、至少60%、至少50%、至少40%、至少30%、至少20%、至少10%或至少5%的多肽片段。在优选实施例中,生物活性是靶序列的结合亲和力和/或切割活性。在某些实施例中,多肽片段可以包括长度为至少5到约1700个氨基酸的氨基酸链。应当理解的是,在某些实施例中,片段长度为至少5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、17个、18个、19个、20个、21个、22个、23个、24个、25个、26个、27个、28个、29个、30个、31个、32个、33个、34个、35个、36个、37个、38个、39个、40个、41个、42个、43个、44个、45个、46个、47个、48个、49个、50个、55个、60个、65个、70个、75个、80个、85个、90个、95个、100个、110个、150个、200个、250个、300个、350个、400个、450个、500个、550个、600个、650个、700个、750个、800个、850个、900个、950个、1000个、1100个、1200个、1300个、1400个、1500个、1600个、1700个或更多个氨基酸。在特定实施例中,多肽包括归巢核酸内切酶变体的生物活性片段。在特定实施例中,本文所示的多肽可以包括一个或多个表示为“X”的氨基酸。如果存在于氨基酸SEQ ID NO中,则“X”指代任何氨基酸。一个或多个“X”残基可以存在于本文所设想的SEQ ID NO所具体阐述的氨基酸序列的N末端和C末端处。如果“X”氨基酸不存在,则SEQ ID NO中所示的剩余氨基酸序列可以被认为是生物活性片段。
在特定实施例中,多肽包括归巢核酸内切酶变体的生物活性片段,例如,SEQ IDNO:89、92、95、98或101或megaTAL(例如,SEQ ID NO:7、10、58、67和76)。生物活性片段可以包括N末端截短和/或C末端截短。在特定实施例中,与对应的野生型归巢核酸内切酶序列相比,生物活性片段缺乏或包括归巢核酸内切酶变体的1个、2个、3个、4个、5个、6个、7个或8个N末端氨基酸的缺失,更优选地与对应的野生型归巢核酸内切酶序列相比归巢核酸内切酶变体的4个N末端氨基酸的缺失。在特定实施例中,与对应的野生型归巢核酸内切酶序列相比,生物活性片段缺乏或包括归巢核酸内切酶变体的1个、2个、3个、4个或5个C末端氨基酸的缺失,更优选地与对应的野生型归巢核酸内切酶序列相比归巢核酸内切酶变体的2个C末端氨基酸的缺失。在特定优选实施例中,与对应的野生型归巢核酸内切酶序列相比,生物活性片段缺乏或包括归巢核酸内切酶变体的4个N末端氨基酸和2个C末端氨基酸的缺失。
在特定实施例中,I-OnuI变体包括:以下N末端氨基酸的1个、2个、3个、4个、5个、6个、7个或8个的缺失:M、A、Y、M、S、R、R、E;和/或以下1个、2个、3个、4个或5个C末端氨基酸的缺失:R、G、S、F、V。
在特定实施例中,I-OnuI变体包括:以下N末端氨基酸的1个、2个、3个、4个、5个、6个、7个或8个的缺失或取代:M、A、Y、M、S、R、R、E;和/或以下1个、2个、3个、4个或5个C末端氨基酸的缺失或取代:R、G、S、F、V。
在特定实施例中,I-OnuI变体包括:以下N末端氨基酸的1个、2个、3个、4个、5个、6个、7个或8个的缺失:M、A、Y、M、S、R、R、E;和/或以下1个或2个C末端氨基酸的缺失:F、V。
在特定实施例中,I-OnuI变体包括:以下N末端氨基酸的1个、2个、3个、4个、5个、6个、7个或8个的缺失或取代:M、A、Y、M、S、R、R、E;和/或以下1个或2个C末端氨基酸的缺失或取代:F、V。
如上所述,多肽可以以多种方法改变,包含氨基酸取代、缺失、截短和插入。用于此类操纵的方法是本领域所公知的。例如,参考多肽的氨基酸序列变体可以通过DNA中的突变来制备。用于诱变和核苷酸序列更改的方法是本领域中公知的。参见,例如,Kunkel(1985,《美国国家科学院院刊》82:488-492);Kunkel等人,(1987,《酶学方法(Methods inEnzymol)》,154:367-382);美国专利第4,873,192号;Watson,J.D.等人,(《基因的分子生物学(Molecular Biology of the Gene)》,第四版,加利福尼亚门洛帕克的本杰明/卡明斯公司(Benjamin/Cummings,Menlo Park,Calif.),1987)以及本文引用的参考文献。关于不影响所关注蛋白质的生物活性的适当氨基酸取代的指导可以在Dayhoff等人,(1978),《蛋白质序列和结构图集(Atlas of Protein Sequence and Structure)》(华盛顿哥伦比亚特区的美国国家生物医学研究基金会(Natl.Biomed.Res.Found.,Washington,D.C.))。
在某些实施例中,变体将含有一个或多个保守取代。“保守取代”是其中氨基酸被具有类似性质的另一个氨基酸取代从而使得肽化学领域的技术人员预期多肽的二级结构和亲水性基本上未改变的取代。可以对特定实施例中所设想的多核苷酸和多肽的结构中进行修饰,多肽包含具有至少约并且仍然获得对具有期望特性的变体或衍生多肽进行编码的功能分子的多肽。当期望更改多肽的氨基酸序列以产生等效的或甚至经改进的变体多肽时,本领域技术人员例如可以例如根据表1改变编码DNA序列的密码子中的一个或多个。
表1-氨基酸密码子
可以使用本领域中公知的计算机程序如DNASTAR、DNA Strider、G、Mac Vector或Vector NTI软件找到关于确定哪些氨基酸残基可以被取代、插入或缺失而不消除生物活性的指南。优选地,本文所公开的蛋白质变体的氨基酸改变是保守氨基酸改变,即,带类似电荷或不带电荷的氨基酸的取代。保守氨基酸改变涉及在侧链方面相关的氨基酸的家族中的一个氨基酸的取代。天然存在的氨基酸通常分为四个家族:酸性氨基酸(天冬氨酸、谷氨酸)、碱性氨基酸(赖氨酸、精氨酸、组氨酸)、非极性氨基酸(丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸、色氨酸)和极性不带电氨基酸(甘氨酸、天冬酰胺、谷胺酰胺、半胱氨酸、丝氨酸、苏氨酸、酪氨酸)。苯丙氨酸、色氨酸和酪氨酸有时被共同分类为芳香族氨基酸。在肽或蛋白质中,适当的氨基酸保守取代对于本领域的技术人员来说是已知的,并且通常可以在不改变所得分子的生物活性的情况下进行。本领域的技术人员认识到,通常,多肽的非必需区中的单个氨基酸取代基本上不会改变生物活性(参见,例如,Watson等人《基因的分子生物学》,第4版,1987,本杰明/卡明斯出版公司(The Benjamin/CummingsPub.Co.),第224页)。
在一个实施例中,在期望表达两个或更多个多肽的情况下,如本文其它地方所公开的,对其进行编码的多核苷酸序列可以通过IRES序列分开。
特定实施例中设想的多肽包含融合多肽,例如SEQ ID NO:7、10、13、19、22、25、28、31、34、37、40、43、46、49、52、55、58、61、64、67、70、73、76、79和82。在优选的实施例中,所述融合多肽包括如SEQ ID NO:46、64、73和82中的任一个中所示的氨基酸序列。在特定实施例中,提供了融合多肽和编码融合多肽的多核苷酸。融合多肽和融合蛋白指代具有至少两个、三个、四个、五个、六个、七个、八个、九个或十个多肽片段的多肽。
在另一个实施例中,可以将两个或更多个多肽表达为包括如本文中其它地方所公开的一个或多个自切割多肽序列的融合蛋白。
在一个实施例中,本文设想的融合蛋白包括一个或多个DNA结合结构域和一个或多个核酸酶,以及一个或多个连接子和/或自切割多肽。
在一个实施例中,本文设想的融合蛋白包括核酸酶变体;连接子或自切割肽;以及包含但不限于5′-3′核酸外切酶、5′-3′碱性核酸外切酶和3′-5′核酸外切酶(例如,Trex2、ExoI或ExoX)的末端加工酶。
融合多肽可以包括:一个或多个多肽结构域或片段,包含但不限于信号肽、细胞渗透性肽结构域(CPP)、DNA结合结构域、核酸酶结构域等;表位标签(例如,麦芽糖结合蛋白(“MBP”)、谷胱甘肽S转移酶(GST)、HIS6、MYC、FLAG、V5、VSV-G和HA);多肽连接子;以及多肽切割信号。融合多肽通常是C末端连接到N末端,但其也可以是C末端连接到C末端、N末端连接到N末端或N末端连接到C末端。在特定实施例中,融合蛋白的多肽可以采取任何顺序。融合多肽或融合蛋白还可以包含保守修饰的变体、多态变体、等位基因、突变体、子序列和种间同系物,只要保持融合多肽的期望活性即可。融合多肽可以通过化学合成法或通过两个部分之间的化学连接产生或者通常可以使用其它标准技术制备。包括融合多肽的连接的DNA序列可操作地连接到如本文其它地方所公开的适合的转录或转译控制元件。
融合多肽可以任选地包括可以用于连接多肽内的一个或多个多肽或结构域的连接子。肽连接子序列可以用于将任何两个或更多个多肽组分分开足够的距离,以确保每个多肽折叠成其适当的二级结构和三级结构,以便使多肽结构域发挥其期望功能。这种肽连接子序列使用本领域中的标准技术并入到融合多肽中。适合的肽连接子序列可以基于以下因素进行选择:(1)其能够采用灵活的延伸构象;(2)其不能采用可以与第一多肽和第二多肽上的功能性表位相互作用的次级结构;以及(3)缺乏可以与多肽功能性表位反应的疏水残基或带电残基。优选的肽连接子序列包含Gly、Asn和Ser残基。还可以在连接子序列中使用其它近中性氨基酸,如Thr和Ala。可以有效地用作连接子的氨基酸序列包含以下文献中公开的那些:Maratea等人,《基因(Gene)》40:39-46,1985;Murphy等人,《美国国家科学院院刊》83:8258-8262,1986;美国专利第4,935,233号以及美国专利第4,751,180号。当特定融合多肽片段包含可以用于将功能结构域分开并防止空间干扰的非必需N末端氨基酸区时,不需要连接子序列。优选的连接子通常是被合成为重组融合蛋白的一部分的柔性氨基酸子序列。连接子多肽可以为长度在1个与200个氨基酸之间、长度在1个与100个氨基酸之间或者长度在1个与50个氨基酸之间,包含其间的所有整数值。
示例性连接子包含但不限于以下氨基酸序列:甘氨酸聚合物(G)n;甘氨酸-丝氨酸聚合物(G1-5S1-5)n,其中n为至少一、二、三、四或五的整数;甘氨酸-丙氨酸聚合物;丙氨酸-丝氨酸聚合物;GGG(SEQ ID NO:113);DGGGS(SEQ ID NO:114);TGEKP(SEQ ID NO:115)(参见例如,Liu等人,《美国国家科学院院刊》5525-5530(1997));GGRR(SEQ ID NO:116)(Pomerantz等人1995,同上);(GGGGS)n,其中n=1、2、3、4或5(SEQ ID NO:117和150-153)(Kim等人,《美国国家科学院院刊》93,1156-1160(1996));EGKSSGSGSESKVD(SEQ ID NO:118)(Chaudhary等人,1990,《美国国家科学院院刊》87:1066-1070);KESGSVSSEQLAQFRSLD(SEQ ID NO:119)(Bird等人,1988,《科学》242:423-426);GGRRGGGS(SEQ ID NO:120);LRQRDGERP(SEQ ID NO:121);LRQKDGGGSERP(SEQ ID NO:122);LRQKD(GGGS)2ERP(SEQ IDNO:123)。可替代地,柔性连接子可以使用能够对DNA结合位点和肽两者自身进行建模的计算机程序(Desjarlais和Berg,《美国国家科学院院刊》90:2256-2260(1993);《美国国家科学院院刊》91:11099-11103(1994))或通过噬菌体展示方法合理地设计。
融合多肽可以进一步包括本文所描述的多肽结构域中的每个多肽结构域之间或内源性开放阅读框与供体修复模板编码的多肽之间的多肽切割信号。另外,可以将多肽切割位点置于任何连接子肽序列中。示例性多肽切割信号包含多肽切割识别位点,如蛋白酶切割位点、核酸酶切割位点(例如,稀有限制酶识别位点、自切割核酶识别位点)和自切割病毒寡肽(参见deFelipe和Ryan,2004,《转运(Traffic)》,5(8),616-26)。
合适的蛋白酶切割位点和自切割肽是技术人员已知的(参见例如,Ryan等人,1997,《普通病毒学杂志(J.Gener.Virol.)》78,699-722;Scymczak等人(2004)《自然生物科技(Nature Biotech.)》5,589-594)。示例性蛋白酶切割位点包含但不限于以下的切割位点:马铃薯Y病毒NIa蛋白酶(例如,烟草蚀斑病毒蛋白酶)、马铃薯Y病毒HC蛋白酶、马铃薯Y病毒P1(P35)蛋白酶、byo病毒NIa蛋白酶、byo病毒RNA-2编码蛋白酶、口疮病毒属L蛋白酶、肠病毒2A蛋白酶、鼻病毒2A蛋白酶、小rna 3C蛋白酶、豇豆花叶病毒24K蛋白酶、线虫传多面体病毒24K蛋白酶、RTSV(水稻东格鲁球状病毒)3C样蛋白酶、PYVF(欧防风黄点病毒)3C样蛋白酶、肝素、凝血酶、因子Xa和肠激酶。由于其高切割严格性,在一个实施例中优选TEV(烟草蚀斑病毒)蛋白酶切割位点,例如,EXXYXQ(G/S)(SEQ ID NO:121),例如,ENLYFQG(SEQ IDNO:122)以及ENLYFQS(SEQ ID NO:123),其中X表示任何氨基酸(在Q与G之间或Q与S之间发生由TEV进行的切割)。
在某些实施例中,自切割多肽位点包括2A或2A样位点、序列或结构域(Donnelly等人,2001《普通病毒学杂志》82:1027-1041)。在一特定实施例中,病毒2A肽是口疮病毒2A肽、马铃薯Y病毒2A肽或心脏病毒2A肽。
在一个实施例中,所述病毒2A肽选自由以下组成的组:口蹄疫病毒(FMDV)2A肽、马鼻炎A型病毒(ERAV)2A肽、明脉扁刺蛾β四体病毒(TaV)2A肽、猪捷申病毒-1(PTV-1)2A肽、泰勒病毒2A肽以及脑心肌炎病毒2A肽。
表2中提供了2A位点的说明性实例。
表2:示例性2A位点包含以下序列:
SEQ ID NO:127 | GSGATNFSLLKQAGDVEENPGP |
SEQ ID NO:128 | ATNFSLLKQAGDVEENPGP |
SEQ ID NO:129 | LLKQAGDVEENPGP |
SEQ ID NO:130 | GSGEGRGSLLTCGDVEENPGP |
SEQ ID NO:131 | EGRGSLLTCGDVEENPGP |
SEQ ID NO:132 | LLTCGDVEENPGP |
SEQ ID NO:133 | GSGQCTNYALLKLAGDVESNPGP |
SEQ ID NO:134 | QCTNYALLKLAGDVESNPGP |
SEQ ID NO:135 | LLKLAGDVESNPGP |
SEQ ID NO:136 | GSGVKQTLNFDLLKLAGDVESNPGP |
SEQ ID NO:137 | VKQTLNFDLLKLAGDVESNPGP |
SEQ ID NO:138 | LLKLAGDVESNPGP |
SEQ ID NO:139 | LLNFDLLKLAGDVESNPGP |
SEQ ID NO:140 | TLNFDLLKLAGDVESNPGP |
SEQ ID NO:141 | LLKLAGDVESNPGP |
SEQ ID NO:142 | NFDLLKLAGDVESNPGP |
SEQ ID NO:143 | QLLNFDLLKLAGDVESNPGP |
SEQ ID NO:144 | APVKQTLNFDLLKLAGDVESNPGP |
SEQ ID NO:145 | VTELLYRMKRAETYCPRPLLAIHPTEARHKQKIVAPVKQT |
SEQ ID NO:146 | LNFDLLKLAGDVESNPGP |
SEQ ID NO:147 | LLAIHPTEARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP |
SEQ ID NO:148 | EARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP |
G.多核苷酸
在特定实施例中,提供了编码本文所设想的一个或多个融合多肽、归巢核酸内切酶变体、megaTAL、末端加工酶和核酸外切酶的多核苷酸。如本文所用,术语“多核苷酸”或“核酸”是指脱氧核糖核酸(DNA)、核糖核酸(RNA)和DNA/RNA杂交体。
在各个实施例中,多核苷酸编码一种或多种融合多肽,例如,SEQ ID NO:5、8、11、17、20、23、26、29、32、35、38、41、44、47、50、53、56、59、62、65、68、71、74、77和80。在一些实施例中,编码一种或多种融合多肽的多核苷酸是RNA多核苷酸。在一些实施例中,编码一种或多种融合多肽的RNA多核苷酸包括SEQ ID NO:6、9、12、18、21、24、27、30、33、36、39、42、45、48、51、54、57、60、63、66、69、72、75、78和81中的任一个中所示的序列。在一些实施例中,所述多核苷酸编码如SEQ ID NO:7、10、13、19、22、25、28、31、34、37、40、43、46、49、52、55、58、61、64、67、70、73、76、79和82中的任一个中所示的氨基酸序列。
多核苷酸可以是单链的或双链的以及重组的、合成的或分离的。多核苷酸包含但不限于:前信使RNA(前mRNA)、信使RNA(mRNA)、合成RNA、合成mRNA、基因组DNA(gDNA)、PCR扩增DNA、互补DNA(cDNA)、合成DNA和重组DNA。多核苷酸是指长度为至少5个、至少10个、至少15个、至少20个、至少25个、至少30个、至少40个、至少50个、至少100个、至少200个、至少300个、至少400个、至少500个、至少1000个、至少5000个、至少10000个或至少15000个或更多个核苷酸以及所有中间长度的核苷酸的聚合形式,或者是核糖核苷酸或脱氧核糖核苷酸或者是任一类型核苷酸的修饰形式。容易理解的是,在此上下文中,“中间长度”意指所引用值之间的任何长度,如6、7、8、9等,101、102、103等;151、152、153等;201、202、203等。在特定实施例中,多核苷酸或变体与参考序列具有至少或约50%、55%、60%、65%、70%、71%、72%、73%、74%、75%、76%、77%、78%、79%、80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%序列同一性。
在特定实施例中,多核苷酸可以是密码子优化的。如本文所用,术语“密码子优化的”指代取代编码多肽的多核苷酸中的密码子以增加多肽的表达、稳定性和/或活性。影响密码子优化的因素包含但不限于以下中的一种或多种:(i)两个或更多个生物体或基因之间的密码子偏倚或者合成地构成的偏倚表格的变化;(ii)生物体、基因或基因组内的密码子偏倚度的变化;(iii)包含背景在内的密码子的系统变化;(iv)密码子根据其解码tRNA的变化;(v)密码子根据GC%在总体上或在三联体中的一个位置处的变化;(vi)与参考序列例如天然存在的序列的相似度的变化;(vii)密码子频率截止的变化;(viii)从DNA序列转录的mRNA的结构性质;(ix)关于设计密码子取代组所基于的DNA序列的功能的先验知识;和/或(x)每个氨基酸的密码子组的系统变化;和/或(xi)假性转译起始位点的经过分离的移除。
如本文所用,术语“核苷酸”是指与磷酸化糖呈N-糖苷连接的杂环含氮碱基。核苷酸被理解成包含天然碱基和多种多样的本领域所认可的经过修饰的碱基。这种碱基通常位于核苷酸糖部分的1′位置。核苷酸通常包括碱、糖和磷酸基团。在核糖核酸(RNA)中,糖为核糖,并且在脱氧核糖核酸(DNA)中,糖为脱氧核糖,即缺少存在于核糖中的羟基的糖。示例性天然含氮碱基包含嘌呤、腺苷(A)和胍(G)以及嘧啶、胞苷(C)和胸苷(T)(或在RNA的背景下,尿嘧啶(U))。脱氧核糖的C-1原子与嘧啶的N-1或嘌呤的N-9键合。核苷酸通常是单磷酸、二磷酸或三磷酸。可以在糖、磷酸和/或碱基部分不修饰或修饰核苷酸(也互换地称为核苷酸类似物、核苷酸衍生物、经过修饰的核苷酸、非天然核苷酸和非标准核苷酸;参见例如,WO92/07065和WO 93/15187)。由Limbach等人(1994,《核酸研究(Nucleic Acids Res.)》22,2183-2196)总结了经修饰的核酸碱基的实例。
核苷酸也可以被认为是核苷的磷酸酯,酯化发生在与糖的C-5附接的羟基上。如本文所用,术语“核苷”指代与糖呈N-糖苷连接的杂环含氮碱基。核苷在本领域中被认为包含天然碱基并且还包含公知的经过修饰的碱基。这种碱基通常位于核苷糖部分的1'位置处。核苷通常包括碱基和糖基。核苷可以是未经过修饰的或者在糖和/或碱基部分进行修饰(也可互换地被称为核苷类似物、核苷衍生物、经过修饰的核苷、非天然核苷或非标准核苷)。也如上文所述,由Limbach等人(1994,《核酸研究》22,2183-2196)总结了经修饰的核酸碱基的实例。
多核苷酸的说明性实例包含但不限于编码SEQ ID NO:7、10、13、16、19、22、25、28、31、34、37、40、43、46、49、52、55、58、61、64、67、70、73、76、79、82、89、92、95、98和101的多核苷酸,以及SEQ ID NO:5、6、8、9、11、12、14、15、17、18、20、21、23、24、26、27、29、30、32、33、35、36、38、39、41、42、44、45、47、48、50、51、53、54、56、57、59、60、62、63、65、66、68、69、71、72、74、75、77、78、80、81、87、88、90、91、93、94、96、97、99和100中所示的多核苷酸序列。
在各个说明性实施例中,本文所设想的多核苷酸包含但不限于编码融合多肽、归巢核酸内切酶变体、megaTAL、末端加工酶、核酸外切酶的多核苷酸以及包括本文所设想的多核苷酸的表达载体、病毒载体和转移质粒。
如本文所用,术语“多核苷酸变体”和“变体”等是指展现出与参考多核苷酸序列或在下文中定义的严格条件下与参考序列杂交的多核苷酸具有实质性序列同一性的多核苷酸。这些术语还涵盖通过添加、缺失、取代或修饰至少一个核苷酸而区别于参考多核苷酸的多核苷酸。因此,术语“多核苷酸变体”和“变体”包含其中已添加或缺失、或修饰或用不同核苷酸替换一个或多个核苷酸的多核苷酸。在此方面,本领域中充分理解的是,可以对参考多核苷酸作出包括突变、添加、缺失以及取代在内的某些改变,由此所改变的多核苷酸保留所述参考多核苷酸的生物功能或活性。
在一个实施例中,多核苷酸包括在严格条件下与靶核酸序列杂交的核苷酸序列。在“严格条件”下杂交描述了其中彼此至少60%相同的核苷酸序列维持杂交的杂交方案。通常,严格条件在限定的离子强度和pH下被选择为比特定序列的热熔点(Tm)低约5℃。Tm是温度(在限定的离子强度、pH和核酸浓度下),在所述温度下,与靶序列互补的探针中的50%在平衡时与靶序列杂交。由于靶序列通常过量存在,所以在Tm下,探针中的50%在平衡时被占据。
如本文所用,陈述“序列同一性”或例如包括“与……50%相同的序列”是指序列在一个比较窗口内在逐核苷酸的基础上或在逐氨基酸的基础上相同的程度。因此,“序列同一性百分比”可以通过以下方式计算:在比较窗内比较两个最佳地比对的序列,测定的一致核酸碱基(例如,A、T、C、G、I)或一致氨基酸残基(例如,Ala、Pro、Ser、Thr、Gly、Val、Leu、Ile、Phe、Tyr、Trp、Lys、Arg、His、Asp、Glu、Asn、Gln、Cys以及Met)在两个序列中出现的位置的数目以得到相配位置的数目,用相配位置的数目除以比较窗中的位置总数(即,窗大小)并将结果乘以100,得到序列同一性百分比。包含与本文所描述的参考序列中的任何参考序列具有至少约50%、55%、60%、65%、70%、75%、80%、85%、90%、95%、96%、97%、98%、99%或100%的序列同一性的核苷酸和多肽,通常其中多肽变体保持参考多肽的至少一种生物活性。
用于描述两个或更多个多核苷酸或多肽之间的序列关系的术语包含“参考序列”、“比较窗口”、“序列同一性”、“序列同一性百分比”以及“实质性同一性”。“参考序列”的长度为至少12个、但通常为15个到18个,并且通常为至少25个单体单元,包含核苷酸和氨基酸残基。因为两个多核苷酸可以各自包括(1)在两个多核苷酸之间类似的序列(即,仅完整多核苷酸序列的一部分);和(2)在两个多核苷酸之间相异的序列,两个(或更多个)多核苷酸之间的序列比较通常通过在“比较窗”内比较两个多核苷酸的序列以鉴别并比较序列局部区域的相似性来进行。“比较窗”是指具有至少6个、通常约50个至约100个、更通常约100个至约150个连续位置的概念性区段,其中在最佳地比对一个序列与具有相同数目连续位置的参考序列之后,比较所述两个序列。为了对两条序列进行最佳比对,比较窗可以包括相较于参考序列(不包括添加或缺失的序列),约20%或更低百分比的添加或缺失(即,空位)。用于比对比较窗口的序列的最佳比对可以通过算法的计算机化实施例(美国威斯康星州麦迪逊的科学大道575号的遗传学计算机集团(Genetics Computer Group,575Science DriveMadison,WI,USA)的威斯康星遗传学软件包7.0版本(Wisconsin Genetics SoftwarePackage Release 7.0)的GAP、BESTFIT、FASTA和TFASTA)或者通过检查和由所选择的各种方法中的任何一种方法生成的最佳比对(即,产生了比较窗口内的最高同源性百分比)来进行。还可以参考如例如通过Altschul等人,1997,《核酸研究》25:3389所公开的BLAST程序家族。序列分析的详细论述可以见于Ausubel等人,《当代分子生物学实验手册》,约翰威立父子公司(John Wiley&Sons,Inc.),1994-1998,第15章的第19.3单元。
如本文所用,“分离的多核苷酸”是指已经从在其两侧处于天然存在状态的序列纯化的多核苷酸,例如,已经从通常与DNA片段相邻的序列移除的片段。在特定实施例中,“分离的多核苷酸”指代互补DNA(cDNA)、重组多核苷酸、合成多核苷酸或并不天然存在但已经通过人工制成的其它多核苷酸。
在各个实施例中,多核苷酸包括编码本文所设想的多肽的mRNA,所述多肽包含但不限于融合多肽、归巢核酸内切酶变体、megaTAL、末端加工酶和核酸外切酶。在某些实施例中,mRNA包括帽、一个或多个核苷酸和/或经修饰的核苷酸和poly(A)尾部。
在特定实施例中,本文所设想的mRNA包括poly(A)尾,以帮助保护mRNA免于核酸外切酶降解、稳定mRNA并且促进翻译。在某些实施例中,mRNA包括3′poly(A)尾结构。
在特定实施例中,poly(A)尾的长度为至少约10个、25个、50个、75个、100个、150个、200个、250个、300个、350个、400个、450个或至少约500个或更多个腺嘌呤核苷酸或者任何中间数量的腺嘌呤核苷酸。在特定实施例中,poly(A)尾的长度为至少约125个、126个、127个、128个、129个、130个、131个、132个、133个、134个、135个、136个、137个、138个、139个、140个、141个、142个、143个、144个、145个、146个、147个、148个、149个、150个、151个、152个、153个、154个、155个、156个、157个、158个、159个、160个、161个、162个、163个、164个、165个、166个、167个、168个、169个、170个、171个、172个、173个、174个、175个、176个、177个、178个、179个、180个、181个、182个、183个、184个、185个、186个、187个、188个、189个、190个、191个、192个、193个、194个、195个、196个、197个、198个、199个、200个、201个、202个、202个、203个、205个、206个、207个、208个、209个、210个、211个、212个、213个、214个、215个、216个、217个、218个、219个、220个、221个、222个、223个、224个、225个、226个、227个、228个、229个、230个、231个、232个、233个、234个、235个、236个、237个、238个、239个、240个、241个、242个、243个、244个、245个、246个、247个、248个、249个、250个、251个、252个、253个、254个、255个、256个、257个、258个、259个、260个、261个、262个、263个、264个、265个、266个、267个、268个、269个、270个、271个、272个、273个、274个或275个或更多个腺嘌呤核苷酸。
在特定实施例中,poly(A)尾的长度为约10个至约500个腺嘌呤核苷酸、约50个至约500个腺嘌呤核苷酸、约100个至约500个腺嘌呤核苷酸、约150个至约500个腺嘌呤核苷酸、约200个至约500个腺嘌呤核苷酸、约250个至约500个腺嘌呤核苷酸、约300个至约500个腺嘌呤核苷酸、约50个约450个腺嘌呤核苷酸、约50个至约400个腺嘌呤核苷酸、约50个至约350个腺嘌呤核苷酸、约100个至约500个腺嘌呤核苷酸、约100个至约450个腺嘌呤核苷酸、约100个至约400个腺嘌呤核苷酸、约100个至约350个腺嘌呤核苷酸、约100个至约300个腺嘌呤核苷酸、约150个至约500个腺嘌呤核苷酸、约150个至约450个腺嘌呤核苷酸、约150个至约400个腺嘌呤核苷酸、约150个至约350个腺嘌呤核苷酸、约150个至约300个腺嘌呤核苷酸、约150个至约250个腺嘌呤核苷酸、约150个至约200个腺嘌呤核苷酸、约200个至约500个腺嘌呤核苷酸、约200个至约450个腺嘌呤核苷酸、约200个至约400个腺嘌呤核苷酸、约200个至约350个腺嘌呤核苷酸、约200个至约300个腺嘌呤核苷酸、约250个至约500个腺嘌呤核苷酸、约250个至约450个腺嘌呤核苷酸、约250个至约400个腺嘌呤核苷酸、约250个至约350个腺嘌呤核苷酸或约250个至约300个腺嘌呤核苷酸或者任何中间范围的腺嘌呤核苷酸。
描述多核苷酸取向的术语包含:5′(通常是具有游离磷酸基团的多核苷酸的末端)和3′(通常是具有游离羟基(OH)基团的多核苷酸的末端)。多核苷酸序列可以以5′-3′取向或3′-5′取向注释。对于DNA和mRNA,5′-3′链被命名为“有义”、“正”或“编码”链,因为其序列与前信使(前mRNA)的序列相同[RNA中的尿嘧啶(U)而非DNA中的胸腺嘧啶(T)除外]。对于DNA和mRNA,作为由RNA聚合酶转录的链的互补3′-5′链被指定为“模板”链、“反义”链、“负”链或“非编码”链。如本文所用,术语“相反取向”是指以3′-5′取向书写的5′-3′序列或以5′-3′取向书写的3′-5′序列。
术语“互补”和“互补性”是指通过碱基配对规则相关的多核苷酸(即,核苷酸序列)。例如,DNA序列5′A G T C A T G 3′的互补链是3′T C A G TA C 5′。后一序列通常被书写为左边是5′末端并且右边是3′末端的反向补体5′C AT G AC T 3′。与其反向补体相等的序列被称为回文序列。互补性可以是“部分的”,其中根据碱基配对规则仅匹配一些核酸碱基。或者,核酸之间可以存在“完全”或“全部”的互补性。
如本文所用的术语“核酸盒”或“表达盒”是指载体内可以表达RNA和随后表达多肽的基因序列。在一个实施例中,核酸盒含有所关注基因,例如,所关注多核苷酸。在另一个实施例中,核酸盒含有一个或多个表达控制序列,例如,启动子、增强子、poly(A)序列和所关注基因,例如,所关注多核苷酸。载体可以包括1个、2个、3个、4个、5个、6个、7个、8个、9个或10个或更多个核酸盒。核酸盒在载体内定位且顺序地朝向,使得可以将盒中的核酸转录成RNA并在必要时转译成蛋白质或多肽、经历在转化细胞中具有活性所需的适当的转译后修饰并且通过将适合的细胞内隔区靶向细胞外隔区或分泌到细胞外隔区而易位到适合的隔区以具有生物活性。优选地,盒使其3′端和5′端适于准备插入到载体中,例如,其在每一端均具有限制性核酸内切酶位点。在优选实施例中,核酸盒含有用于治疗、预防或减轻遗传性病症的治疗基因序列。盒可以作为单个单元移除和插入到质粒或病毒载体中。
多核苷酸包含一个或多个所关注多核苷酸。如本文所用,术语“所关注多核苷酸”是指对多肽或融合多肽进行编码的多核苷酸或充当用于转录抑制性多核苷酸的模板的多核苷酸,如本文所设想的。
此外,本领域的普通技术人员应理解,由于遗传密码的简并性,存在许多可以对多肽或其变体片段进行编码的核苷酸序列,如本文所设想的。这些多核苷酸中的一些与任何天然基因的核苷酸序列具有最小的同源性。尽管如此,在特定实施例中具体地设想了由于密码子使用的差异而变化的多核苷酸,例如针对人和/或灵长类动物密码子选择而优化的多核苷酸。在一个实施例中,提供了包括特定等位基因序列的多核苷酸。等位基因是由于如核苷酸的缺失、添加和/或取代等一种或多种突变而改变的内源性多核苷酸序列。
在某个实施例中,所关注多核苷酸包括供体修复模板。
无论编码序列本身的长度如何,如本文其它地方所公开或如本领域已知的,在特定实施例中设想的多核苷酸可以与其它DNA序列组合,如启动子和/或增强子、未转译区域(UTR)、Kozak序列、多腺苷酸化信号、另外的限制酶位点、多个克隆位点、内部核糖体进入位点(IRES)、重组酶识别位点(例如,LoxP、FRT和Att位点)、终止密码子、转录终止信号、转录后应答元件(例如,土拔鼠肝炎病毒转录后应答元件(WPRE)、乙型肝炎病毒转录后应答元件(HPRE))以及编码自切割多肽的多核苷酸、表位标签,从而使得其整体长度可以显著变化。因此在特定的实施例中设想的是,可以采用几乎任何长度的多核苷酸片段,其总长度优选地受制备的容易性和在预期的重组DNA方案中的使用的限制。
可以使用本领域已知和可获得的各种成熟技术中的任何一种来制备、操纵、表达和/或递送多核苷酸。为了表达期望的多肽,可以将编码多肽的核苷酸序列插入适当的载体中。还可以通过将编码多肽的mRNA递送到细胞中来表达期望的多肽。
载体的说明性实例包含但不限于质粒、自主复制序列和可转座元件,例如睡美人(Sleeping Beauty)、PiggyBac。
载体的另外的说明性实例包含但不限于质粒、噬菌粒、粘粒、人工染色体如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1衍生的人工染色体(PAC)、如λ噬菌体或M13噬菌体等噬菌体以及动物病毒。
可用作载体的病毒的说明性实例包含但不限于逆转录病毒(包含慢病毒)、腺病毒、腺相关病毒、疱疹病毒(例如,单纯性疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒和乳多空病毒(例如,SV40)。
表达载体的说明性实例包含但不限于:用于在哺乳动物细胞中表达的pClneo载体(普洛麦格公司(Promega));用于慢病毒介导的基因转移和在哺乳动物细胞中表达的pLenti4/V5-DESTTM、pLenti6/V5-DESTTM和pLenti6.2/V5-GW/lacZ(英杰公司(Invitrogen))。在特定实施例中,本文所公开的多肽的编码序列可以连接到用于在哺乳动物细胞中表达多肽的此类表达载体中。
在特定实施例中,载体是游离型载体或在染色体外维持的载体。如本文所用,术语“附加型”是指能够复制而不整合到宿主的染色体DNA中并且不会从分裂的宿主细胞中逐渐丧失的载体,这也意味着所述载体在染色体外或附加地复制。
表达载体中存在的“表达控制序列”、“控制元件”或“调控序列”是载体——复制起点——的那些非翻译区、选择盒、启动子、增强子、翻译起始信号(Shine Dalgarno序列或Kozak序列)内含子、转录后调控元件、多腺苷酸化序列、5'和3'非翻译区——其与宿主细胞蛋白相互作用以进行转录和翻译。这些元件的强度和特异性可能不同。根据所利用的载体系统和宿主,可以使用任何数量的合适的转录和转译元件,包含普遍存在的启动子和诱导型启动子。
在特定实施例中,多核苷酸包括载体,所述载体包含但不限于表达载体和病毒载体。载体可以包括一种或多种外源、内源或异源控制序列,如启动子和/或增强子。“内源控制序列”是与基因组中的给定基因天然连接的序列。“外源”控制序列是通过基因操纵(即,分子生物技术)被放置成与基因并置,使得所述基因的转录由所连接的增强子/启动子引导的控制序列。“异源控制序列”是与被基因操纵的细胞来自不同物种的外源序列。“合成”控制序列可以包括一种或多种内源和/或外源序列和/或在体外或计算机中所确定的为特定疗法提供最佳启动子和/或增强子活性的序列的元件。
如本文所用,术语“启动子”指代RNA聚合酶所结合的多核苷酸(DNA或RNA)的识别位点。RNA聚合酶引发并转录可操作地连接到启动子的多核苷酸。在特定实施例中,在哺乳动物细胞中可操作的启动子包括位于转录起始位点上游大约25到30个碱基处的富AT区域和/或在转录起点上游70到80个碱基处所发现的另一序列:CNCAAT区域,其中N可以是任何核苷酸。
术语“增强子”指代含有能够提供增强的转录并且在一些情况下可以不依赖于其相对于另一个控制序列的朝向而起作用的序列的DNA片段。增强子可以与启动子和/或另一个增强子元件协作地或加性地起作用。术语“启动子/增强子”指代含有能够提供启动子功能和增强子功能两者的序列的DNA片段。
术语“可操作地连接”指代并置,其中所描述的组分处于允许所述组分按其预期方式起作用的关系。在一个实施例中,所述术语是指核酸表达控制序列(如启动子和/或增强子)与第二多核苷酸序列,例如,所关注的多核苷酸之间的功能性连接,其中表达控制序列引导对应于第二序列的核酸的转录。
如本文所用,术语“组成型表达控制序列”是指连续地或连续不断地允许可操作地连接的序列的转录的启动子、增强子或启动子/增强子。组成型表达控制序列可以是允许在各种各样的细胞和组织类型中表达的“普遍存在的”启动子、增强子或启动子/增强子或分别允许在受限种类的细胞和组织类型中表达的“细胞特异性”、“细胞类型特异性”、“细胞谱系特异性”或“组织特异性”启动子、增强子或启动子/增强子。
适合于在特定实施例中使用的示例性普遍存在的表达控制序列包含但不限于:巨细胞病毒(CMV)立即早期启动子、病毒猿猴病毒40(SV40)(例如,早期或晚期)、莫洛尼鼠白血病病毒(MoMLV)LTR启动子、Rous肉瘤病毒(RSV)LTR、单纯疱疹病毒(HSV)(胸苷激酶)启动子、H5、来自牛痘病毒的P7.5启动子和P11启动子、短延长因子1-α(EF1a-短)启动子、长延长因子1-α(EF1a-长)启动子、早期生长应答1(EGR1)、铁蛋白H(FerH)、铁蛋白L(FerL)、甘油醛3-磷酸脱氢酶(GAPDH)、真核翻译起始因子4A1(EIF4A1)、热休克70kDa蛋白5(HSPA5)、热休克蛋白90kDaβ、成员1(HSP90B1)、热休克蛋白70kDa(HSP70)、β-驱动蛋白(β-KIN)、人ROSA26基因座(Irions等人,《自然生物技术(Nature Biotechnology)》,25,1477-1482(2007))、泛素C启动子(UBC)、磷酸甘油酸激酶-1(PGK)启动子、巨细胞病毒增强子/鸡β-肌动蛋白(CAG)启动子、β-肌动蛋白启动子和骨髓增殖性肉瘤病毒增强子、阴性对照区缺失的、dl587rev引物结合位点取代的(MND)启动子(Challita等人,《病毒学杂志(J Virol)》69(2):748-55(1995))。
在特定实施例中,可以期望使用细胞、细胞类型、细胞谱系或组织特异性表达控制序列来实现期望的多核苷酸序列的细胞类型特异性、谱系特异性或组织特异性表达(例如,以在仅细胞类型、细胞谱系或组织的子集中或在具体发育阶段期间表达编码多肽的特定核酸)。
如本文所用,“条件表达”可以是指任何类型的条件表达,包含但不限于:诱导型表达;可阻遏型表达;在具有特定生理、生物或疾病状态等的细胞或组织中的表达。此定义不旨在排除细胞类型或组织特异性表达。某些实施例提供了所关注多核苷酸的条件表达,例如,通过使细胞、组织、生物体等经受导致多核苷酸表达或导致由所关注多核苷酸编码的多核苷酸的表达增加或减少的治疗或病状来控制表达。
诱导型启动子/系统的说明性实例包含但不限于类固醇诱导型启动子,如对糖皮质激素或雌性激素受体进行编码的基因的启动子(可通过用相应激素处理来诱导)、巯基金属结合蛋白启动子(可通过用各种重金属处理来诱导)、MX-1启动子(可通过干扰素来诱导)、“基因开关(GeneSwitch)”米非司酮可调系统(Sirin等人,2003,《基因(Gene)》,323:67)、cumate诱导型基因开关(WO 2002/088346)、四环素依赖性调控系统等。
还可以通过使用位点特异性DNA重组酶实现条件表达。根据某些实施例,多核苷酸包括至少一个(通常两个)用于位点特异性重组酶介导的重组的位点。如本文所用,术语“重组酶”或“位点特异性重组酶”包含切除或整合蛋白、酶、辅因子或参与涉及一个或多个重组位点(例如,两个、三个、四个、五个、六个、七个、八个、九个、十个或更多个)的重组反应的相关蛋白,所述蛋白可以是野生型蛋白质(参见Landy,《生物技术当前述评(Current Opinionin Biotechnology)》3:699-707(1993))或突变体、衍生物(例如,含有重组蛋白序列或其片段的融合蛋白)、片段和其变体。适于在特定实施例中使用的重组酶的说明性实例包含但不限于:Cre、Int、IHF、Xis、Flp、Fis、Hin、Gin、ΦC31、Cin、Tn3解离酶、TndX、XerC、XerD、TnpX、Hjc、Gin、SpCCE1和ParA。
多核苷酸可以包括各种各样的位点特异性重组酶中的任何位点特异性重组酶的一个或多个重组位点。应理解,位点特异性重组酶的靶位点是整合载体,例如,逆转录病毒载体或慢病毒载体,所需的任何一个或多个位点的补充。如本文所用,术语“重组序列”、“重组位点”或“位点特异性重组位点”指代重组酶识别和结合的特定核酸序列。
在特定实施例中,本文所设想的多核苷酸包含编码一个或多个多肽的一个或多个所关注多核苷酸。在特定实施例中,为了实现多个多肽中的每一个的有效翻译,可以通过一个或多个IRES序列或编码自切割多肽的多核苷酸序列来分离多核苷酸序列。
如本文所用,“内部核糖体进入位点”或“IRES”是指促进引导内部核糖体进入顺反子(蛋白编码区)的如ATG等起始密码子由此导致基因的帽依赖性翻译的元件。参见例如,Jackson等人,1990,《生物化学科学趋势(Trends Biochem Sci)》15(12):477-83)以及Jackson和Kaminski 1995.《RNA》1(10):985-1000。本领域的技术人员通常采用的IRES的实例包含在美国专利第6,692,736号中所描述的IRES。本领域已知的“IRES”的另外的实例包含但不限于可从小核糖核酸病毒(Jackson等人,1990)获得的IRES以及可从病毒或细胞mRNA源获得的IRES,所述病毒或细胞mRNA源如例如免疫球蛋白重链结合蛋白(BiP)、血管内皮生长因子(VEGF)(Huez等人,1998,《分子与细胞生物学(Mol.Cell.Biol.)》18(11):6178-6190)、成纤维细胞生长因子2(FGF-2)和胰岛素样生长因子(IGFII)、可转译起始因子eIF4G和酵母转录因子TFIID和HAP4、可从诺瓦根公司(Novagen)商购获得的脑心肌炎病毒(EMCV)(Duke等人,1992,《病毒学杂志》66(3):1602-9)以及VEGF IRES(Huez等人,1998,《分子与细胞生物学》18(11):6178-90)。已经报道了小核糖核酸病毒科(Picornaviridae)、二顺反子病毒科(Dicistroviridae)和黄病毒科(Flaviviridae)物种的病毒基因组中以及HCV、弗里德鼠白血病病毒(Friend murine leukemia virus,FrMLV)和莫洛尼氏鼠白血病病毒(Moloney murine leukemia virus,MoMLV)中的IRES。
在特定实施例中,多核苷酸包括具有共有Kozak序列并编码期望多肽的多核苷酸。如本文所用,术语“Kozak序列”是指大大促进mRNA与核糖体的小亚单元的初始结合并增加转译的短核苷酸序列。共有Kozak序列为(GCC)RCCATGG(SEQ ID NO:149),其中R为嘌呤(A或G)(Kozak,1986,《细胞(Cell)》44(2):283-92以及Kozak,1987,《核酸研究》15(20):8125-48)。
引导异源核酸转录物的高效终止和聚腺苷酸化的元件将增加异源基因表达。转录终止信号通常存在于多腺苷酸化信号的下游。在特定实施例中,载体包括对待表达的多肽进行编码的多核苷酸的3'处的多腺苷酸化序列。如本文所用,术语“polyA位点”或“polyA序列”表示通过RNA聚合酶II引导新生RNA转录物的终止和多腺苷酸化二者的DNA序列。聚腺苷酸化序列可以通过向编码序列的3′端添加polyA尾来促进mRNA稳定性,并且因此有助于提高转译效率。切割和聚腺苷酸化由RNA中的poly(A)序列引导。哺乳动物前mRNA的核心poly(A)序列具有位于切割-多腺苷酸化位点两侧的两个识别元件。通常,几乎不变的AAUAAA六聚体位于富含U或GU残基的更加可变元件的上游20-50个核苷酸处。新生转录物的切割发生在这两种元件之间,并且与向5'切割产物添加的多达250个腺苷偶联。在特定实施例中,核心poly(A)序列是理想的polyA序列(例如,AATAAA、ATTAAA、AGTAAA)。在特定实施例中,poly(A)序列是SV40 polyA序列、牛生长激素polyA序列(BGHpA)、兔β-球蛋白polyA序列(rβgpA)、其变体或本领域已知的另一适合的异源或内源polyA序列。
在特定实施例中,编码一种或多种融合多肽、归巢核酸内切酶变体、megaTAL、末端加工酶或核酸外切酶的多核苷酸可以通过非病毒方法和病毒方法两者引入到造血细胞,例如CD34+细胞。在特定实施例中,编码核酸酶和/或供体修复模板的一种或多种多核苷酸的递送可以通过相同的方法或通过不同的方法和/或通过相同的载体或通过不同的载体来提供。
术语“载体”在本文中用于指能够转移或转运另一个核酸分子的核酸分子。所转移的核酸通常连接到,(例如,插入)载体核酸分子。载体可以包含在细胞中引导自主复制的序列,或可以包含足以允许整合到宿主细胞DNA中的序列。在特定实施例中,非病毒载体用于将本文所设想的一个或多个多核苷酸递送到CD34+细胞。
非病毒载体的说明性实例包含但不限于质粒(例如,DNA质粒或RNA质粒)、转座子、粘粒以及细菌人工染色体。
特定实施例中所设想的多核苷酸的非病毒递送的说明性方法包含但不限于:电穿孔、声致穿孔、脂质转染、显微注射、基因枪法、病毒体、脂质体、免疫脂质体、纳米颗粒、聚阳离子或脂质:核酸缀合物、裸DNA、人工病毒体、DEAE-右旋糖酐介导的转移、基因枪和热休克。
在特定实施例中所设想的适合在特定实施例中使用的多核苷酸递送系统的说明性实例包含但不限于由阿马夏生物系统公司(Amaxa Biosystems)、马克赛特公司(Maxcyte,Inc.)、BTX分子递送系统公司(BTX Molecular Delivery Systems)和哥白尼治疗公司(Copernicus Therapeutics Inc.)提供的系统。脂质转染试剂是商业销售的(例如,TransfectamTM和LipofectinTM)。已经在文献中描述了适于多核苷酸的高效受体识别脂质转染的阳离子脂质和中性脂质。参见例如,Liu等人(2003),《基因疗法》10:180-187;以及Balazs等人(2011)《药物递送杂志(Journal of Drug Delivery.)》2011:1-12。在特定实施例中还设想了抗体靶向的、源自细菌的、基于无生命纳米细胞的递送。
在特定实施例中,编码本文所设想的融合多肽的多核苷酸(例如,DNA或RNA)可以例如通过电穿孔直接引入到细胞中。在电穿孔方法中,多核苷酸或定点多肽和多核苷酸的复合物在电穿孔缓冲液中与靶细胞混合以形成悬浮液。然后,此悬浮液在优化的电压下经受电脉冲,这在细胞膜的磷脂双层中产生临时孔,允许DNA和蛋白质等带电分子穿过孔进入细胞中。用于进行电穿孔的试剂和设备是市售的。电穿孔介质可以是本领域已知的任何合适的介质。合适的电穿孔方法是可用的,并且是本领域技术人员已知的。
如下文所描述的,包括在特定实施例中所设想的多核苷酸的病毒载体可以通过向个体患者施用进行体内递送,通常通过全身施用(例如,静脉内、腹膜内、肌肉内、皮下或颅内输注)或局部应用。可替代地,载体可以离体递送到细胞,如从个体患者移植的细胞(例如,动员的外周血、淋巴细胞、骨髓抽吸物、组织活检等)或通用供体造血干细胞,然后将细胞再植入患者体内。
在一个实施例中,直接向生物体施用包括核酸酶变体和/或供体修复模板的病毒载体以在体内转导细胞。可替代地,可以施用裸DNA或mRNA。施用是通过正常用于引入分子与血液或组织细胞最终接触的任何途径,包含(但不限于):注射、输注、局部施用和电穿孔。适合的施用此类核酸的方法是可利用的并且是所属领域的技术人员熟知的,并且虽然可以使用超过一种途径来施用特定的组合物,但特定的途径经常可以提供比另一途径更直接和更有效的反应。
适合于在本文所设想的特定实施例中使用的病毒载体系统的说明性实例包含但不限于腺病毒伴随病毒(AAV)、逆转录病毒、单纯疱疹病毒、腺病毒以及痘苗病毒载体。
H.经基因组编辑的细胞
提供了通过特定实施例中设想的方法制造和/或编辑的经基因组编辑的细胞。在特定实施例中设想的经基因组编辑的细胞可以是自体(autologous)/自体的(autogeneic)(“自我”)或非自体的(“非自我的”,例如同种异体的、同基因的或异种的)。如本文所用,“自体”指代来自同一受试者的细胞。如本文所用,“同种异体”指代同一物种的与相比之下的细胞在基因上有所不同的细胞。如本文所用,“同基因”指代不同受试者的与相比之下的细胞在基因上相同的细胞。如本文所用,“异种”指代与相比之下的细胞属于不同物种的细胞。在优选实施例中,细胞从哺乳动物受试者获得。在更优选实施例中,细胞从灵长类动物受试者获得,任选地为非人类灵长类动物。在最优选实施例中,细胞从人类受试者获得。
“分离的细胞”是指例如已经从体内组织或器官获得并且基本上不含细胞外基质的非天然存在的细胞,例如自然界中不存在的细胞、经过修饰的细胞、工程化细胞等。
在特定实施例中,所述细胞由本文所设想的融合多肽编辑,所述融合多肽例如包括DNA结合结构域和结合并切割细胞中的选定双链DNA(dsDNA)靶位点的归巢核酸内切酶(HE)变体、多肽连接子和核酸外切酶或其生物活性片段的融合多肽。
在各个实施例中,所述细胞包括由本文所设想的融合多肽诱导的定向偏向的缺失。在一些实施例中,至少50%、至少55%、至少60%、至少65%、至少70%、至少75%或至少80%的定向偏向的缺失的缺失中心位置位于HE靶位点中心位置的一侧上。在一些实施例中,相对于所述HE靶位点中心位置,所述缺失中心位置与所述DNA结合结构域靶位点位于同一侧上。在一些实施例中,所述缺失中心位置位于HE靶位点中心位置的5'端。
在各个实施例中,至少50%、至少55%、至少60%、至少65%、至少70%、至少75%或至少80%的缺失的缺失中心距所述HE靶位点中心位置大于4个核苷酸。在各个实施例中,至少10%、至少15%、至少20%、至少25%、至少30%或至少35%的缺失的缺失中心距所述HE靶位点中心位置大于8个核苷酸。
在各个实施例中,至少50%、至少55%、至少60%、至少65%、至少70%、至少75%或至少80%的缺失的长度为6bp或更长。在各个实施例中,至少30%、至少31%、至少32%、至少33%、至少34%、至少35%、至少40%、至少55%或至少60%的缺失的长度为12bp或更长。在特定实施例中,所述定向偏向的缺失的长度包括约10个、约11个、约12个、约13个、约14个、约15个、约16个、约17个、约18个、约19个或约20个核苷酸。
在各个实施例中,所述缺失延伸到DNA结合结构域靶位点中。在一些实施例中,所述缺失中心位置位于所述DNA结合结构域靶位点内。
可以使用本文所设想的组合物和方法编辑其基因组的细胞类型的说明性实例包含但不限于细胞系、原代细胞、干细胞、祖细胞和分化细胞。
术语“干细胞”指代作为能够进行以下的未分化细胞的细胞:(1)长期自我更新或能够产生原始细胞的至少一个相同拷贝;(2)在单细胞水平下分化成多种专门的细胞类型并且在某种情况下仅一种专门的细胞类型;以及(3)在体内功能性地再生组织。干细胞根据其发育潜能被细分为全能的、专能的、多能的、寡能/单能的。“自我更新”指代具有产生未改变的子细胞并产生专门的细胞类型的独特能力(潜能)的细胞。自我更新可以通过两种方式实现。不对称细胞分裂产生一个与亲本细胞相同的子细胞和一个与亲本细胞不同并且是祖细胞或分化细胞的子细胞。对称细胞分裂产生两个相同的子细胞。细胞的“增殖”或“扩增”指代对称分裂的细胞。
如本文所用,术语“祖先”或“祖细胞”指代细胞具有自我更新和分化成更成熟细胞的能力。许多祖细胞沿着单个谱系分化,但可能具有相当广泛的增殖能力。
在特定实施例中,细胞是原代细胞。如本文所用,术语“原代细胞”在本领域中已知用于指代已经从组织中分离并且已经建立用于在体外或离体生长的细胞。对应细胞已经经历了非常少(如果有的话)的群体倍增并且因此更能代表组织的主要功能组分,从而表示体内状态的更具代表性模型,所述相应细胞与连续细胞系相比源自所述组织。用于从各种组织获得样品的方法和用于建立原代细胞系的方法在本领域中是熟知的(参见例如,Jones和Wise,《分子生物学方法(Methods Mol Biol.)》1997)。用于本文所设想的方法的原代细胞源自脐带血、胎盘血、动员的外周血和骨髓。在一个实施例中,原生细胞是造血干细胞或祖细胞。
在一个实施例中,经基因组编辑的细胞是胚胎干细胞。在一个实施例中,经基因组编辑的细胞是成体干细胞或祖细胞。在一个实施例中,经基因组编辑的细胞是原代细胞。
在优选的实施例中,经基因组编辑的细胞是造血细胞,例如造血干细胞、造血祖细胞,如B细胞祖细胞,或包括造血细胞的细胞群体。
如本文所用,术语“细胞群体”指代可以由任何数量和/或组合的同质或异质细胞类型构成的多个细胞,如本文其它地方所描述的。例如,为了转导造血干细胞或造血祖细胞,可以从脐带血、胎盘血、骨髓或动员的外周血中分离或获得细胞群体。细胞群体可以包括约10%、约20%、约30%、约40%、约50%、约60%、约70%、约80%、约90%或约100%要编辑的靶细胞类型。在某些实施例中,可以使用本领域中已知的方法从异质细胞群中分离或纯化造血干细胞或造血祖细胞。
用于获得造血细胞的说明性来源包含但不限于:脐带血、骨髓或动员的外周血。
造血干细胞(HSC)产生能够在生物体的生命周期内产生整个成熟血细胞库的定向造血祖细胞(HPC)。术语“造血干细胞”或“HSC”是指产生生物体的所有血细胞类型的多能干细胞,包含骨髓细胞(例如,单核细胞和巨噬细胞、中性粒细胞、嗜碱性粒细胞、嗜酸性粒细胞、红细胞、巨核细胞/血小板、树突状细胞)和淋巴系细胞(例如,T细胞、B细胞、NK细胞)以及本领域已知的其它细胞(参见Fei,R.等人,美国专利第5,635,387号;McGlave等人,美国专利第5,460,964号;Simmons,P.等人,美国专利第5,677,136号;Tsukamoto等人,美国专利第5,750,397号;Schwartz等人,美国专利第5,759,793号;DiGuisto等人,美国专利第5,681,599号;Tsukamoto等人,美国专利第5,716,827号)。当移植到被致命地照射的动物或人中时,造血干细胞和造血祖细胞可以重新填充红系中性粒细胞-巨噬细胞、巨核细胞和淋巴造血细胞池。
适用于本文所设想的方法和组合物的造血干细胞或祖细胞的另外的说明性实例包含作为CD34+CD38LoCD90+CD45RA-的造血细胞、作为CD34+、CD59+、Thy1/CD90+、CD38Lo/-、C-试剂盒/CD117+和Lin(-)的造血细胞,以及作为CD133+的造血细胞。
在优选的实施例中,造血细胞是CD133+CD90+。在优选的实施例中,造血细胞是CD133+CD34+。在优选的实施例中,造血细胞是CD133+CD90+CD34+。
如本文所用,术语“CD34+细胞”是指在其细胞表面上表达CD34蛋白的细胞。如本文所用,“CD34”是指通常充当细胞—细胞粘附因子并且参与T细胞进入淋巴结的细胞表面糖蛋白(例如,唾液黏蛋白)。CD34+细胞群含有造血干细胞(HSC),其在施用给患者时分化并促成所有造血谱系,包含T细胞、NK细胞、NKT细胞、嗜中性粒细胞和单核细胞/巨噬细胞谱系的细胞。
存在用于表征造血层级的各种方法。一种表征方法是SLAM代码。SLAM(信号传导淋巴细胞活化分子)家族是由基因大多数串联定位在染色体1(鼠)上的单个基因座中、全都属于免疫球蛋白基因超家族的子集并且最初被认为参与T细胞刺激的>10个分子构成的组。这个家族包含CD48、CD150、CD244等,CD150是创始成员,并且因此也被称为slamF1,即,SLAM家族成员1。造血层级的签名SLAM代码为:造血干细胞(HSC)——CD150+CD48-CD244-;多能祖细胞(MPP)——CD150-CD48-CD244+;谱系限制性祖细胞(LRP)——CD150-CD48+CD244+;共同髓系祖细胞(CMP)——lin-SCA-1-c-试剂盒+CD34+CD16/32中;粒细胞-巨噬细胞祖细胞(GMP)——lin-SCA-1-c-试剂盒+CD34+CD16/32高;粒细胞-巨噬细胞祖细胞(GMP)——lin-SCA-1-c-试剂盒+CD34-CD16/32低。
用本文所设想的组合物和方法编辑的优选靶细胞类型包含造血细胞,优选地人造血细胞,更优选地人造血干细胞和人造血祖细胞并且甚至更优选地CD34+人造血干细胞。如本文所用,术语“CD34+细胞”是指在其细胞表面上表达CD34蛋白的细胞。如本文所用的,“CD34”指代常常充当细胞-细胞粘附因子的细胞表面糖蛋白(例如,唾液黏蛋白)。CD34+是造血干细胞和造血祖细胞两者的细胞表面标志物。
在一个实施例中,经基因组编辑的造血细胞是CD150+CD48-CD244-细胞。在一个实施例中,经基因组编辑的造血细胞是CD34+CD133+细胞。在一个实施例中,经基因组编辑的造血细胞是CD133+细胞。在一个实施例中,经基因组编辑的造血细胞是CD34+细胞。
在特定实施例中,本文所设想的融合多肽被引入并在免疫效应细胞中表达。“免疫效应细胞”是免疫系统的任何细胞,其具有一种或多种效应子功能(例如,细胞毒性细胞杀伤活性、细胞因子分泌、ADCC和/或CDC的诱导)。本文所设想的说明性免疫效应细胞为T淋巴细胞,包含但不限于细胞毒性T细胞(CTL;CD8+T细胞)、TIL和辅助T细胞(HTL;CD4+T细胞)。在一特定实施例中,所述细胞包括αβT细胞。在一特定实施例中,所述细胞包括γδT细胞。在一个实施例中,免疫效应细胞包含自然杀伤(NK)细胞。在一个实施例中,免疫效应细胞包含自然杀伤T(NKT)细胞。
免疫效应细胞可以是自体的(autologous/autogeneic)(“自身的”)或非自体的(“非自身的”,例如,同种异体的、同基因的或异种的)。如本文所用,“自体”指代来自同一受试者的细胞。如本文所用,“同种异体”指代同一物种的与相比之下的细胞在基因上有所不同的细胞。如本文所用,“同基因”指代不同受试者的与相比之下的细胞在基因上相同的细胞。如本文所用,“异种”指代与相比之下的细胞属于不同物种的细胞。在优选实施例中,细胞是自体的。
与在特定实施例中所设想的融合多肽一起使用的说明性免疫效应细胞包含T淋巴细胞。术语“T细胞”或“T淋巴细胞”是本领域公认的并且旨在包含胸腺细胞、未成熟T淋巴细胞、成熟T淋巴细胞、静息T淋巴细胞或激活T淋巴细胞。T细胞可以是T辅助(Th)细胞,例如T辅助1(Th1)细胞或T辅助2(Th2)细胞。T细胞可以是辅助T细胞(HTL;CD4+T细胞)CD4+T细胞、细胞毒性T细胞(CTL,CD8+T细胞)、CD4+CD8+T细胞、CD4-CD8-T细胞或任何其它T细胞亚群。适于在特定实施例中使用的其它说明性T细胞群体包含原始T细胞(TN)、T记忆干细胞(TSCM)、中心记忆T细胞(TCM)、效应记忆T细胞(TEM)和效应T细胞(TEFF)。
如本领域的技术人员所理解的,其它细胞还可以用作具有本文的融合多肽的免疫效应细胞。具体地,免疫效应细胞还包含NK细胞、NKT细胞、嗜中性粒细胞和巨噬细胞。免疫效应细胞还包含效应细胞的祖细胞,其中可以在体内或体外诱导这种祖细胞分化成免疫效应细胞。因此,在特定实施例中,免疫效应细胞包含免疫效应细胞的祖细胞,如源自脐带血、骨髓或动员外周血的CD34+细胞群体内含有的造血干细胞(HSC),所述HSC在施用于受试者时分化为成熟免疫效应细胞或者可以在体外诱导以分化为成熟免疫效应细胞。
在特定实施例中,提供了用于制备表达本文所设想的CAR的免疫效应细胞的方法。在一个实施例中,所述方法包括转染或转导从个体分离的免疫效应细胞,使得免疫效应细胞表达如本文所设想的一种或多种CAR。在某些实施例中,免疫效应细胞从个体分离并经基因修饰而无需在体外进一步操纵。然后,可以将这种细胞直接再次施用到个体。在另外的实施例中,免疫效应细胞在基因修饰以表达CAR之前首先在体外被激活和刺激以增殖。在这方面,免疫效应细胞可以在被基因修饰(即,被转导或转染成表达本文所设想的CAR)之前和/或之后培养。
在特定实施例中,在本文所设想的免疫效应细胞的体外操纵或基因修饰之前,从受试者获得细胞来源。在特定实施例中,经修饰免疫效应细胞包括T细胞。
在特定实施例中,可以使用本文所设想的方法直接基因修饰PBMC以表达CAR。在某些实施例中,在PBMC分离之后,将T淋巴细胞进一步分离,并且在某些实施例中,可以在基因修饰和/或扩增之前或之后将细胞毒性和辅助T淋巴细胞分选到原始、记忆和效应T细胞亚群中。
如T细胞等免疫效应细胞可以在分离之后使用已知方法进行基因修饰,或者免疫效应细胞可以在进行基因修饰之前进行体外激活和扩增(或在祖细胞的情况下分化)。在特定实施例中,如T细胞等免疫效应细胞用本文所设想的嵌合抗原受体进行基因修饰(例如,用包括编码CAR或多顺反子信使的核酸的病毒载体进行转导,所述多顺反子信使编码CAR),并且然后在体外进行激活和扩增。在各个实施例中,可以在基因修饰之前或之后使用如例如以下所描述的方法对T细胞进行激活和扩增以表达CAR:美国专利第6,352,694号;第6,534,055号;第6,905,680号;第6,692,964号;第5,858,358号;第6,887,466号;第6,905,681号;第7,144,575号;第7,067,318号;第7,172,869号;第7,232,566号;第7,175,843号;第5,883,223号;第6,905,874号;第6,797,514号;第6,867,041号;以及美国专利申请公开第20060121005号。
在一个实施例中,CD34+细胞用本文所设想的核酸构建体转导。在某些实施例中,经转导的CD34+细胞在施用于受试者,通常是细胞最初从其分离的受试者之后在体内分化成成熟免疫效应细胞。在另一个实施例中,CD34+细胞可以在暴露于以下细胞因子中的一种或多种细胞因子之前或在用如本文所设想的CAR进行基因修饰之后根据前述方法在体外进行刺激:Flt-3配体(FLT3)、干细胞因子(SCF)、巨核细胞生长和分化因子(TPO)、IL-3和IL-6(Asheuer等人,2004;Imren,等人,2004)。
在特定实施例中,用于治疗癌症的修饰的免疫效应细胞群体包括本文所设想的CAR。例如,经修饰的免疫效应细胞群体由从被诊断患有本文所描述的B细胞恶性肿瘤的患者(自体供体)获得的外周血单核细胞(PBMC)制备。PBMC形成可以是CD4+、CD8+或CD4+和CD8+的T淋巴细胞的异质群体。
PBMC还可以包含其它细胞毒性淋巴细胞,如NK细胞或NKT细胞。将携带特定实施例中所设想的CAR的编码序列的表达载体引入到人供体T细胞、NK细胞或NKT细胞的群体中。在特定实施例中,携带表达载体的成功转导的T细胞可以使用流式细胞术分选,以分离CD3阳性T细胞,并且接着除了使用抗CD3抗体和/或抗CD28抗体和IL-2或如本文其它地方所述本领域已知的任何其它方法进行细胞激活之外,进一步增殖以增加这些表达CAR蛋白的T细胞的数目。标准程序可以用于表达CAR蛋白T细胞的T细胞的冷冻保存,所述冷冻保存用于储存和/或制备以用于人类受试者。在一个实施例中,在不存在非人动物衍生产物如胎牛血清(fetal calf serum/fetal bovine serum)的情况下进行T细胞的体外转导、培养和/或扩增。由于PBMC的异质群体是经基因修饰的,因此所得的经转导的细胞是包括BCMA靶向CAR的经修饰的细胞的异质群体,如本文所设想的。
在另外的实施例中,例如,一种、两种、三种、四种、五种或更多种不同表达载体的混合物可用于基因修饰免疫效应细胞的供体群,其中每个载体编码如本文所涵盖的不同嵌合抗原受体蛋白。所得的经修饰免疫效应细胞形成经修饰细胞的混合群体。
可以使用本领域已知的各种方法制造经基因工程化的细胞,包含T细胞,参见例如WO 2016/094304,其通过引用整体并入本文。
I.组合物和调配物
如本文所设想,在特定实例中设想的组合物可以包括一种或多种多肽、多核苷酸、包括相同物质的载体以及基因组编辑组合物和经基因组编辑的细胞组合物。具体实施例中设想的基因组编辑组合物和方法用于编辑细胞或细胞群体中的靶位点。
在各个实施例中,本文所设想的组合物包括融合多肽,所述融合多肽包括DNA结合结构域、归巢核酸内切酶变体和末端加工酶,例如3′-5′核酸外切酶(ExoX)。核酸酶变体可以采用mRNA的形式,所述mRNA通过上文所公开的多核苷酸递送方法例如电穿孔、脂质纳米颗粒等引入到细胞中。在一个实施例中,通过上文所公开的多核苷酸递送方法,将包括编码融合多肽、归巢核酸内切酶变体和3′-5′核酸外切酶(例如,ExoX)的mRNA的组合物引入到细胞中。
在特定实施例中,本文所设想的组合物包括细胞群体、核酸酶变体、以及任选地供体修复模板。在特定实施例中,本文所设想的组合物包括细胞群体、核酸酶变体、末端加工酶、以及任选地供体修复模板。核酸酶变体和/或末端加工酶可以呈通过上文所公开的多核苷酸递送方法引入细胞中的mRNA的形式。也可以通过单独的组合物将供体修复模板引入到细胞中。
在特定实施例中,本文所设想的组合物包括细胞群体、包括DNA结合结构域、归巢核酸内切酶变体、3′-5′核酸外切酶(例如,ExoX)的融合多肽以及任选地供体修复模板。包括DNA结合结构域、归巢核酸内切酶变体和3′-5′核酸外切酶的融合多肽可以呈通过上文所公开的多核苷酸递送方法引入到细胞中的mRNA形式。也可以通过单独的组合物将供体修复模板引入到细胞中。
在具体实施例中,细胞群体包括经基因修饰的造血细胞,所述经基因修饰的造血细胞包含但不限于造血干细胞、造血祖细胞、CD133+细胞、CD34+细胞和免疫效应细胞。
组合物包含但不限于药物组合物。“药物组合物”指代单独地或与一种或多种其它治疗方式组合地施用于细胞或动物的在药学上可接受的或生理学上可接受的溶液中配制的组合物。还应理解,如果需要,组合物也可以与其它药剂组合施用,例如,细胞因子、生长因子、激素、小分子、化学治疗剂、前药、药物、抗体或其它各种药学活性剂。实际上对也可以包含在组合物中的其它组分没有限制,条件是额外的药剂不会不利地影响组合物。
本文采用的短语“药学上可接受的”是指在合理医学判断范围内的那些化合物、材料、组合物和/或剂型,这些是适合用于与人类和动物组织接触使用而没有过多的毒性、刺激、过敏反应或其它问题或并发症,与合理的益处/风险比相称。
术语“药学上可接受的载剂”是指对治疗细胞施用的稀释剂、佐剂、赋形剂或媒剂。药物载体的说明性实例可以是无菌液体,如细胞培养基、水和油,包含石油、动物、植物或合成来源的那些油,如花生油、大豆油、矿物油、芝麻油等。还可以采用盐水溶液和葡萄糖和甘油水溶液作为液体载剂,尤其是用于可注射溶液的液体载剂。在特定实施例中,适合的药物赋形剂包含淀粉、葡萄糖、乳糖、蔗糖、明胶、麦芽、水稻、面粉、白垩、硅胶、硬脂酸钠、单硬脂酸甘油酯、滑石、氯化钠、脱脂奶粉、甘油、丙烯、乙二醇、水、乙醇等。除了任何常规培养基或药剂与活性成分不相容的情况之外,设想了其在药物组合物中的用途。补充性活性成分也可以并入到组合物中。
在一个实施例中,包括药学上可接受的载剂的组合物适于施用于受试者。在特定实施例中,包括载剂的组合物适于肠胃外施用,例如血管内(静脉内或动脉内)、腹膜内或肌内施用。在特定实施例中,包括药学上可接受的载剂的组合物适合于心室内、脊柱内或鞘内施用。药学上可接受的载剂包含无菌水溶液、细胞培养基或分散体。这些介质和药剂用于药物活性物质的用途在本领域中是熟知的。除了任何常规培养基或药剂与经过转导的细胞不相容的情况之外,设想了其在药物组合物中的用途。
在特定实施例中,本文所设想的组合物包括经基因修饰的造血干细胞和/或祖细胞或免疫效应细胞以及药学上可接受的载体,所述细胞包括编码本文所设想的融合多肽的外源性多核苷酸。
包括本文所设想的基于细胞的组合物的组合物可以通过肠胃外施用方法施用。
药学上可接受的载剂必须具有足够高的纯度和足够低的毒性以使其适于施用于正在治疗的人类受试者。药学上可接受的载剂应当保持或增加组合物的稳定性。药学上可接受的载剂可以是液体的或固体的并且在考虑计划的施用方式的情况下被选择以在与组合物的其它组分组合时提供期望的体积、一致性等。例如,药学上可接受的载体可以是但不限于结合剂(例如,预凝胶化的玉米淀粉、聚乙烯吡咯烷酮或羟丙基甲基纤维素等);填充剂(例如,乳糖和其它糖、微晶纤维素、果胶、明胶、硫酸钙、乙基纤维素、聚丙烯酸酯、磷酸氢钙等);润滑剂(例如,硬脂酸镁、滑石、硅石、胶体二氧化硅、硬脂酸、金属硬脂酸盐、氢化植物油、玉米淀粉、聚乙二醇、苯甲酸钠、乙酸钠等);崩解剂(例如,淀粉、羟乙酸淀粉钠等);或润湿剂(例如,十二烷基硫酸钠等)。用于本文所设想的组合物的其它适合的药学上可接受的载剂包含但不限于水、盐溶液、醇、聚乙二醇、明胶、直链淀粉、硬脂酸镁、滑石、硅酸、粘性石蜡、羟甲基纤维素、聚乙烯吡咯烷酮等。
此类载剂溶液还可以含有缓冲剂、稀释剂和其它适合的添加剂。如本文所用,术语“缓冲剂”指代化学成分中和酸或碱而不显著改变pH的溶液或液体。本文所设想的缓冲剂的实例包含但不限于杜尔贝克氏(Dulbecco's)磷酸盐缓冲盐水(PBS)、林格氏(Ringer's)溶液、5%葡萄糖水溶液(D5W)、正常(normal)/生理(physiologic)盐水(0.9% NaCl)。
药学上可接受的载剂可以存在的量足以使组合物的pH保持处于约7。可替代地,组合物的pH的范围为约6.8至约7.4,例如6.8、6.9、7.0、7.1、7.2、7.3和7.4。在仍另一个实施例中,组合物的pH为约7.4。
本文所设想的组合物可以包括无毒的药学上可接受的培养基。组合物可以是悬浮液。如本文所用,术语“悬浮液”指代细胞未连接到固相载体的非粘附性条件。例如,可以搅拌或搅动保持为悬浮液的细胞并且所述细胞未粘附到支持物,如培养皿。
在特定实施例中,本文所设想的组合物在悬浮液中调配,其中经基因组编辑的造血干细胞和/或祖细胞分散在静脉注射(IV)袋等中的可接受的液体培养基或溶液例如盐水或无血清培养基内。可接受的稀释剂包含但不限于水、PlasmaLyte、林格氏溶液、等渗氯化钠(盐水)溶液、无血清细胞培养基和适合于低温储存的培养基,例如培养基。
在某些实施例中,药学上可接受的载剂基本上不含人或动物来源的天然蛋白质并且适于储存包括经基因组编辑的细胞群体,例如造血干细胞和造血祖细胞的组合物。药物组合物旨在施用到人类患者中并且因此基本上不含细胞培养组分,如牛血清白蛋白、马血清和胎牛血清。
在一些实施例中,组合物在药学上可接受的细胞培养基中调配。此类组合物适于施用于人类受试者。在特定实施例中,药学上可接受的细胞培养基是无血清培养基。
无血清培养基相比于含有血清的培养基具有若干优点,包含组合物得到简化且得到更好定义、污染物程度得到减小、可能的传染剂来源得到消除以及成本降低。在各个实施例中,无血清培养基是无动物的并且可以任选地是无蛋白质的。任选地,培养基可以含有生物药学上可接受的重组蛋白。“无动物”培养基是指组合物源自非动物来源的培养基。重组蛋白替代无动物培养基中的天然动物蛋白质,并且营养物从合成的、植物或微生物来源获得。相比之下,“无蛋白质”培养基被定义为基本上不含蛋白质。
特定实施例中使用的无血清培养基的说明性实例包含但不限于QBSF-60(质量生物有限公司(Quality Biological,Inc.))、StemPro-34(生命技术公司(LifeTechnologies))和X-VIVO 10。
在优选的实施例中,在勃脉力中制备包括经基因组编辑的造血干细胞和/或造血祖细胞的组合物。
在各个实施例中,在低温保存培养基中制备包括造血干细胞和/或造血祖细胞的组合物。例如,可以使用具有低温保存药剂的低温保存培养基在解冻后保持高细胞存活率结果。特定实施例中使用的低温保存培养基的说明性实例包含但不限于CryoStor CS10、CryoStor CS5和CryoStor CS2。
在一个实施例中,组合物在包括50:50勃脉力A:CryoStor CS10的溶液中调配。
在特定实施例中,组合物基本上不含支原体、内毒素和微生物污染。关于内毒素,“基本上不含”是指每剂细胞的内毒素含量低于FDA针对生物制剂允许的含量,其为每天5EU/kg体重的总内毒素,针对平均70kg的人而言为每总剂量细胞350EU。在特定实施例中,包括用本文所设想的逆转录病毒载体转导的造血干细胞或造血祖细胞的组合物含有约0.5EU/mL到约5.0EU/mL、或约0.5EU/mL、1.0EU/mL、1.5EU/mL、2.0EU/mL、2.5EU/mL、3.0EU/mL、3.5EU/mL、4.0EU/mL、4.5EU/mL或5.0EU/mL。
在某些实施例中,设想了适合于递送多核苷酸的组合物和配制品,包含但不限于编码一种或多种重编程的核酸酶和任选地末端加工酶的一种或多种mRNA。
用于离体递送的示例性调配物还可以包含使用本领域中已知的各种转染剂,如磷酸钙、电穿孔、热休克和各种脂质体配制品(即,脂质介导的转染)。如下文中更加详细地描述的,脂质体是截留一小部分水性流体的脂质双层。DNA自发缔合到阳离子脂质体的外表面(借助于其电荷),并且这些脂质体将与细胞膜交互。
在特定实施例中,对药学上可接受的载体溶液的调配对本领域的技术人员来说是熟知的,这和开发用于将本文所描述的特定组合物用于各种治疗方案中的适合的给药和治疗方案一样,包含例如,肠内和肠胃外,例如,血管内、静脉内、动脉内、骨内、心室内、脑内、颅内、脊柱内、鞘内和髓内施用和调配。本领域的技术人员应当理解,本文所设想的特定实施例可以包括如制药领域中熟知并且在例如以下各项中描述的调配物等其它调配物:《雷明顿:药学科学与实践(Remington:The Science and Practice of Pharmacy)》,第I卷和第II卷,第22版编辑:Loyd V.Allen Jr,宾夕法尼亚州费城:医药出版社(Philadelphia,PA:Pharmaceutical Press);2012,所述文献通过引用整体并入本文。
J.定点诱变方法
定义给定插入或缺失(插入缺失)的定性性质是:(i)其长度,以插入或缺失的碱基数量表示;(ii)其沿染色体的纵向位置,通常相对于核酸酶的靶位点或断点而言;以及(iii)对于插入,插入的序列的长度和组成。缺失是最显著的结果,通常占观察到的事件的90-95%。它们最常报道的大小特性趋于变小(即,长度为1-20个碱基对,频率偏向所述范围的低端),并且已经发现它们的位置分布是均匀分布的,覆盖了DNA断裂点并且在没有明显偏向的情况下向两个方向向外发散。这些性质的例外经常被假设为由定位于DNA断裂点两侧的微同源物(长度为大约3-6个碱基对的小的重复片段)驱动。在基因组编辑工具的应用期间,很少有关于插入性质的报道。另外地,使每个插入缺失物种与表型相关的基因型特征(例如,它如何影响开放阅读框或它是否破坏转录因子结合基序)对于每个给定的应用来说是潜在巨大的和特异的。
工程化megaTAL核酸酶和包括DNA结合结构域和归巢核酸内切酶的其它融合多肽具有与其它基因编辑平台不同的特性。例如,megaTAL是单体的杂交分子,包括与重编程归巢核酸内切酶(HE)融合的模块化组装的转录激活因子样效应物(TALE)阵列。将HE锚定在靶位点的TALE阵列的大小可以设定为识别大约6个至18个碱基对的结合位点。归巢核酸内切酶识别并切割22个碱基对靶位点。两个靶位点被间隔子区隔开,所述间隔子区的长度可以是0至大约12个碱基对。megaTAL核酸酶的两个独特性质源于其独特的组成:(i)TALE阵列的锚定机制向靶位点的一侧提供了总体结合亲和力的高度偏向分布;以及(ii)由归巢核酸内切酶催化的DNA切割反应的产物是具有长度为4个碱基对的3'突出端的DNA末端。相反,ZFN和TALEN,其中锌指或TALE阵列分别由FokI核酸酶操作,具有相对均匀的结合亲和力分布,并且产生长度为4个碱基对的5'突出端。CRISPR DNA识别和切割的机制在DNA序列识别和亲和力方面是根本不同的,然而DNA切割的产物是平端DNA末端(Cas9)或5'突出端(Cpf1)。
为了评估基因编辑事件的定量和定性方面,本文所设想的融合多肽的发明人测试了各种末端加工酶的共递送,以表征其对编辑的等位基因的影响。此外,发明人设计了许多多部分megaTAL融合蛋白,以评估如何通过在线酶功能操纵编辑结果。
因此,发明人惊奇地发现,当在细胞中表达时,包括DNA结合结构域和归巢核酸内切酶变体(通过连接子结构域(例如,多肽连接子)与核酸外切酶特别是Trex2、ExoI或ExoX连接)的融合多肽,与包括DNA结合结构域和未与核酸外切酶连接的归巢核酸内切酶变体的融合多肽相比,即当DNA结合结构域/归巢核酸内切酶融合体和核酸外切酶分开表达时,产生了延长的和定向偏向的缺失。
因此,提供了在细胞中进行定点诱变的方法,所述方法包括选择双链DNA(dsDNA)靶位点,并且向细胞中引入融合多肽,或编码如本文所设想的融合多肽的多核苷酸、mRNA或载体,其中所述融合肽产生定向偏向的缺失,所述缺失在细胞中的选定dsDNA靶切割位点附近具有缺失中心。
在各个实施例中,所述方法包括引融合多肽,所述融合多肽包括DNA结合结构域和归巢核酸内切酶(HE)变体,所述HE变体结合并且切割细胞中的选定双链DNA(dsDNA)靶位点;连接子结构域;以及核酸外切酶或其生物活性片段。在一些实施例中,所述核酸外切酶是Trex2、ExoI或ExoX。在一些实施例中,所述DNA结合结构域包括TALE DNA结合结构域(例如,megaTAL)或锌指DNA结合结构域。
在各个实施例中,所述ExoX或其生物活性片段包括与如SEQ ID NO:109中所示的氨基酸序列具有至少85%、90%、95%、96%、97%、98%或99%同一性的氨基酸序列。
在各个实施例中,所述ExoI或其生物活性片段包括与如SEQ ID NO:112中所示的氨基酸序列具有至少85%、90%、95%、96%、97%、98%或99%同一性的氨基酸氨基酸序列。
在各个实施例中,所述连接子结构域是肽连接子。在一些实施例中,所述肽连接子是自切割肽连接子。在一些实施例中,所述肽连接子包括约4个至约30个氨基酸。在一些实施例中,所述肽连接子是[g1](GGGGS)1-4连接子(SEQ ID NO:117和150-152)。
在各个实施例中,所述HE变体是LAGLIDADG归巢核酸内切酶(LHE)变体。在一些实施例中,所述HE变体是选自由以下组成的组的LHE的变体:I-AabMI、I-AaeMI、I-AniI、I-ApaMI、I-CapIII、I-CapIV、I-CkaMI、I-CpaMI、I-CpaMII、I-CpaMIII、I-CpaMIV、I-CpaMV、I-CpaV、I-CraMI、I-EjeMI、I-GpeMI、I-GpiI、I-GzeMI、I-GzeMII、I-GzeMIII、I-HjeMI、I-LtrII、I-LtrI、I-LtrWI、I-MpeMI、I-MveMI、I-NcrII、I-Ncrl、I-NcrMI、I-OheMI、I-OnuI、I-OsoMI、I-OsoMII、I-OsoMIII、I-OsoMIV、I-PanMI、I-PanMII、I-PanMIII、I-PnoMI、I-SceI、I-ScuMI、I-SmaMI、I-SscMI和I-Vdi141I。
在各个实施例中,所设想的方法包括引入靶向位点的融合多肽,所述位点位于免疫系统检查点基因、珠蛋白基因、编码有助于抑制γ-珠蛋白基因表达和HbF的多肽的基因或免疫抑制信号传导基因内。在一些实施例中,所述靶位点位于选自由以下组成的组的基因内:程序性细胞死亡蛋白1(PD-1;PDCD1)、淋巴细胞激活基因3蛋白(LAG-3)、T细胞免疫球蛋白结构域和粘蛋白结构域蛋白3(TIM-3)、细胞毒性T淋巴细胞抗原-4(CTLA-4)、带T淋巴细胞弱化因子(BTLA)、T细胞免疫球蛋白和基于免疫受体酪氨酸的抑制基序结构域(TIGIT)、T细胞激活的V结构域Ig抑制因子(VISTA)和杀伤细胞免疫球蛋白样受体(KIR)、CCR5、TRAC(TCRα)、TCRβ、IL10Rα、IL10Rβ、TGFBR1、TGFBR2、CBL-B、PCSK9、AHR、BTK、α-珠蛋白、β-珠蛋白、γ-珠蛋白、BCL11A、KLF1、SOX6、GATA1、LSD1、α叶酸受体(FRα)、αvβ6整合素、B细胞成熟抗原(BCMA)、B7-H3(CD276)、B7-H6、碳酸酐酶IX(CAIX)、CD16、CD19、CD20、CD22、CD30、CD33、CD37、CD38、CD44、CD44v6、CD44v7/8、CD70、CD79a、CD79b、CD123、CD133、CD138、CD171、癌胚抗原(CEA)、C型凝集素样分子-1(CLL-1)、CD2子集1(CS-1)、硫酸软骨素蛋白聚糖4(CSPG4)、皮肤T细胞淋巴瘤相关抗原1(CTAGE1)、表皮生长因子受体(EGFR)、表皮生长因子受体变体III(EGFRvIII)、上皮糖蛋白2(EGP2)、上皮糖蛋白40(EGP40)、上皮细胞粘附分子(EPCAM)、肝配蛋白A型受体2(EPHA2)、成纤维细胞激活蛋白(FAP)、Fc受体样5(FCRL5)、胎儿乙酰胆碱酯酶受体(AchR)、神经节苷脂G2(GD2)、神经节苷脂G3(GD3)、磷脂酰肌醇蛋白聚糖-3(GPC3)、包含ErbB2(HER2)的EGFR家族、IL-11Rα、IL-13Rα2、κ、癌/睾丸抗原2(LAGE-1A)、λ、路易斯-Y(Lewis-Y,LeY)、L1细胞粘附分子(L1-CAM)、黑色素瘤抗原基因(MAGE)-A1、MAGE-A3、MAGE-A4、MAGE-A6、MAGEA10、T细胞识别的黑色素瘤抗原1(MelanA或MART1)、间皮素(MSLN)、MUC1、MUC16、MHC I类链相关蛋白A(MICA)、MHC I类链相关蛋白B(MICB)、神经细胞粘附分子(NCAM)、癌/睾丸抗原1(NY-ESO-1)、聚唾液酸;胎盘特异性1(PLAC1)、黑色素瘤中优先表达的抗原(PRAME)、前列腺干细胞抗原(PSCA)、前列腺特异性膜抗原(PSMA)、受体酪氨酸激酶样孤儿受体1(ROR1)、滑膜肉瘤、X断点2(SSX2)、存活蛋白、肿瘤相关糖蛋白72(TAG72)、肿瘤内皮标志物1(TEM1/CD248)、肿瘤内皮标志物7相关(TEM7R)、TEM5、TEM8、滋养层糖蛋白(TPBG)、UL16结合蛋白(ULBP)1、ULBP2、ULBP3、ULBP4、ULBP5、ULBP6、血管内皮生长因子受体2(VEGFR2)和威尔姆斯瘤1(WT-1)基因。
在一些实施例中,所述靶位点位于选自由以下组成的组的基因内:程序性细胞死亡蛋白1(PD-1;PDCD1)、淋巴细胞激活基因3蛋白(LAG-3)、T细胞免疫球蛋白结构域和粘蛋白结构域蛋白3(TIM-3)、细胞毒性T淋巴细胞抗原-4(CTLA-4)、带T淋巴细胞弱化因子(BTLA)、T细胞免疫球蛋白和基于免疫受体酪氨酸的抑制基序结构域(TIGIT)、T细胞激活的V结构域Ig抑制因子(VISTA)和杀伤细胞免疫球蛋白样受体(KIR)、CCR5、TRAC(TCRα)、IL10Rα、TGFBR2、CBL-B、PCSK9、AHR、BTK、α-珠蛋白、β-珠蛋白、γ-珠蛋白和BCL11A基因。
在各个实施例中,所述靶位点位于TRAC(TCRα)基因、CBL-B基因或PDCD1(PD-1)基因内。在特定实施例中,所述TCRα基因靶位点包括SEQ ID NO:1中所示的氨基酸序列。在特定实施例中,所述CBL-B基因靶位点包括SEQ ID NO:2中所示的氨基酸序列。在特定实施例中,所述PD-1基因靶位点包括SEQ ID NO:3中所示的氨基酸序列。
如整个公开内容所设想的,融合多肽和相关方法在细胞中在选定dsDNA靶位点处产生定向偏向的缺失。在各个实施例中,相对于所述HE靶位点中心位置,所述缺失中心位置与所述DNA结合结构域靶位点位于同一侧上。在特定实施例中,所述缺失中心位置位于HE靶位点中心位置的5'端。
在各个实施例中,大于50%的所述定向偏向的缺失的缺失中心位置位于所述HE靶位点中心位置的一侧上。在一些实施例中,大于51%的所述定向偏向的缺失的缺失中心位置位于所述HE靶位点中心位置的一侧上。在一些实施例中,大于52%的所述定向偏向的缺失的缺失中心位置位于所述HE靶位点中心位置的一侧上。在一些实施例中,大于53%的所述定向偏向的缺失的缺失中心位置位于所述HE靶位点中心位置的一侧上。在一些实施例中,大于54%的所述定向偏向的缺失的缺失中心位置位于所述HE靶位点中心位置的一侧上。在一些实施例中,大于55%的所述定向偏向的缺失的缺失中心位置位于所述HE靶位点中心位置的一侧上。在一些实施例中,大于56%的所述定向偏向的缺失的缺失中心位置位于所述HE靶位点中心位置的一侧上。在一些实施例中,大于57%的所述定向偏向的缺失的缺失中心位置位于所述HE靶位点中心位置的一侧上。在一些实施例中,大于58%的所述定向偏向的缺失的缺失中心位置位于所述HE靶位点中心位置的一侧上。在一些实施例中,大于59%的所述定向偏向的缺失的缺失中心位置位于所述HE靶位点中心位置的一侧上。在一些实施例中,大于60%的所述定向偏向的缺失的缺失中心位置位于所述HE靶位点中心位置的一侧上。在一些实施例中,大于65%的所述定向偏向的缺失的缺失中心位置位于所述HE靶位点中心位置的一侧上。在一些实施例中,大于70%的所述定向偏向的缺失的缺失中心位置位于所述HE靶位点中心位置的一侧上。在一些实施例中,大于75%的所述定向偏向的缺失的缺失中心位置位于所述HE靶位点中心位置的一侧上。在一些实施例中,大于80%的所述定向偏向的缺失的缺失中心位置位于所述HE靶位点中心位置的一侧上。
在各个实施例中,至少50%的缺失的缺失中心距所述HE靶位点中心位置大于4个核苷酸。在一些实施例中,至少51%的缺失的缺失中心距所述HE靶位点中心位置大于4个核苷酸。在一些实施例中,至少52%的缺失的缺失中心距所述HE靶位点中心位置大于4个核苷酸。在一些实施例中,至少53%的缺失的缺失中心距所述HE靶位点中心位置大于4个核苷酸。在一些实施例中,至少54%的缺失的缺失中心距所述HE靶位点中心位置大于4个核苷酸。在一些实施例中,至少55%的缺失的缺失中心距所述HE靶位点中心位置大于4个核苷酸。在一些实施例中,至少56%的缺失的缺失中心距所述HE靶位点中心位置大于4个核苷酸。在一些实施例中,至少57%的缺失的缺失中心距所述HE靶位点中心位置大于4个核苷酸。在一些实施例中,至少58%的缺失的缺失中心距所述HE靶位点中心位置大于4个核苷酸。在一些实施例中,至少59%的缺失的缺失中心距所述HE靶位点中心位置大于4个核苷酸。在一些实施例中,至少60%的缺失的缺失中心距所述HE靶位点中心位置大于4个核苷酸。在一些实施例中,至少65%的缺失的缺失中心距所述HE靶位点中心位置大于4个核苷酸。在一些实施例中,至少70%的缺失的缺失中心距所述HE靶位点中心位置大于4个核苷酸。在一些实施例中,至少75%的缺失的缺失中心距所述HE靶位点中心位置大于4个核苷酸。在一些实施例中,至少80%的缺失的缺失中心距所述HE靶位点中心位置大于4个核苷酸。
在各个实施例中,至少10%的缺失的缺失中心距所述HE靶位点中心位置大于8个核苷酸。在一些实施例中,至少11%的缺失的缺失中心距所述HE靶位点中心位置大于8个核苷酸。在一些实施例中,至少12%的缺失的缺失中心距所述HE靶位点中心位置大于8个核苷酸。在一些实施例中,至少13%的缺失的缺失中心距所述HE靶位点中心位置大于8个核苷酸。在一些实施例中,至少14%的缺失的缺失中心距所述HE靶位点中心位置大于8个核苷酸。在一些实施例中,至少15%的缺失的缺失中心距所述HE靶位点中心位置大于8个核苷酸。在一些实施例中,至少16%的缺失的缺失中心距所述HE靶位点中心位置大于8个核苷酸。在一些实施例中,至少17%的缺失的缺失中心距所述HE靶位点中心位置大于8个核苷酸。在一些实施例中,至少18%的缺失的缺失中心距所述HE靶位点中心位置大于8个核苷酸。在一些实施例中,至少19%的缺失的缺失中心距所述HE靶位点中心位置大于8个核苷酸。在一些实施例中,至少20%的缺失的缺失中心距所述HE靶位点中心位置大于8个核苷酸。在一些实施例中,至少25%的缺失的缺失中心距所述HE靶位点中心位置大于8个核苷酸。在一些实施例中,至少30%的缺失的缺失中心距所述HE靶位点中心位置大于8个核苷酸。在一些实施例中,至少35%的缺失的缺失中心距所述HE靶位点中心位置大于8个核苷酸。
在各个实施例中,至少50%的缺失的长度为6bp或更长。在一些实施例中,至少51%的缺失的长度为6bp或更长。在一些实施例中,至少52%的缺失的长度为6bp或更长。在一些实施例中,至少53%的缺失的长度为6bp或更长。在一些实施例中,至少54%的缺失的长度为6bp或更长。在一些实施例中,至少55%的缺失的长度为6bp或更长。在一些实施例中,至少56%的缺失的长度为6bp或更长。在一些实施例中,至少57%的缺失的长度为6bp或更长。在一些实施例中,至少58%的缺失的长度为6bp或更长。在一些实施例中,至少59%的缺失的长度为6bp或更长。在一些实施例中,至少60%的缺失的长度为6bp或更长。在一些实施例中,至少65%的缺失的长度为6bp或更长。在一些实施例中,至少70%的缺失的长度为6bp或更长。在一些实施例中,至少75%的缺失的长度为6bp或更长。在一些实施例中,至少80%的缺失的长度为6bp或更长。
在各个实施例中,至少30%的缺失的长度为12bp或更长。在一些实施例中,至少31%的缺失的长度为12bp或更长。在一些实施例中,至少32%的缺失的长度为12bp或更长。在一些实施例中,至少33%的缺失的长度为12bp或更长。在一些实施例中,至少34%的缺失的长度为12bp或更长。在一些实施例中,至少35%的缺失的长度为12bp或更长。在一些实施例中,至少36%的缺失的长度为12bp或更长。在一些实施例中,至少37%的缺失的长度为12bp或更长。在一些实施例中,至少38%的缺失的长度为12bp或更长。在一些实施例中,至少39%的缺失的长度为12bp或更长。在一些实施例中,至少40%的缺失的长度为12bp或更长。在一些实施例中,至少45%的缺失的长度为12bp或更长。在一些实施例中,至少50%的缺失的长度为12bp或更长。在一些实施例中,至少55%的缺失的长度为12bp或更长。在一些实施例中,至少60%的缺失的长度为12bp或更长。
在各个实施例中,所述定向偏向的缺失的长度包括约10个核苷酸。在一些实施例中,所述定向偏向的缺失的长度包括约11个核苷酸。在一些实施例中,所述定向偏向的缺失的长度包括约12个核苷酸。在一些实施例中,所述定向偏向的缺失的长度包括约13个核苷酸。在一些实施例中,所述定向偏向的缺失的长度包括约14个核苷酸。在一些实施例中,所述定向偏向的缺失的长度包括约15个核苷酸。在一些实施例中,所述定向偏向的缺失的长度包括约16个核苷酸。在一些实施例中,所述定向偏向的缺失的长度包括约17个核苷酸。在一些实施例中,所述定向偏向的缺失的长度包括约18个核苷酸。在一些实施例中,所述定向偏向的缺失的长度包括约19个核苷酸。在一些实施例中,所述定向偏向的缺失的长度包括约20个核苷酸。
在各个实施例中,所述缺失延伸到DNA结合结构域靶位点中。在各个实施例中,所述缺失中心位置位于所述DNA结合结构域靶位点内。
在各个实施例中,除了融合多肽之外,所述方法进一步包括向细胞中引入末端加工酶或其生物活性片段,或编码末端加工酶(例如,核酸外切酶)的多核苷酸、RNA或载体,或者。在一些实施例中,末端加工酶或其生物活性片段选自由以下组成的组:Trex2、Trex1、不含跨膜结构域的Trex1、Apollo、Artemis、DNA2、ExoI、ExoT、ExoIII、ExoX、Fen1、Fan1、MreII、Rad2、Rad9、TdT(末端脱氧核苷酸转移酶)、PNKP、RecE、RecJ、RecQ、λ核酸外切酶、Sox、牛痘DNA聚合酶、核酸外切酶I、核酸外切酶III、核酸外切酶VII、NDK1、NDK5、NDK7、NDK8、WRN、T7-核酸外切酶基因6、禽类成髓细胞瘤病毒整合蛋白(IN)、Bloom、热敏磷酸酶、碱性磷酸酶、多核苷酸激酶(PNK)、ApeI、绿豆核酸酶、Hex1、TTRAP(TDP2)、Sgs1、Sae2、CUP、Polμ、Polλ、MUS81、EME1、EME2、SLX1、SLX4以及UL-12。在一些实施例中,末端加工酶是核酸外切酶。在特定实施例中,核酸外切酶是Trex2或其生物活性片段。
在特定实施例中,提供了在细胞中进行定点诱变的方法,所述方法包括选择双链DNA(dsDNA)靶位点,并且向细胞中引入融合多肽,或编码如本文所设想的融合多肽的多核苷酸、mRNA或载体,其中所述融合肽产生定向偏向的缺失,所述缺失在细胞中的选定dsDNA靶切割位点附近具有缺失中心。
在特定实施例中,所述方法包括选择双链DNA(dsDNA)靶位点;向细胞中引入融合多肽,所述融合多肽包括DNA结合结构域和结合并切割细胞中的选定双链DNA(dsDNA)靶位点的归巢核酸内切酶(HE)变体、连接子结构域和ExoI或其生物活性片段;以及引入核酸外切酶(例如,Trex2);其中所述方法产生定向偏向的缺失,所述定向偏向的缺失的缺失中心靠近所述细胞中的选定dsDNA靶切割位点。
在各个实施例中,所述方法为体外方法。在各个实施例中,所述方法为离体方法。在各个实施例中,所述方法为体内方法。
K.治疗方法
本文所设想的融合多肽、经基因编辑的细胞、组合物和/或相关的基因编辑方法可以用于预防、治疗和改善疾病或病症,或改善疾病病状或与其相关的症状。在一些实施例中,本文所设想的融合多肽、经基因编辑的细胞、组合物和/或相关的基因编辑方法可以用于治疗、预防或抑制疾病(例如,癌症、局部缺血、糖尿病性视网膜病变、黄斑变性、类风湿性关节炎、银屑病、HIV感染、镰状细胞性贫血、阿尔茨海默氏病、肌营养不良症、神经退行性疾病、血管疾病、囊性纤维化、中风、高IGE综合征、血友病)或者改善疾病病状或与疾病相关的症状,所述疾病如癌症、局部缺血、糖尿病性视网膜病变、黄斑变性、类风湿性关节炎、银屑病、HIV感染、镰状细胞性贫血、阿尔茨海默氏病、肌营养不良症、神经退行性疾病、血管疾病、囊性纤维化、中风、高IGE综合征、血友病。
在一些实施例中,本文所设想的融合多肽、经基因编辑的细胞、组合物和/或相关的基因编辑方法可用于治疗、预防或抑制常染色体显性遗传病,如软骨发育不全、假软骨发育不全、多发性骨骺发育不良、软骨发育不良、成骨不全、马凡综合征(Marfan syndrome)、多趾畸形、遗传性运动感觉神经病I和II(夏柯-马利-杜斯氏病(Charcot-Marie-Toothdisease))、肌强直性营养不良和神经纤维瘤病,或者改善与常染色体显性遗传病相关的疾病病状或症状,所述常染色体显性遗传病如软骨发育不全、假软骨发育不全、多发性骨骺发育不良、软骨发育不良、成骨不全、马凡综合征、多趾畸形、遗传性运动感觉神经病I和II(夏柯-马利-杜斯氏病)、肌强直性营养不良和神经纤维瘤病。在一些实施例中,本文所设想的融合多肽、经基因编辑的细胞、组合物和/或相关的基因编辑方法可用于治疗、预防或抑制由基因错调引起的疾病。
在优选的实施例中,本文所设想的融合多肽、经基因编辑的细胞、组合物和/或相关的基因编辑方法可以用于预防、治疗免疫病症或癌症和/或改善与免疫病症或癌症相关的至少一种症状。
“免疫病症”是指引起免疫系统应答的疾病。在特定实施例中,术语“免疫障碍”是指癌症、移植物抗宿主病、自身免疫性疾病或免疫缺陷。在一个实施例中,免疫障碍涵盖传染病。
如本文所用,术语“癌症”通常涉及异常细胞在没有控制的情况下分裂并且可能侵入附近组织的一类疾病或病状。
如本文所用,术语“恶性”是指一组肿瘤细胞显示出不受控制的生长(即,超出正常限度的分裂)、侵入(即,侵入和破坏相邻组织)和转移(即,经由淋巴或血液扩散到体内的其它位置)中的一种或多种的癌症。
如本文所用,术语“转移”是指癌症从身体的一个部位扩散到另一个部位。由已经扩散的细胞形成的肿瘤被称为“转移性肿瘤”或“转移”。转移性肿瘤含有与原始(原发性)肿瘤中的细胞相似的细胞。
如本文所用,术语“良性”或“非恶性”是指可以长得更大但不扩散到身体的其它部位的肿瘤。良性肿瘤是自限性的并且通常不会侵入或转移。
“癌细胞(cancer cell)”或“肿瘤细胞(tumor cell)”是指癌性生长或组织的单个细胞。肿瘤通常是指由细胞异常生长形成的肿胀或病变,其可以是良性的、恶化前的或恶性的。大多数癌症形成肿瘤,但是一些例如白血病不一定形成肿瘤。对于形成肿瘤的那些癌症,术语癌症(细胞)和肿瘤(细胞)可互换使用。个体中肿瘤的量是可以测量为肿瘤的数目、体积或重量的“肿瘤负荷”。
“移植物抗宿主病”或“GVHD”是指在细胞、组织或实体器官移植后可能发生的并发症。GVHD可以在干细胞或骨髓移植后发生,其中移植的供体细胞攻击移植接受者的身体。人类急性GVHD在移植后约60天内发生,并且通过细胞溶解性淋巴细胞的作用导致对皮肤、肝脏和肠道的损害。慢性GVHD发生较晚并且是主要影响皮肤的系统性自身免疫疾病,其导致B细胞的多克隆活化和Ig和自身抗体的生产过剩。实体器官移植移植物抗宿主病(SOT-GVHD)以两种形式发生。更常见的类型是抗体介导的,其中来自具有O型血的供体的抗体攻击具有A型血、B型血或AB型血的接受者的接受者的红细胞,从而导致轻度短暂的溶血性贫血。第二种形式的SOT-GVHD是与高死亡率相关联的细胞类型,其中供体衍生的T细胞对免疫不同的宿主组织产生免疫攻击,最常见于皮肤、肝脏、胃肠道和骨髓,从而导致这些器官中的并发症。
“移植物抗白血病(graft-versus-leukemia)”或“GVL”是指存在于供体的移植组织如骨髓或外周血中的免疫细胞对人的白血病细胞的免疫应答。
“自身免疫疾病”指代身体对其自身组织的某个成分产生免疫原性(即,免疫系统)应答的疾病。换言之,免疫系统失去其将身体内的某个组织或系统识别为“自身”的能力并且靶向并攻击所述组织或系统,好像所述组织或系统是外来的一样。自身免疫性疾病的说明性实例包含但不限于:关节炎、炎性肠病、桥本氏甲状腺炎(Hashimoto's thyroiditis)、格雷夫氏病(Grave'sdisease)、狼疮、多发性硬化症、风湿性关节炎、溶血性贫血、抗免疫性甲状腺炎、全身性红斑狼疮、乳糜泻、克罗恩氏病(Crohn's disease)、结肠炎、糖尿病、硬皮病、牛皮癣等。
“免疫缺陷”意指其免疫系统已经被疾病或化学品施用损害的患者的状态。这一病状使系统缺乏防御外来物质所需的数目和类型的血细胞。免疫缺陷病症或疾病是本领域已知的,并且包含例如AIDS(获得性免疫缺陷综合征)、SCID(严重联合免疫缺陷病)、选择性IgA缺乏症、常见变异型免疫缺陷、X连锁性无丙种球蛋白血症、慢性肉芽肿病、高IgM综合征、威斯科特-奥尔德里奇综合征(Wiskott-Aldrich Syndrome)(WAS)和糖尿病。
“传染病”是指可以从人到人或从生物体到生物体传播并且由微生物或病毒剂引起的疾病(例如,普通感冒)。传染病在本领域中是已知的并且包含例如肝炎、性传播疾病(例如,衣原体、淋病)、结核病、HIV/AIDS、白喉、B型肝炎、C型肝炎、霍乱和流感。
如本文所用,术语“个体”和“受试者”经常可互换使用并且是指展现出可以用本文中其它地方所设想的融合多肽、经基因编辑的细胞、组合物和/或相关的基因编辑方法治疗的免疫病症的症状的任何动物。适合的受试者(例如,患者)包含实验用动物(如小鼠、大鼠、兔或豚鼠)、农场动物和家养动物或宠物(如猫或狗)。包含非人灵长类动物以及优选地人类受试者。典型的受试者包含患有、已经被诊断为患有或有风险患有免疫障碍的人类患者。
如本文所用,术语“患者”是指已经被诊断患有可以用本文中其它地方所设想的融合多肽、经基因编辑的细胞、组合物和/或相关的基因编辑方法治疗的免疫病症的受试者。
如本文所用,“治疗(treatment)”或“治疗(treating)”包含对疾病或病理病症的症状或病理的任何有益或期望的效果,并且甚至可以包含所治疗的疾病或病症例如癌症、GVHD、传染病、自身免疫疾病、炎性疾病和免疫缺陷的一个或多个可测量标记物的最小减少。治疗可以任选地涉及延迟疾病或病症的进展。“治疗”不一定表示完全根除或治愈疾病或病症或其相关症状。
如本文所用,“预防(prevent)”和类似的词语如“预防(prevention)”、“预防(prevented)”、“预防(preventing)”等指示用于预防、抑制或减少疾病或病症例如癌症、GVHD、传染病、自身免疫疾病、炎性疾病和免疫缺陷发生或复发的可能性的方法。预防还是指延迟疾病或病状的发作或复发或者延迟疾病或病状的症状的发生或复发。如本文所用,“预防(prevention)”和类似词语还包含在疾病或病状发作或复发之前减少疾病或病状的强度、效果、症状和/或负担。
如本文所用,短语“改善其至少一种症状”是指减少针对其而正对受试者进行治疗的疾病或病症例如癌症、GVHD、传染病、自身免疫疾病、炎性疾病和免疫缺陷的一种或多种症状。在特定实施例中,被治疗的疾病或病状是癌症,其中所减轻的所述一种或多种症状包含但不限于虚弱、疲劳、呼吸短促、容易挫伤和出血、频繁感染、淋巴结肿大、腹部肿胀或疼痛(由于腹部器官肿大)、骨骼或关节疼痛、骨折、意外体重减轻、食欲不振、盗汗、持续性轻度发烧以及排尿减少(由于肾功能受损)。
在一个实施例中,本文所设想的融合多肽、经基因编辑的细胞、组合物和/或相关的基因编辑方法用于治疗癌症的方法中。在特定实施例中,本文所设想的融合多肽、经基因编辑的细胞、组合物和/或相关的基因编辑方法用于治疗实体瘤或癌症的方法中。
在特定实施例中,本文所设想的融合多肽、基因编辑的细胞、组合物和/或相关的基因编辑方法用于治疗实体瘤或癌症,所述实体瘤或癌症包含但不限于:肾上腺癌、肾上腺皮质癌、肛门癌、阑尾癌、星形细胞瘤、非典型畸胎样/横纹肌样瘤、基底细胞癌、胆管癌、膀胱癌、骨癌、脑/CNS癌、乳腺癌、支气管肿瘤、心脏肿瘤、宫颈癌、胆管癌、软骨肉瘤、脊索瘤、结肠癌、结直肠癌、颅咽管瘤、原位导管癌(DCIS)子宫内膜癌、室管膜瘤、食管癌、鼻腔神经胶质瘤、尤文氏肉瘤、颅外生殖细胞肿瘤、性腺外生殖细胞肿瘤、眼癌、输卵管癌、纤维组织肉瘤、纤维肉瘤、胆囊癌、胃癌、胃肠道类癌、胃肠道间质瘤(GIST)、生殖细胞肿瘤、胶质瘤、胶质母细胞瘤、头颈癌、血管母细胞瘤、肝细胞癌、下咽癌、眼内黑色素瘤、卡波西肉瘤、肾癌、喉癌、平滑肌肉瘤、唇癌、脂肪肉瘤、肝癌、肺癌、非小细胞肺癌、肺类癌、恶性间皮瘤、髓样癌、成神经管细胞瘤、脑膜瘤、黑色素瘤、梅克尔细胞癌、中线癌、口腔癌、粘膜肉瘤、骨髓增生异常综合征、骨髓增生性肿瘤、鼻腔和鼻窦癌、鼻咽癌、神经母细胞瘤、少突神经胶质瘤、口腔癌、口腔癌、口咽癌、骨肉瘤、卵巢癌、胰腺癌、胰岛细胞肿瘤、乳头状癌、副神经节瘤、甲状旁腺癌、阴茎癌、咽癌、嗜铬细胞瘤、松果体瘤、垂体瘤、胸膜肺母细胞瘤、原发性腹膜癌、前列腺癌、直肠癌、视网膜母细胞瘤、肾细胞癌、肾盂和输尿管癌、横纹肌肉瘤、唾液腺癌、皮脂腺癌、皮肤癌、软组织肉瘤、鳞状细胞癌、小细胞肺癌、小肠癌、胃癌、汗腺癌、滑膜瘤、睾丸癌、咽喉癌、胸腺癌、甲状腺癌,尿道癌症、子宫癌、子宫肉瘤、阴道癌、血管癌、外阴癌和威尔姆斯肿瘤。
在特定实施例中,本文所设想的融合多肽、基因编辑的细胞、组合物和/或相关的基因编辑方法用于治疗实体瘤或癌症,包含但不限于肝癌、胰腺癌、肺癌、乳腺癌、膀胱癌、脑癌、骨癌、甲状腺癌、肾癌或皮肤癌。
在特定实施例中,本文所设想的融合多肽、经基因编辑的细胞、组合物和/或相关的基因编辑方法用于治疗各种癌症,包含但不限于胰腺癌、膀胱癌和肺癌。
在特定实施例中,本文所设想的融合多肽、经基因编辑的细胞、组合物和/或相关的基因编辑方法用于治疗液体癌或血液癌。
在特定实施例中,本文所设想的融合多肽、经基因编辑的细胞、组合物和/或相关的基因编辑方法用于治疗B细胞恶性肿瘤,包含但不限于白血病、淋巴瘤和多发性骨髓瘤。
在特定实施例中,本文所设想的融合多肽、基因编辑的细胞、组合物和/或相关的基因编辑方法用于治疗液体癌症,包含但不限于白血病、淋巴瘤和多发性骨髓瘤:急性淋巴细胞白血病(ALL)、急性髓性白血病(AML)、成髓细胞白血病、早幼粒细胞白血病、髓单核细胞白血病、单核细胞白血病、红白血病、毛细胞白血病(HCL)、慢性淋巴细胞白血病(CLL)和慢性粒细胞白血病(CML)、慢性髓单核细胞白血病(CMML)和真性红细胞增多症、霍奇金淋巴瘤(Hodgkin lymphoma)、结节性淋巴细胞为主的霍奇金淋巴瘤、伯基特淋巴瘤(Burkittlymphoma)、小淋巴细胞淋巴瘤(SLL)、弥漫性大B细胞淋巴瘤、滤泡性淋巴瘤、免疫母细胞性大细胞淋巴瘤、前体B淋巴母细胞淋巴瘤、套细胞淋巴瘤、边缘区淋巴瘤、蕈样真菌病、间变性大细胞淋巴瘤、塞扎里氏综合征(Sézary syndrome)、前体T淋巴母细胞淋巴瘤、多发性骨髓瘤、明显多发性骨髓瘤、冒烟型多发性骨髓瘤、浆细胞白血病、非分泌性骨髓瘤、IgD骨髓瘤、骨硬化性骨髓瘤、骨孤立性浆细胞瘤和髓外浆细胞瘤。
在特定实施例中,一种方法包括向有需要的患者施用治疗有效量的融合多肽、组合物和/或经基因编辑的细胞。
在某些实施例中,本文所设想的融合多肽、经基因编辑的细胞、组合物和/或相关的基因编辑方法用于治疗处于患癌症的风险的患者。因此,具体实施例包括治疗或预防或改善癌症的至少一种症状,包括向有需要的患者施用治疗有效量的本文设想的融合多肽、经基因组编辑的细胞和/或组合物。
在特定实施例中,提供了一种治疗、预防或改善疾病或与其相关的病状的至少一种症状的方法,所述方法包括:从受试者采集细胞群体;根据本文所提供的基因编辑/诱变方法编辑所述细胞群体;以及将经编辑的细胞群体施用于有需要的受试者(例如,患有癌症的受试者)。
施用的数量和频率将由如患者的病状以及患者疾病的类型和严重性等因素确定,尽管适当的剂量可以通过临床试验确定。
在一个实施例中,向受试者施用的组合物中的基因编辑的细胞的量为至少0.1×105个细胞、至少0.5×105个细胞、至少1×105个细胞、至少5×105个细胞、至少1×106个细胞、至少0.5×107个细胞、至少1×107个细胞、至少0.5×108个细胞、至少1×108个细胞、至少0.5×109个细胞、至少1×109个细胞、至少2×109个细胞、至少3×109个细胞、至少4×109个细胞、至少5×109个细胞或至少1×1010个细胞。
在特定实施例中,向受试者施用约1×107个细胞至约1×109个细胞、约2×107个细胞至约0.9×109个细胞、约3×107个细胞至约0.8×109个细胞、约4×107个细胞至约0.7×109个细胞、约5×107个细胞至约0.6×109个细胞或约5×107个细胞至约0.5×109个细胞。
在一个实施例中,向受试者施用的组合物中的经基因编辑的细胞的量为至少0.1×104个细胞/kg体重、至少0.5×104个细胞/kg体重、至少1×104个细胞/kg体重、至少5×104个细胞/kg体重、至少1×105个细胞/kg体重、至少0.5×106个细胞/kg体重、至少1×106个细胞/kg体重、至少0.5×107个细胞/kg体重、至少1×107个细胞/kg体重、至少0.5×108个细胞/kg体重、至少1×108个细胞/kg体重、至少2×108个细胞/kg体重、至少3×108个细胞/kg体重、至少4×108个细胞/kg体重、至少5×108个细胞/kg体重或至少1×109个细胞/kg体重。
在特定实施例中,向受试者施用约1×106个细胞/kg体重至约1×108个细胞/kg体重、约2×106个细胞/kg体重至约0.9×108个细胞/kg体重、约3×106个细胞/kg体重至约0.8×108个细胞/kg体重、约4×106个细胞/kg体重至约0.7×108个细胞/kg体重、约5×106个细胞/kg体重至约0.6×108个细胞/kg体重、或约5×106个细胞/kg体重至约0.5×108个细胞/kg体重。
本领域普通技术人员将认识到,可能需要多次施用特定实施例中设想的组合物以影响期望的疗法。例如,组合物可以在1周、2周、3周、1个月、2个月、3个月、4个月、5个月、6个月、1年、2年、5年、10年或更长时间的跨度内施用1次、2次、3次、4次、5次、6次、7次、8次、9次或10次或更多次。
可以任何常规方式执行在特定实施例中所设想的组合物的施用,包含通过雾化吸入、注射、摄取、输血、植入或移植。在一优选实施例中,组合物是经鼻、口服、肠内或肠胃外施用的。如本文所用的短语“肠胃外施用(parenteral administration和administeredparenterally)”意指除了肠内施用和局部施用以外的通常通过注射进行的施用方式,并且包含但不限于:血管内、静脉内、肌肉内、动脉内、鞘内、囊内、眼眶内、瘤内、心内、真皮内、腹膜内、经气管、皮下、表皮下、关节内、囊下、蛛网膜下、脊柱内以及胸骨内注射和输注。在一个实施例中,本文所设想的组合物通过直接注射到肿瘤、淋巴结或感染部位向受试者施用。
在一个实施例中,向有需要的受试者施用有效量的组合物以增加对受试者中癌症的细胞免疫应答。免疫应答可以包含由能够杀死感染细胞、调节性T细胞和辅助T细胞应答的细胞毒性T细胞介导的细胞免疫应答。还可以诱导主要由能够活化B细胞从而导致抗体产生的辅助T细胞介导的体液免疫应答。可以使用在本领域中详细描述的多种技术来分析由组合物诱导的免疫应答的类型;例如,《当代免疫学手册(Current Protocols inImmunology)》,由:John E.Coligan,Ada M.Kruisbeek,David H.Margulies,EthanM.Shevach,Warren Strober编辑(2001)纽约州纽约市的约翰威立父子公司(John Wiley&Sons,NY,N.Y.)。
本说明书中引用的所有出版物、专利申请和经发布专利通过引用并入本文,如同每个单独的出版物、专利申请或经发布专利具体地且单独地指示通过引用并入一样。
尽管已经出于清楚理解的目的通过说明和举例的方式稍为详细地描述了前述实施例,但是根据本文设想的教导,本领域的普通技术人员将容易清楚的是,可以在不脱离随附权利要求的精神或范围的情况下对其进行某些改变和修改。提供以下实例仅作为说明并且不具有限制性。本领域的技术人员将容易认识到可以被改变或修改以产生本质上类似的结果的各种非关键参数。
实例
实例1
MEGATAL诱导的插入缺失长度和分布的可重复性
使用TRAC基因座(SEQ ID NO:1)处进行的基因编辑作为模型系统来评估由megaTAL核酸酶产生的插入缺失事件的性质。在这些研究中使用了低/中等效率的megaTAL(例如SEQ ID NO:5-7)以便为假设改变编辑速率的任何下游操纵提供动态范围。使用从不同的PBMC供体激活和扩增的原代人T细胞进行了若干个独立的实验。PBMC解冻、激活和培养3-4天的时段后,对体外转录、加帽和聚腺苷酸化的mRNA进行电穿孔,所述mRNA编码TRAC基因座的megaTAL靶向外显子1(TCRαmegaTAL)。将所得细胞培养7-10天的另外生长期,以允许稀释和降解递送的mRNA并完成编辑过程。聚合酶链式反应(PCR)用于扩增TRAC基因座,并且随后对PCR扩增子的深度测序使得能够表征由megaTAL引起的批量编辑事件。记录编辑事件大小的频率直方图如图1B所示。最常观察到的插入缺失长度为2个碱基对长(累计占事件总群体的约40-45%),同时也观察到高频率的1个碱基对缺失(约20%)和3个、8个和9个碱基对缺失(各5-10%)。这些插入缺失大小群体的相对频率在技术复制和独立实验中具有高度可重复性,说明了在megaTAL基因编辑期间插入缺失长度分布的一致性。
图1B所示的直方图中的每个条表示经编辑的等位基因群体,所述经编辑的等位基因群体具有相同的插入缺失长度,但相对于megaTAL靶位点的位置可能不同。图2示出了根据每个独特的经编辑的物种相对于megaTAL靶位点断点中心的定位位置而绘制的所述物种。这些“指纹”根据每个经编辑的物种的长度(y轴)、位置(x轴)和频率(圆圈大小)绘制所述物种,从而捕捉每个经编辑的物种的定量和定性性质两者。缺失的位置计算为22个碱基对归巢核酸内切酶靶位点的中心的其中点5'(负)或3'(正)的定位。以此方式,相对于断点中心的缺失物种的分布可以监测频率和位置两者的变化。使用不同PBMC供体的独立实验,以及使用具有低(例如SEQ ID NO:5-7)编辑效率或高(SEQ ID NO:8-10)编辑效率的TCRαmegaTAL构建体的独立实验,说明了单独的缺失物种——特定组成的每个重复缺失事件——以可重复的频率出现。因此,整个megaTAL缺失物种具有相对的定性和定量性质,所述性质既高度一致又不依赖于核酸内切酶反应的酶促速率。
实例2
TREX2定性地改变MEGATAL编辑的等位基因
三素修复核酸外切酶-2(Trex2)与基因编辑核酸酶的共递送先前已显示出增强了基因编辑效率,特别是通过megaTAL,这是由于其3'突出端核酸内切酶反应产物。然而,Trex2共递送对编辑结果的定性影响尚未得到彻底评估。因此,对两种不同的共递送模式的插入缺失性质进行了评估;Trex2与megaTAL的C末端直接融合;以及Trex2和megaTAL作为独立多肽的共表达。
用体外转录、加帽和聚腺苷酸化的编码TCRαmegaTAL的mRNA(图3A)、编码TCRαmegaTAL-Trex2融合体的mRNA(图3B)或两种不同的mRNA(一种编码TCRαmegaTAL,另一种编码Trex2)对激活的原代人T细胞样品(图3C)进行电穿孔。将所得细胞培养7-10天的另外生长期,以允许稀释和降解递送的mRNA并完成编辑过程。聚合酶链式反应(PCR)用于扩增TRAC基因座,并且随后对PCR扩增子的深度测序使得能够表征由megaTAL引起的批量编辑事件。记录编辑事件大小的频率直方图如图3A-3C所示。Trex2核酸外切酶与TCRαmegaTAL(例如,SEQ ID NO:11-13)的直接融合导致缺失长度分布的改变,其中长度高达12个碱基对的缺失占插入缺失物种的显著百分比。TCRαmegaTAL和Trex2核酸外切酶(例如,SEQ ID NO:14-16)的独立共表达导致插入缺失物种朝向长度为1个至4个碱基对的缺失变窄,这与偏好单链3'突出端的分布核酸外切酶活性模型一致。在每个Trex2递送场景中,相对于单独的TCRαmegaTAL递送,总体编辑速率和缺失与插入的比率增加。
图4示出了图3A-3C中每个样品的缺失指纹图,捕获了每个经编辑的物种的定量和定性性质。如在图2中,缺失的位置计算为22个碱基对归巢核酸内切酶靶位点的中心的其中点5'(负)或3'(正)的定位。缺失物种相对于断点中心的分布显示出TCRαmegaTAL、TCRαmegaTAL-Trex2融合体与TCRαmegaTAL加Trex2共递送编辑结果之间的显著差异(参见表3-5)。在megaTAL-Trex2融合体中,观察到缺失物种的出现,所述物种的位置在5'方向严重倾斜。此模式意味着在DNA断点末端处存在优先的核酸外切酶活性,所述DNA断点末端与TALE阵列保持顺式或位于断点的同一侧上。
另外地,观察到与TALE阵列结合位点的3'边缘相对应的缺失长度和位置急剧下降。尽管如此,惊奇地观察到延伸到TALE结合位点的缺失长度。这进一步暗示了在核酸外切酶活性与其结合位点处存在的TALE之间存在相互排斥的关系,进一步巩固了TALE阵列在定位Trex2以执行其核酸外切酶功能中的作用。然而,缺失中心位于TALE结合位点之外。相反,作为单独多肽的Trex2共表达不会引起任何实质性的方向偏斜,尽管改变了物种组成,使缺失群体以DNA断点为中心,大小在1-4bp范围内。
表3
表4
多肽 | 大于6bp的缺失% | 大于12bp的缺失% |
TCRa megaTAL | 17.97% | 7.17% |
TCRa megaTAL-Trex2融合体 | 35.49% | 14.21% |
TCRa megaTAL+Trex2共表达 | 4.66% | 0.83% |
表5
实例3
对TREX2同源物编辑的等位基因的评估
为了证实Trex2的酶活性是编辑结果中观察到的变化的直接原因,而不是与DNA修复机制的潜在混杂相互作用,将若干Trex2同源物ORF与TCRαmegaTAL(例如,SEQ ID NO:17-34)融合。用体外转录的、加帽的和聚腺苷酸化的编码这些融合蛋白的mRNA对激活的原代人T细胞样品进行电穿孔,然后通过流式细胞术分析TCR复合物的表达和通过表征插入缺失性质来监测靶位点编辑。
图5示出了许多Trex2同源物融合蛋白将整体编辑增强到与人Trex2ORF相似的程度。使用流式细胞术染色细胞表面与TCRα和β链组合的CD3组分来评估编辑效率。因此,失去CD3染色的细胞频率与TCRα基因座处的编辑速率相关。如图6所示,对插入缺失物种的分布的分析进一步证实了鸭嘴兽、负鼠、犰狳和小鼠Trex2蛋白引起的编辑结果具有与人Trex2ORF与TCRαmegaTAL融合时所引起的性质一致的性质。值得注意的是,绵羊Trex2引起了独特的插入缺失谱,富含1-4bp缺失,以及令人惊讶地,高频率的1bp插入。
如图7中展示的指纹图所示,TCRαmegaTAL-Trex2同源物融合蛋白的缺失性质反映了在人Trex2蛋白中观察到的性质。不希望受任何特定理论的束缚,此数据表明Trex2核酸外切酶活性是所观察到的缺失性质的主要决定因素。因此,延长的缺失长度结果可能不依赖于与其它DNA修复机制的相互作用。在绵羊Trex2同源物中观察到的插入发生率的增加表明此变体可能具有独特的酶促机制,如潜在的残留模板非依赖性聚合酶活性。最后,缺失大小和位置的高度一致和清晰的分界进一步表明碱基的定向切除可能受到TALE阵列与其结合位点相互作用的缔合和解离动力学和/或空间性质的限制。
实例4
鉴定唯一影响MEGATAL编辑的核酸外切酶
为了确定具有不同底物特异性和/或进行性性质的其它核酸外切酶是否可以独特地改变基因编辑结果,选择核酸外切酶或核酸外切酶结构域ORF与TCRαmegaTAL(例如,SEQID NO:35-55)融合,并且检测它们对基因编辑结果的影响。检测了作为独立TCRαmegaTAL融合蛋白以及与Trex2共表达期间的编辑速率。在这两种场景中进行测试的基本原理有两个方面:(i)允许Trex2活性为测试核酸外切酶产生独特的和潜在优先的底物;以及(ii)寻找Trex2介导的编辑速率增强的损失,作为测试核酸外切酶有意义地加工末端的替代。图8描述了核酸外切酶筛选的结果,其中每个样品一式两份运行,并且在不存在(左)或存在(右)Trex2共表达的情况下进行测试。
在测试的核酸外切酶中,大多数没有明显改变Trex2的编辑速率或编辑速率增强(测量为Trex2样品中CD3阴性细胞与单独递送megaTAL-核酸外切酶融合体的比率)。若干核酸外切酶显著降低了整体编辑,表明它们可能显著影响了蛋白质稳定性和/或表达。TCRαmegaTAL与ExoI的融合导致Trex2增强率适度增加,表明这两种核酸外切酶可能协同修饰DNA断裂加工事件。值得注意的是,TCRαmegaTAL与ExoX融合导致Trex2增强作用完全丧失。
实例5
对MEGATAL-EXOI和MEGATAL-EXOX编辑图谱的评估
为了进一步理解图8中描述的基因编辑事件的定性方面,如先前所描述进行扩增子测序和缺失指纹分析。那些不影响TCRαmegaTAL编辑速率模式的核酸外切酶也未能显示指纹变化(数据未显示)。然而,图9示出了,虽然TCRαmegaTAL与ExoI的融合相对于单独的TCRαmegaTAL没有改变缺失模式,但当Trex2共表达时,出现了一系列长的定向偏向的缺失事件。
megaTAL-ExoI融合体产生在TALE阵列的方向上偏向的缺失事件。与界限分明的megaTAL-Trex2融合缺失事件相比之下,megaTAL-ExoI缺失事件似乎具有更长的长度,并且因此延伸超过TALE阵列结合位点。
TCRαmegaTAL与ExoX的融合导致了与通过megaTAL-ExoI融合观察到的不同的缺失图谱。在不存在Trex2的情况下,TCRαmegaTAL-ExoX融合体高效地产生长的定向偏向的缺失。与megaTAL-Trex2融合蛋白不同,观察到的缺失中心更大,以至于它们确实以可观的频率超过TALE阵列结合位点。相对于megaTAL-Trex2融合蛋白,megaTAL-ExoX融合体可能具有独特的核酸外切酶机制,产生更长的缺失,其中缺失中心远离切割位点。
在图9中,当Trex2与TCRαmegaTAL-ExoX融合蛋白共表达时,编辑事件变得几乎不可检测,并且缺失了megaTAL-ExoX融合体中明显存在的长的定向偏向的缺失。
实例6
对MEGATAL-EXOX融合蛋白编辑图谱的进一步评估
考虑到在评估TCRαmegaTAL-ExoX融合蛋白时观察到的缺失模式,测试了另外若干种靶向不同遗传基因座的megaTAL-ExoX融合蛋白。用体外转录、加帽和聚腺苷酸化的mRNA对激活的原代人T细胞样品进行电穿孔,所述mRNA编码高活性CBL-B靶向megaTAL、高活性CBL-B megaTAL-Trex2融合体或高活性CBL-B megaTAL-ExoX融合体(例如,SEQ ID NO:56-64)。将所得细胞培养7-10天的另外生长期,以允许稀释和降解递送的mRNA并完成编辑过程。聚合酶链式反应(PCR)用于扩增CBL-B基因座,并且随后对PCR扩增子的深度测序使得能够表征靠近CBL-B靶位点(SEQ ID NO:3)的编辑速率和插入缺失性质。图10示出了针对这三种高活性CBL-B megaTAL格式中的每一种观察到的缺失指纹(也参见表6-8)。高活性CBL-BmegaTAL产生了与TCRαmegaTAL相似的缺失物种分布,其中最频繁的事件是1-5bp缺失,并且没有显著的方向偏向。同样,高活性CBL-BmegaTAL-Trex2融合体延长了缺失物种,并且使它们在5'方向上严重倾斜,证实了对于正交megaTAL靶标,优先的核酸外切酶活性发生在DNA断点末端处,所述断点末端与TALE阵列保持顺式或与所述阵列位于断点的相同侧上。在高活性CBL-B megaTAL-Trex2融合体中还重复出现的是与TALE阵列结合位点的3'边缘相对应的缺失长度和位置的急剧下降。
表6
表7
表8
图11示出了用相似系列的CBL-B megaTAL、megaTAL-Trex2和megaTAL-ExoX融合蛋白处理的T细胞的代表性缺失指纹,然而这些融合蛋白采用了低若干倍活性的megaTAL(例如,SEQ ID NO:65-73)。还参见表9-11。当将这些低活性CBL-B megaTAL和megaTAL融合蛋白与其高活性对应物进行比较时,观察到的缺失物种的谱有很大的相似性,如图10所显示。这说明当这两种核酸外切酶融合成megaTAL时观察到的独特缺失结果不依赖于核酸内切酶反应的酶促速率。这些结果既表明每个核酸内切酶产生的断裂具有高效的核酸外切酶加工速率,又进一步说明Trex2和ExoX通过定性不同的机制明显影响基因编辑结果,并且不仅仅是以不同效率发生的相同机制。
表9
表10
多肽 | 大于6bp的缺失% | 大于12bp的缺失% |
低活性CBL-B megaTAL | 36.82% | 26.71% |
低活性CBL-B megaTAL-Trex2融合体 | 68.01% | 30.58% |
低活性CBL-B megaTAL-ExoX融合体 | 50.57% | 37.93% |
表11
第三个系列的酶是靶向PD-1基因座构建的。图12示出了来自T细胞电穿孔的mRNA的代表性缺失指纹,所述mRNA编码PD-1megaTAL、PD-1megaTAL-Trex2融合体或PD-1megaTAL-ExoX融合体(例如,SEQ ID NO:74-82)。还参见表12-14。正如针对TCRα和CBL-BmegaTAL以及核酸外切酶融合蛋白所描述的,针对三个PD-1megaTAL样品中的每个样品观察到的缺失物种遵循了独立PD-1megaTAL中出现非定向的小缺失物种的一致趋势,当靶标暴露于PD-1megaTAL-Trex2融合蛋白时,出现5'定向和更长的物种,并且用PD-1megaTAL-ExoX融合蛋白处理产生的种类仍更长。这些观察到的模式在若干测试的megaTAL中的持续性说明了在megaTAL与不同核酸外切酶结构域融合时发生的不同编辑结果的稳健性。
表12
表13
表14
实例7
对PD-1MEGATAL-EXOX融合蛋白编辑图谱的评估
图13描绘了PD-1megaTAL靶位点,所述靶位点位于PDCD1基因的ATG起始密码子附近。22bp PD-1HE靶位点(SEQ ID NO:2)大约以编码异亮氨酸残基的第三个密码子为中心,所述异亮氨酸残基是靶向PD-1以在质膜上进行表达的信号序列的一部分。TALE阵列结合位点(SEQ ID NO:4)位于ATG起始密码子的5'端。此靶位点朝向增加了不同的megaTAL或megaTAL-核酸外切酶融合体组合物根据其相应的缺失物种的分布不同地影响PD-1表达的可能性。
根据其对PD-1ORF的影响来划分缺失物种:ATG缺失;框内;框架2;或框架3;图13示出了每个物种的示例缺失类型。对于图12中描述的PD-1megaTAL和the megaTAL-核酸外切酶融合蛋白样品,对每个缺失物种的框架特性进行了生物信息学分析。图14示出了将基因编辑物种的框架类别叠加到缺失指纹图上进行的该分析的结果。因为PD-1megaTAL产生了广谱的缺失物种,所以框架特性在缺失指纹分析中稍微均匀地分布,其中ATG缺失的物种占据了图的上部,在所述上部,长缺失被表示。相反,PD-1megaTAL-Trex2融合蛋白引起框内、框架2和框架3物种的位置的移位。当比较每个类别的物种的归一化的分数时,如图15所示,PD-1megaTAL和PD-1megaTAL-Trex2融合体没有显著差异。这是因为通过ATG起始密码子的长的定向的缺失的出现虽然有所增加,但已经有效地替代了在使用PD-1megaTAL时发生的少量长的(但非定向的)缺失。
相反,由PD-1megaTAL-ExoX融合蛋白引起的缺失长度的增加显著增加了消除ATG起始密码子的缺失物种的分数比例。ATG缺失的等位基因的增加是以产生属于其它三个类别的物种的缺失为代价的,并且进一步表明PD-1megaTAL-ExoX融合体可能不成比例地删除不期望的PD-1基因编辑的等位基因,如信号序列的N末端的框内单个氨基酸缺失,所述缺失可能不影响PD-1表面表达和功能。为了评估框内缺失可能不会在功能上使PDCD1基因座失活的可能性,用体外转录的、加帽的和聚腺苷酸化的mRNA对激活的原代人T细胞样品进行电穿孔,所述mRNA编码以下中的任一者或两者:用于追踪转染效率的青色荧光蛋白(CFP),以及三种可能的阅读框中的每种阅读框中的野生型或模拟物编辑的PD-1等位基因。然后进行流式细胞术以评估转染效率和PD-1蛋白表达,如图16所描述。虽然野生型PD-1mRNA(SEQ IDNO:83)电穿孔导致高水平蛋白质表达,但编码1个或2个碱基对缺失的人工mRNA(SEQ IDNO:84和85)(分别模拟框架2或框架3缺失物种)并不促进PD-1蛋白质表达,如编码框外蛋白质转录物的mRNA所预期的那样。相比之下,编码PD-1megaTAL靶位点处的3个碱基对缺失的人工mRNA(SEQ ID NO:86)产生高水平的PD-1表面表达,原则上证实了一些PD-1基因编辑等位基因物种在表型上是非渗透性的。
实例8
对脱靶基因座处进行的编辑的评估
为了进一步了解脱靶位点处的基因编辑事件的定性方面,在对低和高编辑TCRαmegaTAL具有特异性的脱靶位点处进行了扩增子测序和缺失指纹分析。用编码低编辑TCRαmegaTAL、高编辑TCRαmegaTAL(TCRα2.2)以及各自与Trex2的直接融合和各自与ExoX的直接融合的1.5ug mRNA电穿孔从PBMC激活和扩增的原代人T细胞。每个构建体都在中靶基因座(图17;一式两份;仅示出了一个复制品),或其脱靶基因座(一式两份,仅示出了一个复制品)处进行评估。在7-10天的生长期后,采集细胞用于基因组DNA分离。PCR在大约300个碱基对区域内进行,所述区域涵盖KAT2B脱靶位点(易受低编辑TCRαmegaTAL的编辑,图18)或2.2脱靶位点AC016700.3(易受高编辑TCRαmegaTAL的编辑,图19)。然后对PCR扩增子进行下一代测序(NGS),并且通过根据其频率(圆圈大小)以及其长度(y轴)和其相对于megaTAL引起的断点的纵向位置(x轴)两者将缺失事件列表显示来进行分析。此分析排除了插入。TCRα2.2与Trex2融合未能编辑,并且因此未显示指纹。那些影响每个TCRαmegaTAL编辑的模式的核酸外切酶导致了相对于每个TCRαmegaTAL的中靶编辑效率非常低的编辑速率,并且显示了指纹的变化。因为TALE阵列缺少底物,所以在测试的任何megaTAL融合体中似乎没有任何偏向的亲和力分布。
序列表
<110> 2赛文缇生物公司
乔丹·贾儒尔
安妮瑞秋·克罗斯塔格
<120> 用于定点诱变的组合物和方法
<130> BLUE-132.PC
<150> US 63/128,391
<151> 2020-12-21
<160> 164
<170> PatentIn 3.5版
<210> 1
<211> 22
<212> DNA
<213> 智人(Homo sapiens)
<400> 1
tgtctgccta ttcaccgatt tt 22
<210> 2
<211> 22
<212> DNA
<213> 智人(Homo sapiens)
<400> 2
ggcatgcaga tcccacaggc gc 22
<210> 3
<211> 22
<212> DNA
<213> 智人(Homo sapiens)
<400> 3
ctgtaagata ttcccatccc ca 22
<210> 4
<211> 14
<212> DNA
<213> 智人(Homo sapiens)
<400> 4
tggtggggct gctc 14
<210> 5
<211> 2637
<212> DNA
<213> 人工序列
<220>
<223> 在实验室中制备-低活性TCRα megaTAL DNA构建体
<400> 5
atgggatcct gcaggccacc taagaagaaa cgcaaagtcg tggatctacg cacgctcggc 60
tacagtcagc agcagcaaga gaagatcaaa ccgaaggtgc gttcgacagt ggcgcagcac 120
cacgaggcac tggtgggcca tgggtttaca cacgcgcaca tcgttgcgct cagccaacac 180
ccggcagcgt tagggaccgt cgctgtcacg tatcagcaca taatcacggc gttgccagag 240
gcgacacacg aagacatcgt tggcgtcggc aaacagtggt ccggcgcacg cgccctggag 300
gccttgctca cggatgcggg ggagttgaga ggtccgccgt tacagttgga cacaggccaa 360
cttgtgaaga ttgcaaaacg tggcggcgtg accgcaatgg aggcagtgca tgcatcgcgc 420
aatgcactga cgggtgcccc cctgaacctg accccggacc aagtggtggc tatcgccagc 480
aacattggcg gcaagcaagc gctcgaaacg gtgcagcggc tgttgccggt gctgtgccag 540
gaccatggcc tgaccccgga ccaagtggtg gctatcgcca gcaacattgg cggcaagcaa 600
gcgctcgaaa cggtgcagcg gctgttgccg gtgctgtgcc aggaccatgg cctgaccccg 660
gaccaagtgg tggctatcgc cagcaacatt ggcggcaagc aagcgctcga aacggtgcag 720
cggctgttgc cggtgctgtg ccaggaccat ggcctgaccc cggaccaagt ggtggctatc 780
gccagcaacg gtggcggcaa gcaagcgctc gaaacggtgc agcggctgtt gccggtgctg 840
tgccaggacc atggcctgac tccggaccaa gtggtggcta tcgccagcca cgatggcggc 900
aagcaagcgc tcgaaacggt gcagcggctg ttgccggtgc tgtgccagga ccatggcctg 960
actccggacc aagtggtggc tatcgccagc cacgatggcg gcaagcaagc gctcgaaacg 1020
gtgcagcggc tgttgccggt gctgtgccag gaccatggcc tgaccccgga ccaagtggtg 1080
gctatcgcca gcaacattgg cggcaagcaa gcgctcgaaa cggtgcagcg gctgttgccg 1140
gtgctgtgcc aggaccatgg cctgaccccg gaccaagtgg tggctatcgc cagcaacaat 1200
ggcggcaagc aagcgctcga aacggtgcag cggctgttgc cggtgctgtg ccaggaccat 1260
ggcctgaccc cggaccaagt ggtggctatc gccagcaacg gtggcggcaa gcaagcgctc 1320
gaaacggtgc agcggctgtt gccggtgctg tgccaggacc atggcctgac cccggaccaa 1380
gtggtggcta tcgccagcaa caatggcggc aagcaagcgc tcgaaacggt gcagcggctg 1440
ttgccggtgc tgtgccagga ccatggcctg accccggacc aagtggtggc tatcgccagc 1500
aacattggcg gcaagcaagc gctcgaaagc attgtggccc agctgagccg gcctgatccg 1560
gcgttggccg cgttgaccaa cgaccacctc gtcgccttgg cctgcctcgg cggacgtcct 1620
gccatggatg cagtgaaaaa gggattgccg cacgcgccgg aattgatcag aagagtcaat 1680
cgccgtattg gcgaacgcac gtcccatcgc gttgcgatat ctagagtggg aggaagctct 1740
cgcagagagt ccatcaaccc atggattctg actggtttcg ctgatgccga aggatcattc 1800
atactagaca tccgcaaccg aaacaacgaa agcaacagat accgaacttc gctgagattc 1860
cagatcaccc tgcacaacaa ggacaaatcg attctggaga atatccagtc cacttggaag 1920
gtcggcaaga tcacaaacag cagtgacaga gccgtcatgc tgagggtcac ccgtttcgaa 1980
gatttgaaag tgattatcga ccacttcgag aaatatccgc tgattaccca gaaattgggc 2040
gattacaagt tgtttaaaca ggcattcagc gtcatggaga ataaagaaca tcttaaggag 2100
aatgggatta aggagctcgt acgaatcaaa gctaagatga attggggtct caatgacgaa 2160
ttgaaaaaag catttccaga gaacattagc aaagagcgcc cccttatcaa taagaacatt 2220
ccgaatttca aatggctggc tggattcaca gctggtgaag gctacttcgg cgtgaatcta 2280
aaaaaggtaa agggcaccgc aaaggtatac gtgggactga gattctcaat ctcacagcac 2340
atcagagaca agaacctgat gaattcattg ataacatacc taggctgtgg ttccatctgg 2400
gagaagaaca agtctgagtt cagatggctc gagttcgtcg taaccaaatt cagcgatatc 2460
aacgacaaga tcattccggt attccaggaa aatactctga ttggcgtcaa actcgaggac 2520
tttgaagatt ggtgcaaggt tgccaaattg atcaaagaga agaaacacct gaccgaatcc 2580
ggtttggatg agattaagaa aatcaagctg aacatgaaca aaggtcgtgt cttctga 2637
<210> 6
<211> 2637
<212> RNA
<213> 人工序列
<220>
<223> 在实验室中制备-低活性TCRα megaTAL RNA构建体
<400> 6
augggauccu gcaggccacc uaagaagaaa cgcaaagucg uggaucuacg cacgcucggc 60
uacagucagc agcagcaaga gaagaucaaa ccgaaggugc guucgacagu ggcgcagcac 120
cacgaggcac uggugggcca uggguuuaca cacgcgcaca ucguugcgcu cagccaacac 180
ccggcagcgu uagggaccgu cgcugucacg uaucagcaca uaaucacggc guugccagag 240
gcgacacacg aagacaucgu uggcgucggc aaacaguggu ccggcgcacg cgcccuggag 300
gccuugcuca cggaugcggg ggaguugaga gguccgccgu uacaguugga cacaggccaa 360
cuugugaaga uugcaaaacg uggcggcgug accgcaaugg aggcagugca ugcaucgcgc 420
aaugcacuga cgggugcccc ccugaaccug accccggacc aagugguggc uaucgccagc 480
aacauuggcg gcaagcaagc gcucgaaacg gugcagcggc uguugccggu gcugugccag 540
gaccauggcc ugaccccgga ccaaguggug gcuaucgcca gcaacauugg cggcaagcaa 600
gcgcucgaaa cggugcagcg gcuguugccg gugcugugcc aggaccaugg ccugaccccg 660
gaccaagugg uggcuaucgc cagcaacauu ggcggcaagc aagcgcucga aacggugcag 720
cggcuguugc cggugcugug ccaggaccau ggccugaccc cggaccaagu gguggcuauc 780
gccagcaacg guggcggcaa gcaagcgcuc gaaacggugc agcggcuguu gccggugcug 840
ugccaggacc auggccugac uccggaccaa gugguggcua ucgccagcca cgauggcggc 900
aagcaagcgc ucgaaacggu gcagcggcug uugccggugc ugugccagga ccauggccug 960
acuccggacc aagugguggc uaucgccagc cacgauggcg gcaagcaagc gcucgaaacg 1020
gugcagcggc uguugccggu gcugugccag gaccauggcc ugaccccgga ccaaguggug 1080
gcuaucgcca gcaacauugg cggcaagcaa gcgcucgaaa cggugcagcg gcuguugccg 1140
gugcugugcc aggaccaugg ccugaccccg gaccaagugg uggcuaucgc cagcaacaau 1200
ggcggcaagc aagcgcucga aacggugcag cggcuguugc cggugcugug ccaggaccau 1260
ggccugaccc cggaccaagu gguggcuauc gccagcaacg guggcggcaa gcaagcgcuc 1320
gaaacggugc agcggcuguu gccggugcug ugccaggacc auggccugac cccggaccaa 1380
gugguggcua ucgccagcaa caauggcggc aagcaagcgc ucgaaacggu gcagcggcug 1440
uugccggugc ugugccagga ccauggccug accccggacc aagugguggc uaucgccagc 1500
aacauuggcg gcaagcaagc gcucgaaagc auuguggccc agcugagccg gccugauccg 1560
gcguuggccg cguugaccaa cgaccaccuc gucgccuugg ccugccucgg cggacguccu 1620
gccauggaug cagugaaaaa gggauugccg cacgcgccgg aauugaucag aagagucaau 1680
cgccguauug gcgaacgcac gucccaucgc guugcgauau cuagaguggg aggaagcucu 1740
cgcagagagu ccaucaaccc auggauucug acugguuucg cugaugccga aggaucauuc 1800
auacuagaca uccgcaaccg aaacaacgaa agcaacagau accgaacuuc gcugagauuc 1860
cagaucaccc ugcacaacaa ggacaaaucg auucuggaga auauccaguc cacuuggaag 1920
gucggcaaga ucacaaacag cagugacaga gccgucaugc ugagggucac ccguuucgaa 1980
gauuugaaag ugauuaucga ccacuucgag aaauauccgc ugauuaccca gaaauugggc 2040
gauuacaagu uguuuaaaca ggcauucagc gucauggaga auaaagaaca ucuuaaggag 2100
aaugggauua aggagcucgu acgaaucaaa gcuaagauga auuggggucu caaugacgaa 2160
uugaaaaaag cauuuccaga gaacauuagc aaagagcgcc cccuuaucaa uaagaacauu 2220
ccgaauuuca aauggcuggc uggauucaca gcuggugaag gcuacuucgg cgugaaucua 2280
aaaaagguaa agggcaccgc aaagguauac gugggacuga gauucucaau cucacagcac 2340
aucagagaca agaaccugau gaauucauug auaacauacc uaggcugugg uuccaucugg 2400
gagaagaaca agucugaguu cagauggcuc gaguucgucg uaaccaaauu cagcgauauc 2460
aacgacaaga ucauuccggu auuccaggaa aauacucuga uuggcgucaa acucgaggac 2520
uuugaagauu ggugcaaggu ugccaaauug aucaaagaga agaaacaccu gaccgaaucc 2580
gguuuggaug agauuaagaa aaucaagcug aacaugaaca aaggucgugu cuucuga 2637
<210> 7
<211> 878
<212> PRT
<213> 人工序列
<220>
<223> 在实验室中制备-低活性TCRα megaTAL蛋白质构建体
<400> 7
Met Gly Ser Cys Arg Pro Pro Lys Lys Lys Arg Lys Val Val Asp Leu
1 5 10 15
Arg Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys
20 25 30
Val Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val Gly His Gly
35 40 45
Phe Thr His Ala His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu
50 55 60
Gly Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr Ala Leu Pro Glu
65 70 75 80
Ala Thr His Glu Asp Ile Val Gly Val Gly Lys Gln Trp Ser Gly Ala
85 90 95
Arg Ala Leu Glu Ala Leu Leu Thr Asp Ala Gly Glu Leu Arg Gly Pro
100 105 110
Pro Leu Gln Leu Asp Thr Gly Gln Leu Val Lys Ile Ala Lys Arg Gly
115 120 125
Gly Val Thr Ala Met Glu Ala Val His Ala Ser Arg Asn Ala Leu Thr
130 135 140
Gly Ala Pro Leu Asn Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
145 150 155 160
Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
165 170 175
Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
180 185 190
Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
195 200 205
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val
210 215 220
Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
225 230 235 240
Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln
245 250 255
Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr
260 265 270
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro
275 280 285
Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu
290 295 300
Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu
305 310 315 320
Thr Pro Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln
325 330 335
Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His
340 345 350
Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly
355 360 365
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
370 375 380
Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Asn
385 390 395 400
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu
405 410 415
Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
420 425 430
Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
435 440 445
Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
450 455 460
Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
465 470 475 480
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val
485 490 495
Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Ser Ile Val
500 505 510
Ala Gln Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu Thr Asn Asp
515 520 525
His Leu Val Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala Met Asp Ala
530 535 540
Val Lys Lys Gly Leu Pro His Ala Pro Glu Leu Ile Arg Arg Val Asn
545 550 555 560
Arg Arg Ile Gly Glu Arg Thr Ser His Arg Val Ala Ile Ser Arg Val
565 570 575
Gly Gly Ser Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr Gly
580 585 590
Phe Ala Asp Ala Glu Gly Ser Phe Ile Leu Asp Ile Arg Asn Arg Asn
595 600 605
Asn Glu Ser Asn Arg Tyr Arg Thr Ser Leu Arg Phe Gln Ile Thr Leu
610 615 620
His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp Lys
625 630 635 640
Val Gly Lys Ile Thr Asn Ser Ser Asp Arg Ala Val Met Leu Arg Val
645 650 655
Thr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys Tyr
660 665 670
Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln Ala
675 680 685
Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile Lys
690 695 700
Glu Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly Leu Asn Asp Glu
705 710 715 720
Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Pro Leu Ile
725 730 735
Asn Lys Asn Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe Thr Ala Gly
740 745 750
Glu Gly Tyr Phe Gly Val Asn Leu Lys Lys Val Lys Gly Thr Ala Lys
755 760 765
Val Tyr Val Gly Leu Arg Phe Ser Ile Ser Gln His Ile Arg Asp Lys
770 775 780
Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Ser Ile Trp
785 790 795 800
Glu Lys Asn Lys Ser Glu Phe Arg Trp Leu Glu Phe Val Val Thr Lys
805 810 815
Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn Thr
820 825 830
Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val Ala
835 840 845
Lys Leu Ile Lys Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp Glu
850 855 860
Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Val Phe
865 870 875
<210> 8
<211> 2730
<212> DNA
<213> 人工序列
<220>
<223> 在实验室中制备-低活性TCRα megaTAL DNA构建体
<400> 8
atgggatccg cgccacctaa gaagaaacgc aaagtcgtgg atctacgcac gctcggctac 60
agtcagcagc agcaagagaa gatcaaaccg aaggtgcgtt cgacagtggc gcagcaccac 120
gaggcactgg tgggccatgg gtttacacac gcgcacatcg ttgcgctcag ccaacacccg 180
gcagcgttag ggaccgtcgc tgtcacgtat cagcacataa tcacggcgtt gccagaggcg 240
acacacgaag acatcgttgg cgtcggcaaa cagtggtccg gcgcacgcgc cctggaggcc 300
ttgctcacgg atgcggggga gttgagaggt ccgccgttac agttggacac aggccaactt 360
gtgaagattg caaaacgtgg cggcgtgacc gcaatggagg cagtgcatgc atcgcgcaat 420
gcactgacgg gtgcccccct gaacctgacc ccggaccaag tggtggctat cgccagcaac 480
attggcggca agcaagcgct cgaaacggtg cagcggctgt tgccggtgct gtgccaggac 540
catggcctga ccccggacca agtggtggct atcgccagca acattggcgg caagcaagcg 600
ctcgaaacgg tgcagcggct gttgccggtg ctgtgccagg accatggcct gaccccggac 660
caagtggtgg ctatcgccag caacattggc ggcaagcaag cgctcgaaac ggtgcagcgg 720
ctgttgccgg tgctgtgcca ggaccatggc ctgaccccgg accaagtggt ggctatcgcc 780
agcaacggtg gcggcaagca agcgctcgaa acggtgcagc ggctgttgcc ggtgctgtgc 840
caggaccatg gcctgactcc ggaccaagtg gtggctatcg ccagccacga tggcggcaag 900
caagcgctcg aaacggtgca gcggctgttg ccggtgctgt gccaggacca tggcctgact 960
ccggaccaag tggtggctat cgccagccac gatggcggca agcaagcgct cgaaacggtg 1020
cagcggctgt tgccggtgct gtgccaggac catggcctga ccccggacca agtggtggct 1080
atcgccagca acattggcgg caagcaagcg ctcgaaacgg tgcagcggct gttgccggtg 1140
ctgtgccagg accatggcct gaccccggac caagtggtgg ctatcgccag caacaatggc 1200
ggcaagcaag cgctcgaaac ggtgcagcgg ctgttgccgg tgctgtgcca ggaccatggc 1260
ctgaccccgg accaagtggt ggctatcgcc agcaacggtg gcggcaagca agcgctcgaa 1320
acggtgcagc ggctgttgcc ggtgctgtgc caggaccatg gcctgacccc ggaccaagtg 1380
gtggctatcg ccagcaacaa tggcggcaag caagcgctcg aaacggtgca gcggctgttg 1440
ccggtgctgt gccaggacca tggcctgacc ccggaccaag tggtggctat cgccagcaac 1500
attggcggca agcaagcgct cgaaaccgta caacgtctcc tcccagtact ttgtcaagac 1560
cacgggttga ctccggatca agtcgtcgcg atcgcgagcc atgatggggg gaagcaggcg 1620
ctggaaagca ttgtggccca gctgagccgg cctgatccgg cgttggccgc gttgaccaac 1680
gaccacctcg tcgccttggc ctgcctcggc ggacgtcctg ccatggatgc agtgaaaaag 1740
ggattgccgc acgcgccgga attgatcaga agagtcaatc gccgtattgg cgaacgcacg 1800
tcccatcgcg ttgcgatatc tagagtggga ggaagctcgc gcagagagtc catcaaccca 1860
tggattctga ctggtttcgc tgatgccgaa ggatcattca tactagacat ccgcaaccga 1920
aacaacgaaa gcaacagata ccgaacttcg ctgagattcc agatcaccct gcacaacaag 1980
gacaaatcga ttctggagaa tatccagtcc acttggaagg tcggcaagat cacaaacagc 2040
agtgacagag ccgtcatgct gagggtcacc cgtttcgaag atttgaaagt gattatcgac 2100
cacttcgaga aatatccgct gattacccag aaattgggcg attacaagtt gtttaaacag 2160
gcattcagcg tcatggagaa taaagaacat cttaaggaga atgggattaa ggagctcgta 2220
cgaatcaaag ctaagatgaa ttggggtctc aatgacgaat tgaaaaaagc atttccagag 2280
aacattagca aagagcgccc ccttatcaat aagaacattc cgaatttcaa atggctggct 2340
ggattcacag ctggtgatgg ccatttcggc gtgaatctaa aaaaggtaaa gggcaccgca 2400
aaggtatacg tgggactgag attcgctatc tcacagcaca tcagagacaa gaacctgatg 2460
aattcattga taacatacct aggctgtggt tccatcagag agaagaacaa gtctgagttc 2520
agatggctcg agttcgaagt aaccaaattc agcgatatca acgacaagat cattccggta 2580
ttccaggaaa atactctgat tggcgtcaaa ctcgaggact ttgaagattg gtgcaaggtt 2640
gccaaattga tcgaagagaa gaaacacctg accgaatccg gtttggatga gattaagaaa 2700
atcaagctga acatgaacaa aggtcgttga 2730
<210> 9
<211> 2730
<212> RNA
<213> 人工序列
<220>
<223> 在实验室中制备-低活性TCRα megaTAL RNA构建体
<400> 9
augggauccg cgccaccuaa gaagaaacgc aaagucgugg aucuacgcac gcucggcuac 60
agucagcagc agcaagagaa gaucaaaccg aaggugcguu cgacaguggc gcagcaccac 120
gaggcacugg ugggccaugg guuuacacac gcgcacaucg uugcgcucag ccaacacccg 180
gcagcguuag ggaccgucgc ugucacguau cagcacauaa ucacggcguu gccagaggcg 240
acacacgaag acaucguugg cgucggcaaa cagugguccg gcgcacgcgc ccuggaggcc 300
uugcucacgg augcggggga guugagaggu ccgccguuac aguuggacac aggccaacuu 360
gugaagauug caaaacgugg cggcgugacc gcaauggagg cagugcaugc aucgcgcaau 420
gcacugacgg gugccccccu gaaccugacc ccggaccaag ugguggcuau cgccagcaac 480
auuggcggca agcaagcgcu cgaaacggug cagcggcugu ugccggugcu gugccaggac 540
cauggccuga ccccggacca agugguggcu aucgccagca acauuggcgg caagcaagcg 600
cucgaaacgg ugcagcggcu guugccggug cugugccagg accauggccu gaccccggac 660
caaguggugg cuaucgccag caacauuggc ggcaagcaag cgcucgaaac ggugcagcgg 720
cuguugccgg ugcugugcca ggaccauggc cugaccccgg accaaguggu ggcuaucgcc 780
agcaacggug gcggcaagca agcgcucgaa acggugcagc ggcuguugcc ggugcugugc 840
caggaccaug gccugacucc ggaccaagug guggcuaucg ccagccacga uggcggcaag 900
caagcgcucg aaacggugca gcggcuguug ccggugcugu gccaggacca uggccugacu 960
ccggaccaag ugguggcuau cgccagccac gauggcggca agcaagcgcu cgaaacggug 1020
cagcggcugu ugccggugcu gugccaggac cauggccuga ccccggacca agugguggcu 1080
aucgccagca acauuggcgg caagcaagcg cucgaaacgg ugcagcggcu guugccggug 1140
cugugccagg accauggccu gaccccggac caaguggugg cuaucgccag caacaauggc 1200
ggcaagcaag cgcucgaaac ggugcagcgg cuguugccgg ugcugugcca ggaccauggc 1260
cugaccccgg accaaguggu ggcuaucgcc agcaacggug gcggcaagca agcgcucgaa 1320
acggugcagc ggcuguugcc ggugcugugc caggaccaug gccugacccc ggaccaagug 1380
guggcuaucg ccagcaacaa uggcggcaag caagcgcucg aaacggugca gcggcuguug 1440
ccggugcugu gccaggacca uggccugacc ccggaccaag ugguggcuau cgccagcaac 1500
auuggcggca agcaagcgcu cgaaaccgua caacgucucc ucccaguacu uugucaagac 1560
cacggguuga cuccggauca agucgucgcg aucgcgagcc augauggggg gaagcaggcg 1620
cuggaaagca uuguggccca gcugagccgg ccugauccgg cguuggccgc guugaccaac 1680
gaccaccucg ucgccuuggc cugccucggc ggacguccug ccauggaugc agugaaaaag 1740
ggauugccgc acgcgccgga auugaucaga agagucaauc gccguauugg cgaacgcacg 1800
ucccaucgcg uugcgauauc uagaguggga ggaagcucgc gcagagaguc caucaaccca 1860
uggauucuga cugguuucgc ugaugccgaa ggaucauuca uacuagacau ccgcaaccga 1920
aacaacgaaa gcaacagaua ccgaacuucg cugagauucc agaucacccu gcacaacaag 1980
gacaaaucga uucuggagaa uauccagucc acuuggaagg ucggcaagau cacaaacagc 2040
agugacagag ccgucaugcu gagggucacc cguuucgaag auuugaaagu gauuaucgac 2100
cacuucgaga aauauccgcu gauuacccag aaauugggcg auuacaaguu guuuaaacag 2160
gcauucagcg ucauggagaa uaaagaacau cuuaaggaga augggauuaa ggagcucgua 2220
cgaaucaaag cuaagaugaa uuggggucuc aaugacgaau ugaaaaaagc auuuccagag 2280
aacauuagca aagagcgccc ccuuaucaau aagaacauuc cgaauuucaa auggcuggcu 2340
ggauucacag cuggugaugg ccauuucggc gugaaucuaa aaaagguaaa gggcaccgca 2400
aagguauacg ugggacugag auucgcuauc ucacagcaca ucagagacaa gaaccugaug 2460
aauucauuga uaacauaccu aggcuguggu uccaucagag agaagaacaa gucugaguuc 2520
agauggcucg aguucgaagu aaccaaauuc agcgauauca acgacaagau cauuccggua 2580
uuccaggaaa auacucugau uggcgucaaa cucgaggacu uugaagauug gugcaagguu 2640
gccaaauuga ucgaagagaa gaaacaccug accgaauccg guuuggauga gauuaagaaa 2700
aucaagcuga acaugaacaa aggucguuga 2730
<210> 10
<211> 909
<212> PRT
<213> 人工序列
<220>
<223> 在实验室中制备-低活性TCRα megaTAL蛋白质构建体
<400> 10
Met Gly Ser Ala Pro Pro Lys Lys Lys Arg Lys Val Val Asp Leu Arg
1 5 10 15
Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys Val
20 25 30
Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val Gly His Gly Phe
35 40 45
Thr His Ala His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu Gly
50 55 60
Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr Ala Leu Pro Glu Ala
65 70 75 80
Thr His Glu Asp Ile Val Gly Val Gly Lys Gln Trp Ser Gly Ala Arg
85 90 95
Ala Leu Glu Ala Leu Leu Thr Asp Ala Gly Glu Leu Arg Gly Pro Pro
100 105 110
Leu Gln Leu Asp Thr Gly Gln Leu Val Lys Ile Ala Lys Arg Gly Gly
115 120 125
Val Thr Ala Met Glu Ala Val His Ala Ser Arg Asn Ala Leu Thr Gly
130 135 140
Ala Pro Leu Asn Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn
145 150 155 160
Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
165 170 175
Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala
180 185 190
Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
195 200 205
Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala
210 215 220
Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
225 230 235 240
Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val
245 250 255
Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val
260 265 270
Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp
275 280 285
Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu
290 295 300
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr
305 310 315 320
Pro Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala
325 330 335
Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly
340 345 350
Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys
355 360 365
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp
370 375 380
His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Asn Gly
385 390 395 400
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
405 410 415
Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn
420 425 430
Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
435 440 445
Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala
450 455 460
Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
465 470 475 480
Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala
485 490 495
Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
500 505 510
Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val
515 520 525
Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Ser Ile
530 535 540
Val Ala Gln Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu Thr Asn
545 550 555 560
Asp His Leu Val Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala Met Asp
565 570 575
Ala Val Lys Lys Gly Leu Pro His Ala Pro Glu Leu Ile Arg Arg Val
580 585 590
Asn Arg Arg Ile Gly Glu Arg Thr Ser His Arg Val Ala Ile Ser Arg
595 600 605
Val Gly Gly Ser Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr
610 615 620
Gly Phe Ala Asp Ala Glu Gly Ser Phe Ile Leu Asp Ile Arg Asn Arg
625 630 635 640
Asn Asn Glu Ser Asn Arg Tyr Arg Thr Ser Leu Arg Phe Gln Ile Thr
645 650 655
Leu His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp
660 665 670
Lys Val Gly Lys Ile Thr Asn Ser Ser Asp Arg Ala Val Met Leu Arg
675 680 685
Val Thr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys
690 695 700
Tyr Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln
705 710 715 720
Ala Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile
725 730 735
Lys Glu Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly Leu Asn Asp
740 745 750
Glu Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Pro Leu
755 760 765
Ile Asn Lys Asn Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe Thr Ala
770 775 780
Gly Asp Gly His Phe Gly Val Asn Leu Lys Lys Val Lys Gly Thr Ala
785 790 795 800
Lys Val Tyr Val Gly Leu Arg Phe Ala Ile Ser Gln His Ile Arg Asp
805 810 815
Lys Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Ser Ile
820 825 830
Arg Glu Lys Asn Lys Ser Glu Phe Arg Trp Leu Glu Phe Glu Val Thr
835 840 845
Lys Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn
850 855 860
Thr Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val
865 870 875 880
Ala Lys Leu Ile Glu Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp
885 890 895
Glu Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg
900 905
<210> 11
<211> 3372
<212> DNA
<213> 人工序列
<220>
<223> 在实验室中制备-低活性TCRα megaTAL-Trex2 DNA构建体
<400> 11
atgggatcct gcaggccacc taagaagaaa cgcaaagtcg tggatctacg cacgctcggc 60
tacagtcagc agcagcaaga gaagatcaaa ccgaaggtgc gttcgacagt ggcgcagcac 120
cacgaggcac tggtgggcca tgggtttaca cacgcgcaca tcgttgcgct cagccaacac 180
ccggcagcgt tagggaccgt cgctgtcacg tatcagcaca taatcacggc gttgccagag 240
gcgacacacg aagacatcgt tggcgtcggc aaacagtggt ccggcgcacg cgccctggag 300
gccttgctca cggatgcggg ggagttgaga ggtccgccgt tacagttgga cacaggccaa 360
cttgtgaaga ttgcaaaacg tggcggcgtg accgcaatgg aggcagtgca tgcatcgcgc 420
aatgcactga cgggtgcccc cctgaacctg accccggacc aagtggtggc tatcgccagc 480
aacattggcg gcaagcaagc gctcgaaacg gtgcagcggc tgttgccggt gctgtgccag 540
gaccatggcc tgaccccgga ccaagtggtg gctatcgcca gcaacattgg cggcaagcaa 600
gcgctcgaaa cggtgcagcg gctgttgccg gtgctgtgcc aggaccatgg cctgaccccg 660
gaccaagtgg tggctatcgc cagcaacatt ggcggcaagc aagcgctcga aacggtgcag 720
cggctgttgc cggtgctgtg ccaggaccat ggcctgaccc cggaccaagt ggtggctatc 780
gccagcaacg gtggcggcaa gcaagcgctc gaaacggtgc agcggctgtt gccggtgctg 840
tgccaggacc atggcctgac tccggaccaa gtggtggcta tcgccagcca cgatggcggc 900
aagcaagcgc tcgaaacggt gcagcggctg ttgccggtgc tgtgccagga ccatggcctg 960
actccggacc aagtggtggc tatcgccagc cacgatggcg gcaagcaagc gctcgaaacg 1020
gtgcagcggc tgttgccggt gctgtgccag gaccatggcc tgaccccgga ccaagtggtg 1080
gctatcgcca gcaacattgg cggcaagcaa gcgctcgaaa cggtgcagcg gctgttgccg 1140
gtgctgtgcc aggaccatgg cctgaccccg gaccaagtgg tggctatcgc cagcaacaat 1200
ggcggcaagc aagcgctcga aacggtgcag cggctgttgc cggtgctgtg ccaggaccat 1260
ggcctgaccc cggaccaagt ggtggctatc gccagcaacg gtggcggcaa gcaagcgctc 1320
gaaacggtgc agcggctgtt gccggtgctg tgccaggacc atggcctgac cccggaccaa 1380
gtggtggcta tcgccagcaa caatggcggc aagcaagcgc tcgaaacggt gcagcggctg 1440
ttgccggtgc tgtgccagga ccatggcctg accccggacc aagtggtggc tatcgccagc 1500
aacattggcg gcaagcaagc gctcgaaagc attgtggccc agctgagccg gcctgatccg 1560
gcgttggccg cgttgaccaa cgaccacctc gtcgccttgg cctgcctcgg cggacgtcct 1620
gccatggatg cagtgaaaaa gggattgccg cacgcgccgg aattgatcag aagagtcaat 1680
cgccgtattg gcgaacgcac gtcccatcgc gttgcgatat ctagagtggg aggaagctct 1740
cgcagagagt ccatcaaccc atggattctg actggtttcg ctgatgccga aggatcattc 1800
atactagaca tccgcaaccg aaacaacgaa agcaacagat accgaacttc gctgagattc 1860
cagatcaccc tgcacaacaa ggacaaatcg attctggaga atatccagtc cacttggaag 1920
gtcggcaaga tcacaaacag cagtgacaga gccgtcatgc tgagggtcac ccgtttcgaa 1980
gatttgaaag tgattatcga ccacttcgag aaatatccgc tgattaccca gaaattgggc 2040
gattacaagt tgtttaaaca ggcattcagc gtcatggaga ataaagaaca tcttaaggag 2100
aatgggatta aggagctcgt acgaatcaaa gctaagatga attggggtct caatgacgaa 2160
ttgaaaaaag catttccaga gaacattagc aaagagcgcc cccttatcaa taagaacatt 2220
ccgaatttca aatggctggc tggattcaca gctggtgaag gctacttcgg cgtgaatcta 2280
aaaaaggtaa agggcaccgc aaaggtatac gtgggactga gattctcaat ctcacagcac 2340
atcagagaca agaacctgat gaattcattg ataacatacc taggctgtgg ttccatctgg 2400
gagaagaaca agtctgagtt cagatggctc gagttcgtcg taaccaaatt cagcgatatc 2460
aacgacaaga tcattccggt attccaggaa aatactctga ttggcgtcaa actcgaggac 2520
tttgaagatt ggtgcaaggt tgccaaattg atcaaagaga agaaacacct gaccgaatcc 2580
ggtttggatg agattaagaa aatcaagctg aacatgaaca aaggtcgtgg tggaggcggt 2640
agcggaggcg gagggtcggc tagctccgaa gcaccgagag ccgaaacctt cgtgttcctc 2700
gacctggaag ccactggcct gcccagtgtg gagcccgaga ttgccgagct gtccctgttc 2760
gccgttcatc ggtcctccct ggaaaaccct gagcacgacg agagcggagc actggtgctg 2820
ccaagggtgc tggacaagct gaccctgtgc atgtgtcccg agcgtccctt cactgccaag 2880
gcatccgaaa tcactggtct gagttcggaa gggctggctc gttgtagaaa ggctggattc 2940
gatggtgccg tggttcggac tttgcaggca ttcctgtcca ggcaggcagg tccaatctgc 3000
ttggtcgccc acaacggttt tgactacgac ttcccactgc tctgtgccga actgcgtcgc 3060
ctgggagcac gccttcctcg ggataccgtg tgcctggaca ccctacccgc tctgcgtgga 3120
ctggacagag cccactcaca cggcactagg gcgagaggac gccagggtta cagcctcggt 3180
tcactgttcc atcggtattt cagggcagag ccttccgcag cccattcagc cgaaggcgat 3240
gtccacacct tgcttctgat cttcctgcat cgtgcagcag aactgctcgc ctgggcagat 3300
gagcaggctc gtggatgggc tcacattgag ccgatgtact tgccacctga tgaccctagc 3360
ctggaagcct ga 3372
<210> 12
<211> 3372
<212> RNA
<213> 人工序列
<220>
<223> 在实验室中制备-低活性TCRα megaTAL-Trex2 RNA构建体
<400> 12
augggauccu gcaggccacc uaagaagaaa cgcaaagucg uggaucuacg cacgcucggc 60
uacagucagc agcagcaaga gaagaucaaa ccgaaggugc guucgacagu ggcgcagcac 120
cacgaggcac uggugggcca uggguuuaca cacgcgcaca ucguugcgcu cagccaacac 180
ccggcagcgu uagggaccgu cgcugucacg uaucagcaca uaaucacggc guugccagag 240
gcgacacacg aagacaucgu uggcgucggc aaacaguggu ccggcgcacg cgcccuggag 300
gccuugcuca cggaugcggg ggaguugaga gguccgccgu uacaguugga cacaggccaa 360
cuugugaaga uugcaaaacg uggcggcgug accgcaaugg aggcagugca ugcaucgcgc 420
aaugcacuga cgggugcccc ccugaaccug accccggacc aagugguggc uaucgccagc 480
aacauuggcg gcaagcaagc gcucgaaacg gugcagcggc uguugccggu gcugugccag 540
gaccauggcc ugaccccgga ccaaguggug gcuaucgcca gcaacauugg cggcaagcaa 600
gcgcucgaaa cggugcagcg gcuguugccg gugcugugcc aggaccaugg ccugaccccg 660
gaccaagugg uggcuaucgc cagcaacauu ggcggcaagc aagcgcucga aacggugcag 720
cggcuguugc cggugcugug ccaggaccau ggccugaccc cggaccaagu gguggcuauc 780
gccagcaacg guggcggcaa gcaagcgcuc gaaacggugc agcggcuguu gccggugcug 840
ugccaggacc auggccugac uccggaccaa gugguggcua ucgccagcca cgauggcggc 900
aagcaagcgc ucgaaacggu gcagcggcug uugccggugc ugugccagga ccauggccug 960
acuccggacc aagugguggc uaucgccagc cacgauggcg gcaagcaagc gcucgaaacg 1020
gugcagcggc uguugccggu gcugugccag gaccauggcc ugaccccgga ccaaguggug 1080
gcuaucgcca gcaacauugg cggcaagcaa gcgcucgaaa cggugcagcg gcuguugccg 1140
gugcugugcc aggaccaugg ccugaccccg gaccaagugg uggcuaucgc cagcaacaau 1200
ggcggcaagc aagcgcucga aacggugcag cggcuguugc cggugcugug ccaggaccau 1260
ggccugaccc cggaccaagu gguggcuauc gccagcaacg guggcggcaa gcaagcgcuc 1320
gaaacggugc agcggcuguu gccggugcug ugccaggacc auggccugac cccggaccaa 1380
gugguggcua ucgccagcaa caauggcggc aagcaagcgc ucgaaacggu gcagcggcug 1440
uugccggugc ugugccagga ccauggccug accccggacc aagugguggc uaucgccagc 1500
aacauuggcg gcaagcaagc gcucgaaagc auuguggccc agcugagccg gccugauccg 1560
gcguuggccg cguugaccaa cgaccaccuc gucgccuugg ccugccucgg cggacguccu 1620
gccauggaug cagugaaaaa gggauugccg cacgcgccgg aauugaucag aagagucaau 1680
cgccguauug gcgaacgcac gucccaucgc guugcgauau cuagaguggg aggaagcucu 1740
cgcagagagu ccaucaaccc auggauucug acugguuucg cugaugccga aggaucauuc 1800
auacuagaca uccgcaaccg aaacaacgaa agcaacagau accgaacuuc gcugagauuc 1860
cagaucaccc ugcacaacaa ggacaaaucg auucuggaga auauccaguc cacuuggaag 1920
gucggcaaga ucacaaacag cagugacaga gccgucaugc ugagggucac ccguuucgaa 1980
gauuugaaag ugauuaucga ccacuucgag aaauauccgc ugauuaccca gaaauugggc 2040
gauuacaagu uguuuaaaca ggcauucagc gucauggaga auaaagaaca ucuuaaggag 2100
aaugggauua aggagcucgu acgaaucaaa gcuaagauga auuggggucu caaugacgaa 2160
uugaaaaaag cauuuccaga gaacauuagc aaagagcgcc cccuuaucaa uaagaacauu 2220
ccgaauuuca aauggcuggc uggauucaca gcuggugaag gcuacuucgg cgugaaucua 2280
aaaaagguaa agggcaccgc aaagguauac gugggacuga gauucucaau cucacagcac 2340
aucagagaca agaaccugau gaauucauug auaacauacc uaggcugugg uuccaucugg 2400
gagaagaaca agucugaguu cagauggcuc gaguucgucg uaaccaaauu cagcgauauc 2460
aacgacaaga ucauuccggu auuccaggaa aauacucuga uuggcgucaa acucgaggac 2520
uuugaagauu ggugcaaggu ugccaaauug aucaaagaga agaaacaccu gaccgaaucc 2580
gguuuggaug agauuaagaa aaucaagcug aacaugaaca aaggucgugg uggaggcggu 2640
agcggaggcg gagggucggc uagcuccgaa gcaccgagag ccgaaaccuu cguguuccuc 2700
gaccuggaag ccacuggccu gcccagugug gagcccgaga uugccgagcu gucccuguuc 2760
gccguucauc gguccucccu ggaaaacccu gagcacgacg agagcggagc acuggugcug 2820
ccaagggugc uggacaagcu gacccugugc augugucccg agcgucccuu cacugccaag 2880
gcauccgaaa ucacuggucu gaguucggaa gggcuggcuc guuguagaaa ggcuggauuc 2940
gauggugccg ugguucggac uuugcaggca uuccugucca ggcaggcagg uccaaucugc 3000
uuggucgccc acaacgguuu ugacuacgac uucccacugc ucugugccga acugcgucgc 3060
cugggagcac gccuuccucg ggauaccgug ugccuggaca cccuacccgc ucugcgugga 3120
cuggacagag cccacucaca cggcacuagg gcgagaggac gccaggguua cagccucggu 3180
ucacuguucc aucgguauuu cagggcagag ccuuccgcag cccauucagc cgaaggcgau 3240
guccacaccu ugcuucugau cuuccugcau cgugcagcag aacugcucgc cugggcagau 3300
gagcaggcuc guggaugggc ucacauugag ccgauguacu ugccaccuga ugacccuagc 3360
cuggaagccu ga 3372
<210> 13
<211> 1123
<212> PRT
<213> 人工序列
<220>
<223> 在实验室中制备-低活性TCRα megaTAL-Trex2蛋白质构建体
<400> 13
Met Gly Ser Cys Arg Pro Pro Lys Lys Lys Arg Lys Val Val Asp Leu
1 5 10 15
Arg Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys
20 25 30
Val Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val Gly His Gly
35 40 45
Phe Thr His Ala His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu
50 55 60
Gly Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr Ala Leu Pro Glu
65 70 75 80
Ala Thr His Glu Asp Ile Val Gly Val Gly Lys Gln Trp Ser Gly Ala
85 90 95
Arg Ala Leu Glu Ala Leu Leu Thr Asp Ala Gly Glu Leu Arg Gly Pro
100 105 110
Pro Leu Gln Leu Asp Thr Gly Gln Leu Val Lys Ile Ala Lys Arg Gly
115 120 125
Gly Val Thr Ala Met Glu Ala Val His Ala Ser Arg Asn Ala Leu Thr
130 135 140
Gly Ala Pro Leu Asn Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
145 150 155 160
Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
165 170 175
Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
180 185 190
Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
195 200 205
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val
210 215 220
Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
225 230 235 240
Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln
245 250 255
Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr
260 265 270
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro
275 280 285
Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu
290 295 300
Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu
305 310 315 320
Thr Pro Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln
325 330 335
Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His
340 345 350
Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly
355 360 365
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
370 375 380
Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Asn
385 390 395 400
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu
405 410 415
Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
420 425 430
Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
435 440 445
Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
450 455 460
Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
465 470 475 480
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val
485 490 495
Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Ser Ile Val
500 505 510
Ala Gln Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu Thr Asn Asp
515 520 525
His Leu Val Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala Met Asp Ala
530 535 540
Val Lys Lys Gly Leu Pro His Ala Pro Glu Leu Ile Arg Arg Val Asn
545 550 555 560
Arg Arg Ile Gly Glu Arg Thr Ser His Arg Val Ala Ile Ser Arg Val
565 570 575
Gly Gly Ser Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr Gly
580 585 590
Phe Ala Asp Ala Glu Gly Ser Phe Ile Leu Asp Ile Arg Asn Arg Asn
595 600 605
Asn Glu Ser Asn Arg Tyr Arg Thr Ser Leu Arg Phe Gln Ile Thr Leu
610 615 620
His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp Lys
625 630 635 640
Val Gly Lys Ile Thr Asn Ser Ser Asp Arg Ala Val Met Leu Arg Val
645 650 655
Thr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys Tyr
660 665 670
Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln Ala
675 680 685
Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile Lys
690 695 700
Glu Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly Leu Asn Asp Glu
705 710 715 720
Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Pro Leu Ile
725 730 735
Asn Lys Asn Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe Thr Ala Gly
740 745 750
Glu Gly Tyr Phe Gly Val Asn Leu Lys Lys Val Lys Gly Thr Ala Lys
755 760 765
Val Tyr Val Gly Leu Arg Phe Ser Ile Ser Gln His Ile Arg Asp Lys
770 775 780
Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Ser Ile Trp
785 790 795 800
Glu Lys Asn Lys Ser Glu Phe Arg Trp Leu Glu Phe Val Val Thr Lys
805 810 815
Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn Thr
820 825 830
Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val Ala
835 840 845
Lys Leu Ile Lys Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp Glu
850 855 860
Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Gly Gly Gly Gly
865 870 875 880
Ser Gly Gly Gly Gly Ser Ala Ser Ser Glu Ala Pro Arg Ala Glu Thr
885 890 895
Phe Val Phe Leu Asp Leu Glu Ala Thr Gly Leu Pro Ser Val Glu Pro
900 905 910
Glu Ile Ala Glu Leu Ser Leu Phe Ala Val His Arg Ser Ser Leu Glu
915 920 925
Asn Pro Glu His Asp Glu Ser Gly Ala Leu Val Leu Pro Arg Val Leu
930 935 940
Asp Lys Leu Thr Leu Cys Met Cys Pro Glu Arg Pro Phe Thr Ala Lys
945 950 955 960
Ala Ser Glu Ile Thr Gly Leu Ser Ser Glu Gly Leu Ala Arg Cys Arg
965 970 975
Lys Ala Gly Phe Asp Gly Ala Val Val Arg Thr Leu Gln Ala Phe Leu
980 985 990
Ser Arg Gln Ala Gly Pro Ile Cys Leu Val Ala His Asn Gly Phe Asp
995 1000 1005
Tyr Asp Phe Pro Leu Leu Cys Ala Glu Leu Arg Arg Leu Gly Ala
1010 1015 1020
Arg Leu Pro Arg Asp Thr Val Cys Leu Asp Thr Leu Pro Ala Leu
1025 1030 1035
Arg Gly Leu Asp Arg Ala His Ser His Gly Thr Arg Ala Arg Gly
1040 1045 1050
Arg Gln Gly Tyr Ser Leu Gly Ser Leu Phe His Arg Tyr Phe Arg
1055 1060 1065
Ala Glu Pro Ser Ala Ala His Ser Ala Glu Gly Asp Val His Thr
1070 1075 1080
Leu Leu Leu Ile Phe Leu His Arg Ala Ala Glu Leu Leu Ala Trp
1085 1090 1095
Ala Asp Glu Gln Ala Arg Gly Trp Ala His Ile Glu Pro Met Tyr
1100 1105 1110
Leu Pro Pro Asp Asp Pro Ser Leu Glu Ala
1115 1120
<210> 14
<211> 747
<212> DNA
<213> 智人(Homo sapiens)
<400> 14
atgggatcct gcaggccacc taagaagaaa cgcaaagtct ccgaagcacc gagagccgaa 60
accttcgtgt tcctcgacct ggaagccact ggcctgccca gtgtggagcc cgagattgcc 120
gagctgtccc tgttcgccgt tcatcggtcc tccctggaaa accctgagca cgacgagagc 180
ggagcactgg tgctgccaag ggtgctggac aagctgaccc tgtgcatgtg tcccgagcgt 240
cccttcactg ccaaggcatc cgaaatcact ggtctgagtt cggaagggct ggctcgttgt 300
agaaaggctg gattcgatgg tgccgtggtt cggactttgc aggcattcct gtccaggcag 360
gcaggtccaa tctgcttggt cgcccacaac ggttttgact acgacttccc actgctctgt 420
gccgaactgc gtcgcctggg agcacgcctt cctcgggata ccgtgtgcct ggacacccta 480
cccgctctgc gtggactgga cagagcccac tcacacggca ctagggcgag aggacgccag 540
ggttacagcc tcggttcact gttccatcgg tatttcaggg cagagccttc cgcagcccat 600
tcagccgaag gcgatgtcca caccttgctt ctgatcttcc tgcatcgtgc agcagaactg 660
ctcgcctggg cagatgagca ggctcgtgga tgggctcaca ttgagccgat gtacttgcca 720
cctgatgacc ctagcctgga agcctga 747
<210> 15
<211> 747
<212> RNA
<213> 智人(Homo sapiens)
<400> 15
augggauccu gcaggccacc uaagaagaaa cgcaaagucu ccgaagcacc gagagccgaa 60
accuucgugu uccucgaccu ggaagccacu ggccugccca guguggagcc cgagauugcc 120
gagcuguccc uguucgccgu ucaucggucc ucccuggaaa acccugagca cgacgagagc 180
ggagcacugg ugcugccaag ggugcuggac aagcugaccc ugugcaugug ucccgagcgu 240
cccuucacug ccaaggcauc cgaaaucacu ggucugaguu cggaagggcu ggcucguugu 300
agaaaggcug gauucgaugg ugccgugguu cggacuuugc aggcauuccu guccaggcag 360
gcagguccaa ucugcuuggu cgcccacaac gguuuugacu acgacuuccc acugcucugu 420
gccgaacugc gucgccuggg agcacgccuu ccucgggaua ccgugugccu ggacacccua 480
cccgcucugc guggacugga cagagcccac ucacacggca cuagggcgag aggacgccag 540
gguuacagcc ucgguucacu guuccaucgg uauuucaggg cagagccuuc cgcagcccau 600
ucagccgaag gcgaugucca caccuugcuu cugaucuucc ugcaucgugc agcagaacug 660
cucgccuggg cagaugagca ggcucgugga ugggcucaca uugagccgau guacuugcca 720
ccugaugacc cuagccugga agccuga 747
<210> 16
<211> 248
<212> PRT
<213> 智人(Homo sapiens)
<400> 16
Met Gly Ser Cys Arg Pro Pro Lys Lys Lys Arg Lys Val Ser Glu Ala
1 5 10 15
Pro Arg Ala Glu Thr Phe Val Phe Leu Asp Leu Glu Ala Thr Gly Leu
20 25 30
Pro Ser Val Glu Pro Glu Ile Ala Glu Leu Ser Leu Phe Ala Val His
35 40 45
Arg Ser Ser Leu Glu Asn Pro Glu His Asp Glu Ser Gly Ala Leu Val
50 55 60
Leu Pro Arg Val Leu Asp Lys Leu Thr Leu Cys Met Cys Pro Glu Arg
65 70 75 80
Pro Phe Thr Ala Lys Ala Ser Glu Ile Thr Gly Leu Ser Ser Glu Gly
85 90 95
Leu Ala Arg Cys Arg Lys Ala Gly Phe Asp Gly Ala Val Val Arg Thr
100 105 110
Leu Gln Ala Phe Leu Ser Arg Gln Ala Gly Pro Ile Cys Leu Val Ala
115 120 125
His Asn Gly Phe Asp Tyr Asp Phe Pro Leu Leu Cys Ala Glu Leu Arg
130 135 140
Arg Leu Gly Ala Arg Leu Pro Arg Asp Thr Val Cys Leu Asp Thr Leu
145 150 155 160
Pro Ala Leu Arg Gly Leu Asp Arg Ala His Ser His Gly Thr Arg Ala
165 170 175
Arg Gly Arg Gln Gly Tyr Ser Leu Gly Ser Leu Phe His Arg Tyr Phe
180 185 190
Arg Ala Glu Pro Ser Ala Ala His Ser Ala Glu Gly Asp Val His Thr
195 200 205
Leu Leu Leu Ile Phe Leu His Arg Ala Ala Glu Leu Leu Ala Trp Ala
210 215 220
Asp Glu Gln Ala Arg Gly Trp Ala His Ile Glu Pro Met Tyr Leu Pro
225 230 235 240
Pro Asp Asp Pro Ser Leu Glu Ala
245
<210> 17
<211> 3384
<212> DNA
<213> 人工序列
<220>
<223> 在实验室中制备-低活性TCRα megaTAL-Trex2鸭嘴兽DNA构建体
<400> 17
atgggatcct gcaggccacc taagaagaaa cgcaaagtcg tggatctacg cacgctcggc 60
tacagtcagc agcagcaaga gaagatcaaa ccgaaggtgc gttcgacagt ggcgcagcac 120
cacgaggcac tggtgggcca tgggtttaca cacgcgcaca tcgttgcgct cagccaacac 180
ccggcagcgt tagggaccgt cgctgtcacg tatcagcaca taatcacggc gttgccagag 240
gcgacacacg aagacatcgt tggcgtcggc aaacagtggt ccggcgcacg cgccctggag 300
gccttgctca cggatgcggg ggagttgaga ggtccgccgt tacagttgga cacaggccaa 360
cttgtgaaga ttgcaaaacg tggcggcgtg accgcaatgg aggcagtgca tgcatcgcgc 420
aatgcactga cgggtgcccc cctgaacctg accccggacc aagtggtggc tatcgccagc 480
aacattggcg gcaagcaagc gctcgaaacg gtgcagcggc tgttgccggt gctgtgccag 540
gaccatggcc tgaccccgga ccaagtggtg gctatcgcca gcaacattgg cggcaagcaa 600
gcgctcgaaa cggtgcagcg gctgttgccg gtgctgtgcc aggaccatgg cctgaccccg 660
gaccaagtgg tggctatcgc cagcaacatt ggcggcaagc aagcgctcga aacggtgcag 720
cggctgttgc cggtgctgtg ccaggaccat ggcctgaccc cggaccaagt ggtggctatc 780
gccagcaacg gtggcggcaa gcaagcgctc gaaacggtgc agcggctgtt gccggtgctg 840
tgccaggacc atggcctgac tccggaccaa gtggtggcta tcgccagcca cgatggcggc 900
aagcaagcgc tcgaaacggt gcagcggctg ttgccggtgc tgtgccagga ccatggcctg 960
actccggacc aagtggtggc tatcgccagc cacgatggcg gcaagcaagc gctcgaaacg 1020
gtgcagcggc tgttgccggt gctgtgccag gaccatggcc tgaccccgga ccaagtggtg 1080
gctatcgcca gcaacattgg cggcaagcaa gcgctcgaaa cggtgcagcg gctgttgccg 1140
gtgctgtgcc aggaccatgg cctgaccccg gaccaagtgg tggctatcgc cagcaacaat 1200
ggcggcaagc aagcgctcga aacggtgcag cggctgttgc cggtgctgtg ccaggaccat 1260
ggcctgaccc cggaccaagt ggtggctatc gccagcaacg gtggcggcaa gcaagcgctc 1320
gaaacggtgc agcggctgtt gccggtgctg tgccaggacc atggcctgac cccggaccaa 1380
gtggtggcta tcgccagcaa caatggcggc aagcaagcgc tcgaaacggt gcagcggctg 1440
ttgccggtgc tgtgccagga ccatggcctg accccggacc aagtggtggc tatcgccagc 1500
aacattggcg gcaagcaagc gctcgaaagc attgtggccc agctgagccg gcctgatccg 1560
gcgttggccg cgttgaccaa cgaccacctc gtcgccttgg cctgcctcgg cggacgtcct 1620
gccatggatg cagtgaaaaa gggattgccg cacgcgccgg aattgatcag aagagtcaat 1680
cgccgtattg gcgaacgcac gtcccatcgc gttgcgatat ctagagtggg aggaagctct 1740
cgcagagagt ccatcaaccc atggattctg actggtttcg ctgatgccga aggatcattc 1800
atactagaca tccgcaaccg aaacaacgaa agcaacagat accgaacttc gctgagattc 1860
cagatcaccc tgcacaacaa ggacaaatcg attctggaga atatccagtc cacttggaag 1920
gtcggcaaga tcacaaacag cagtgacaga gccgtcatgc tgagggtcac ccgtttcgaa 1980
gatttgaaag tgattatcga ccacttcgag aaatatccgc tgattaccca gaaattgggc 2040
gattacaagt tgtttaaaca ggcattcagc gtcatggaga ataaagaaca tcttaaggag 2100
aatgggatta aggagctcgt acgaatcaaa gctaagatga attggggtct caatgacgaa 2160
ttgaaaaaag catttccaga gaacattagc aaagagcgcc cccttatcaa taagaacatt 2220
ccgaatttca aatggctggc tggattcaca gctggtgaag gctacttcgg cgtgaatcta 2280
aaaaaggtaa agggcaccgc aaaggtatac gtgggactga gattctcaat ctcacagcac 2340
atcagagaca agaacctgat gaattcattg ataacatacc taggctgtgg ttccatctgg 2400
gagaagaaca agtctgagtt cagatggctc gagttcgtcg taaccaaatt cagcgatatc 2460
aacgacaaga tcattccggt attccaggaa aatactctga ttggcgtcaa actcgaggac 2520
tttgaagatt ggtgcaaggt tgccaaattg atcaaagaga agaaacacct gaccgaatcc 2580
ggtttggatg agattaagaa aatcaagctg aacatgaaca aaggtcgtgg tggaggcggt 2640
agcggaggcg gagggtcggc tagctccgaa tcacagaaat gcgaaacctt cgtgttcctc 2700
gacctggaag ccactggcct gcccgatgcg tctccccaga ttgccgaaat ctccctgttc 2760
gccgttcatc ggttctccct ggaacaccct gcgtgggacg atagcggagt accgctgctg 2820
ccaagggtgc tggacaagct gaccctgtgc atgaatcccg agcaaccctt cactcccaag 2880
gcagccaaga tcactggtct gaataacaga tccctgactg ataatagaaa ggcttgtttc 2940
gatgggaccg tggttaaggc tttgcgggag ttcctgtcca ggcaggcaag tccaatctgc 3000
ttggtcgccc acaacggttt tgcctacgac ttcccactgc tcaggaccga actgggtcgc 3060
ctggatgcaa accttcctga gaatacctat tgcctggaca ccctacccgc tctgaaagga 3120
ctggacaaag cccacgatca cggcactagg tcgaatcatg gcaagtctta ccgcctcggt 3180
gatctctaca ggcagtattt cggtgacgat cctaaagcag cccattcagc cgaaggcgat 3240
gtctacacct tggttatggt cttcctgcat cgggcaccag aactgctccg ttgggcaggt 3300
gcggaggctc gttcatggga tgagattagg ccgatgtact ccccaggtcc tcgtagatca 3360
ggtggcagaa ggcatgatgc ctga 3384
<210> 18
<211> 3384
<212> RNA
<213> 人工序列
<220>
<223> 在实验室中制备-低活性TCRα megaTAL-Trex2鸭嘴兽RNA构建体
<400> 18
augggauccu gcaggccacc uaagaagaaa cgcaaagucg uggaucuacg cacgcucggc 60
uacagucagc agcagcaaga gaagaucaaa ccgaaggugc guucgacagu ggcgcagcac 120
cacgaggcac uggugggcca uggguuuaca cacgcgcaca ucguugcgcu cagccaacac 180
ccggcagcgu uagggaccgu cgcugucacg uaucagcaca uaaucacggc guugccagag 240
gcgacacacg aagacaucgu uggcgucggc aaacaguggu ccggcgcacg cgcccuggag 300
gccuugcuca cggaugcggg ggaguugaga gguccgccgu uacaguugga cacaggccaa 360
cuugugaaga uugcaaaacg uggcggcgug accgcaaugg aggcagugca ugcaucgcgc 420
aaugcacuga cgggugcccc ccugaaccug accccggacc aagugguggc uaucgccagc 480
aacauuggcg gcaagcaagc gcucgaaacg gugcagcggc uguugccggu gcugugccag 540
gaccauggcc ugaccccgga ccaaguggug gcuaucgcca gcaacauugg cggcaagcaa 600
gcgcucgaaa cggugcagcg gcuguugccg gugcugugcc aggaccaugg ccugaccccg 660
gaccaagugg uggcuaucgc cagcaacauu ggcggcaagc aagcgcucga aacggugcag 720
cggcuguugc cggugcugug ccaggaccau ggccugaccc cggaccaagu gguggcuauc 780
gccagcaacg guggcggcaa gcaagcgcuc gaaacggugc agcggcuguu gccggugcug 840
ugccaggacc auggccugac uccggaccaa gugguggcua ucgccagcca cgauggcggc 900
aagcaagcgc ucgaaacggu gcagcggcug uugccggugc ugugccagga ccauggccug 960
acuccggacc aagugguggc uaucgccagc cacgauggcg gcaagcaagc gcucgaaacg 1020
gugcagcggc uguugccggu gcugugccag gaccauggcc ugaccccgga ccaaguggug 1080
gcuaucgcca gcaacauugg cggcaagcaa gcgcucgaaa cggugcagcg gcuguugccg 1140
gugcugugcc aggaccaugg ccugaccccg gaccaagugg uggcuaucgc cagcaacaau 1200
ggcggcaagc aagcgcucga aacggugcag cggcuguugc cggugcugug ccaggaccau 1260
ggccugaccc cggaccaagu gguggcuauc gccagcaacg guggcggcaa gcaagcgcuc 1320
gaaacggugc agcggcuguu gccggugcug ugccaggacc auggccugac cccggaccaa 1380
gugguggcua ucgccagcaa caauggcggc aagcaagcgc ucgaaacggu gcagcggcug 1440
uugccggugc ugugccagga ccauggccug accccggacc aagugguggc uaucgccagc 1500
aacauuggcg gcaagcaagc gcucgaaagc auuguggccc agcugagccg gccugauccg 1560
gcguuggccg cguugaccaa cgaccaccuc gucgccuugg ccugccucgg cggacguccu 1620
gccauggaug cagugaaaaa gggauugccg cacgcgccgg aauugaucag aagagucaau 1680
cgccguauug gcgaacgcac gucccaucgc guugcgauau cuagaguggg aggaagcucu 1740
cgcagagagu ccaucaaccc auggauucug acugguuucg cugaugccga aggaucauuc 1800
auacuagaca uccgcaaccg aaacaacgaa agcaacagau accgaacuuc gcugagauuc 1860
cagaucaccc ugcacaacaa ggacaaaucg auucuggaga auauccaguc cacuuggaag 1920
gucggcaaga ucacaaacag cagugacaga gccgucaugc ugagggucac ccguuucgaa 1980
gauuugaaag ugauuaucga ccacuucgag aaauauccgc ugauuaccca gaaauugggc 2040
gauuacaagu uguuuaaaca ggcauucagc gucauggaga auaaagaaca ucuuaaggag 2100
aaugggauua aggagcucgu acgaaucaaa gcuaagauga auuggggucu caaugacgaa 2160
uugaaaaaag cauuuccaga gaacauuagc aaagagcgcc cccuuaucaa uaagaacauu 2220
ccgaauuuca aauggcuggc uggauucaca gcuggugaag gcuacuucgg cgugaaucua 2280
aaaaagguaa agggcaccgc aaagguauac gugggacuga gauucucaau cucacagcac 2340
aucagagaca agaaccugau gaauucauug auaacauacc uaggcugugg uuccaucugg 2400
gagaagaaca agucugaguu cagauggcuc gaguucgucg uaaccaaauu cagcgauauc 2460
aacgacaaga ucauuccggu auuccaggaa aauacucuga uuggcgucaa acucgaggac 2520
uuugaagauu ggugcaaggu ugccaaauug aucaaagaga agaaacaccu gaccgaaucc 2580
gguuuggaug agauuaagaa aaucaagcug aacaugaaca aaggucgugg uggaggcggu 2640
agcggaggcg gagggucggc uagcuccgaa ucacagaaau gcgaaaccuu cguguuccuc 2700
gaccuggaag ccacuggccu gcccgaugcg ucuccccaga uugccgaaau cucccuguuc 2760
gccguucauc gguucucccu ggaacacccu gcgugggacg auagcggagu accgcugcug 2820
ccaagggugc uggacaagcu gacccugugc augaaucccg agcaacccuu cacucccaag 2880
gcagccaaga ucacuggucu gaauaacaga ucccugacug auaauagaaa ggcuuguuuc 2940
gaugggaccg ugguuaaggc uuugcgggag uuccugucca ggcaggcaag uccaaucugc 3000
uuggucgccc acaacgguuu ugccuacgac uucccacugc ucaggaccga acugggucgc 3060
cuggaugcaa accuuccuga gaauaccuau ugccuggaca cccuacccgc ucugaaagga 3120
cuggacaaag cccacgauca cggcacuagg ucgaaucaug gcaagucuua ccgccucggu 3180
gaucucuaca ggcaguauuu cggugacgau ccuaaagcag cccauucagc cgaaggcgau 3240
gucuacaccu ugguuauggu cuuccugcau cgggcaccag aacugcuccg uugggcaggu 3300
gcggaggcuc guucauggga ugagauuagg ccgauguacu ccccaggucc ucguagauca 3360
gguggcagaa ggcaugaugc cuga 3384
<210> 19
<211> 1127
<212> PRT
<213> 人工序列
<220>
<223> 在实验室中制备-低活性TCRα megaTAL-Trex2鸭嘴兽蛋白质构建体
<400> 19
Met Gly Ser Cys Arg Pro Pro Lys Lys Lys Arg Lys Val Val Asp Leu
1 5 10 15
Arg Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys
20 25 30
Val Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val Gly His Gly
35 40 45
Phe Thr His Ala His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu
50 55 60
Gly Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr Ala Leu Pro Glu
65 70 75 80
Ala Thr His Glu Asp Ile Val Gly Val Gly Lys Gln Trp Ser Gly Ala
85 90 95
Arg Ala Leu Glu Ala Leu Leu Thr Asp Ala Gly Glu Leu Arg Gly Pro
100 105 110
Pro Leu Gln Leu Asp Thr Gly Gln Leu Val Lys Ile Ala Lys Arg Gly
115 120 125
Gly Val Thr Ala Met Glu Ala Val His Ala Ser Arg Asn Ala Leu Thr
130 135 140
Gly Ala Pro Leu Asn Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
145 150 155 160
Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
165 170 175
Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
180 185 190
Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
195 200 205
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val
210 215 220
Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
225 230 235 240
Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln
245 250 255
Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr
260 265 270
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro
275 280 285
Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu
290 295 300
Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu
305 310 315 320
Thr Pro Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln
325 330 335
Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His
340 345 350
Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly
355 360 365
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
370 375 380
Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Asn
385 390 395 400
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu
405 410 415
Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
420 425 430
Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
435 440 445
Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
450 455 460
Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
465 470 475 480
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val
485 490 495
Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Ser Ile Val
500 505 510
Ala Gln Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu Thr Asn Asp
515 520 525
His Leu Val Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala Met Asp Ala
530 535 540
Val Lys Lys Gly Leu Pro His Ala Pro Glu Leu Ile Arg Arg Val Asn
545 550 555 560
Arg Arg Ile Gly Glu Arg Thr Ser His Arg Val Ala Ile Ser Arg Val
565 570 575
Gly Gly Ser Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr Gly
580 585 590
Phe Ala Asp Ala Glu Gly Ser Phe Ile Leu Asp Ile Arg Asn Arg Asn
595 600 605
Asn Glu Ser Asn Arg Tyr Arg Thr Ser Leu Arg Phe Gln Ile Thr Leu
610 615 620
His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp Lys
625 630 635 640
Val Gly Lys Ile Thr Asn Ser Ser Asp Arg Ala Val Met Leu Arg Val
645 650 655
Thr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys Tyr
660 665 670
Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln Ala
675 680 685
Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile Lys
690 695 700
Glu Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly Leu Asn Asp Glu
705 710 715 720
Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Pro Leu Ile
725 730 735
Asn Lys Asn Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe Thr Ala Gly
740 745 750
Glu Gly Tyr Phe Gly Val Asn Leu Lys Lys Val Lys Gly Thr Ala Lys
755 760 765
Val Tyr Val Gly Leu Arg Phe Ser Ile Ser Gln His Ile Arg Asp Lys
770 775 780
Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Ser Ile Trp
785 790 795 800
Glu Lys Asn Lys Ser Glu Phe Arg Trp Leu Glu Phe Val Val Thr Lys
805 810 815
Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn Thr
820 825 830
Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val Ala
835 840 845
Lys Leu Ile Lys Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp Glu
850 855 860
Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Gly Gly Gly Gly
865 870 875 880
Ser Gly Gly Gly Gly Ser Ala Ser Ser Glu Ser Gln Lys Cys Glu Thr
885 890 895
Phe Val Phe Leu Asp Leu Glu Ala Thr Gly Leu Pro Asp Ala Ser Pro
900 905 910
Gln Ile Ala Glu Ile Ser Leu Phe Ala Val His Arg Phe Ser Leu Glu
915 920 925
His Pro Ala Trp Asp Asp Ser Gly Val Pro Leu Leu Pro Arg Val Leu
930 935 940
Asp Lys Leu Thr Leu Cys Met Asn Pro Glu Gln Pro Phe Thr Pro Lys
945 950 955 960
Ala Ala Lys Ile Thr Gly Leu Asn Asn Arg Ser Leu Thr Asp Asn Arg
965 970 975
Lys Ala Cys Phe Asp Gly Thr Val Val Lys Ala Leu Arg Glu Phe Leu
980 985 990
Ser Arg Gln Ala Ser Pro Ile Cys Leu Val Ala His Asn Gly Phe Ala
995 1000 1005
Tyr Asp Phe Pro Leu Leu Arg Thr Glu Leu Gly Arg Leu Asp Ala
1010 1015 1020
Asn Leu Pro Glu Asn Thr Tyr Cys Leu Asp Thr Leu Pro Ala Leu
1025 1030 1035
Lys Gly Leu Asp Lys Ala His Asp His Gly Thr Arg Ser Asn His
1040 1045 1050
Gly Lys Ser Tyr Arg Leu Gly Asp Leu Tyr Arg Gln Tyr Phe Gly
1055 1060 1065
Asp Asp Pro Lys Ala Ala His Ser Ala Glu Gly Asp Val Tyr Thr
1070 1075 1080
Leu Val Met Val Phe Leu His Arg Ala Pro Glu Leu Leu Arg Trp
1085 1090 1095
Ala Gly Ala Glu Ala Arg Ser Trp Asp Glu Ile Arg Pro Met Tyr
1100 1105 1110
Ser Pro Gly Pro Arg Arg Ser Gly Gly Arg Arg His Asp Ala
1115 1120 1125
<210> 20
<211> 3387
<212> DNA
<213> 人工序列
<220>
<223> 在实验室中制备-低活性TCRα megaTAL-Trex2负鼠DNA构建体
<400> 20
atgggatcct gcaggccacc taagaagaaa cgcaaagtcg tggatctacg cacgctcggc 60
tacagtcagc agcagcaaga gaagatcaaa ccgaaggtgc gttcgacagt ggcgcagcac 120
cacgaggcac tggtgggcca tgggtttaca cacgcgcaca tcgttgcgct cagccaacac 180
ccggcagcgt tagggaccgt cgctgtcacg tatcagcaca taatcacggc gttgccagag 240
gcgacacacg aagacatcgt tggcgtcggc aaacagtggt ccggcgcacg cgccctggag 300
gccttgctca cggatgcggg ggagttgaga ggtccgccgt tacagttgga cacaggccaa 360
cttgtgaaga ttgcaaaacg tggcggcgtg accgcaatgg aggcagtgca tgcatcgcgc 420
aatgcactga cgggtgcccc cctgaacctg accccggacc aagtggtggc tatcgccagc 480
aacattggcg gcaagcaagc gctcgaaacg gtgcagcggc tgttgccggt gctgtgccag 540
gaccatggcc tgaccccgga ccaagtggtg gctatcgcca gcaacattgg cggcaagcaa 600
gcgctcgaaa cggtgcagcg gctgttgccg gtgctgtgcc aggaccatgg cctgaccccg 660
gaccaagtgg tggctatcgc cagcaacatt ggcggcaagc aagcgctcga aacggtgcag 720
cggctgttgc cggtgctgtg ccaggaccat ggcctgaccc cggaccaagt ggtggctatc 780
gccagcaacg gtggcggcaa gcaagcgctc gaaacggtgc agcggctgtt gccggtgctg 840
tgccaggacc atggcctgac tccggaccaa gtggtggcta tcgccagcca cgatggcggc 900
aagcaagcgc tcgaaacggt gcagcggctg ttgccggtgc tgtgccagga ccatggcctg 960
actccggacc aagtggtggc tatcgccagc cacgatggcg gcaagcaagc gctcgaaacg 1020
gtgcagcggc tgttgccggt gctgtgccag gaccatggcc tgaccccgga ccaagtggtg 1080
gctatcgcca gcaacattgg cggcaagcaa gcgctcgaaa cggtgcagcg gctgttgccg 1140
gtgctgtgcc aggaccatgg cctgaccccg gaccaagtgg tggctatcgc cagcaacaat 1200
ggcggcaagc aagcgctcga aacggtgcag cggctgttgc cggtgctgtg ccaggaccat 1260
ggcctgaccc cggaccaagt ggtggctatc gccagcaacg gtggcggcaa gcaagcgctc 1320
gaaacggtgc agcggctgtt gccggtgctg tgccaggacc atggcctgac cccggaccaa 1380
gtggtggcta tcgccagcaa caatggcggc aagcaagcgc tcgaaacggt gcagcggctg 1440
ttgccggtgc tgtgccagga ccatggcctg accccggacc aagtggtggc tatcgccagc 1500
aacattggcg gcaagcaagc gctcgaaagc attgtggccc agctgagccg gcctgatccg 1560
gcgttggccg cgttgaccaa cgaccacctc gtcgccttgg cctgcctcgg cggacgtcct 1620
gccatggatg cagtgaaaaa gggattgccg cacgcgccgg aattgatcag aagagtcaat 1680
cgccgtattg gcgaacgcac gtcccatcgc gttgcgatat ctagagtggg aggaagctct 1740
cgcagagagt ccatcaaccc atggattctg actggtttcg ctgatgccga aggatcattc 1800
atactagaca tccgcaaccg aaacaacgaa agcaacagat accgaacttc gctgagattc 1860
cagatcaccc tgcacaacaa ggacaaatcg attctggaga atatccagtc cacttggaag 1920
gtcggcaaga tcacaaacag cagtgacaga gccgtcatgc tgagggtcac ccgtttcgaa 1980
gatttgaaag tgattatcga ccacttcgag aaatatccgc tgattaccca gaaattgggc 2040
gattacaagt tgtttaaaca ggcattcagc gtcatggaga ataaagaaca tcttaaggag 2100
aatgggatta aggagctcgt acgaatcaaa gctaagatga attggggtct caatgacgaa 2160
ttgaaaaaag catttccaga gaacattagc aaagagcgcc cccttatcaa taagaacatt 2220
ccgaatttca aatggctggc tggattcaca gctggtgaag gctacttcgg cgtgaatcta 2280
aaaaaggtaa agggcaccgc aaaggtatac gtgggactga gattctcaat ctcacagcac 2340
atcagagaca agaacctgat gaattcattg ataacatacc taggctgtgg ttccatctgg 2400
gagaagaaca agtctgagtt cagatggctc gagttcgtcg taaccaaatt cagcgatatc 2460
aacgacaaga tcattccggt attccaggaa aatactctga ttggcgtcaa actcgaggac 2520
tttgaagatt ggtgcaaggt tgccaaattg atcaaagaga agaaacacct gaccgaatcc 2580
ggtttggatg agattaagaa aatcaagctg aacatgaaca aaggtcgtgg tggaggcggt 2640
agcggaggcg gagggtcggc tagcgccgaa ttaccgacat gcgaaacctt cgtgttcctc 2700
gacctggaag ccactggcct gcccaatgct catcccgaga ttgccgaaat ctccctgttc 2760
gccattcatc ggttctccct ggaacaccct gagagagacg agagcggaac acttcaactg 2820
ccaagggtgc tggacaagct gaccctgtgc atgtgtcccg agcagaactt cactcccaag 2880
gcatccgaaa tcactggtct gagtaatcag aacctggctg ataatcacaa ggctggattc 2940
aatggtgccg tgattcgggc tttgcgggag ttcctgaaga ggcagaaaag tccaatctgc 3000
ttggtcgccc acaacggttt tgactacgac ttcccactgc tcaggaccga acttcaacgc 3060
ctgggagcag accttcctgg aggaaccgtg tgcctggaca ccctacccgc tctgcgtgga 3120
ctggacaagg tccaccatca cagcactagg gcgtctcatg gaaagaagtc ttacagcctc 3180
ggtaatctct acaggcggta tttcgatgac gagcctcaag cagcccattc agccgaaggc 3240
gatgtctacg ccttggttat ggtcttcctg catcgggcac aagaactgct ccaatgcgta 3300
cagacggagg ctcagaattg ggatgagatt cagccgatgt ataccccacg taagcgtaga 3360
ctgtccggcg gtagcctgga tgcctga 3387
<210> 21
<211> 3387
<212> RNA
<213> 人工序列
<220>
<223> 在实验室中制备-低活性TCRα megaTAL-Trex2负鼠RNA构建体
<400> 21
augggauccu gcaggccacc uaagaagaaa cgcaaagucg uggaucuacg cacgcucggc 60
uacagucagc agcagcaaga gaagaucaaa ccgaaggugc guucgacagu ggcgcagcac 120
cacgaggcac uggugggcca uggguuuaca cacgcgcaca ucguugcgcu cagccaacac 180
ccggcagcgu uagggaccgu cgcugucacg uaucagcaca uaaucacggc guugccagag 240
gcgacacacg aagacaucgu uggcgucggc aaacaguggu ccggcgcacg cgcccuggag 300
gccuugcuca cggaugcggg ggaguugaga gguccgccgu uacaguugga cacaggccaa 360
cuugugaaga uugcaaaacg uggcggcgug accgcaaugg aggcagugca ugcaucgcgc 420
aaugcacuga cgggugcccc ccugaaccug accccggacc aagugguggc uaucgccagc 480
aacauuggcg gcaagcaagc gcucgaaacg gugcagcggc uguugccggu gcugugccag 540
gaccauggcc ugaccccgga ccaaguggug gcuaucgcca gcaacauugg cggcaagcaa 600
gcgcucgaaa cggugcagcg gcuguugccg gugcugugcc aggaccaugg ccugaccccg 660
gaccaagugg uggcuaucgc cagcaacauu ggcggcaagc aagcgcucga aacggugcag 720
cggcuguugc cggugcugug ccaggaccau ggccugaccc cggaccaagu gguggcuauc 780
gccagcaacg guggcggcaa gcaagcgcuc gaaacggugc agcggcuguu gccggugcug 840
ugccaggacc auggccugac uccggaccaa gugguggcua ucgccagcca cgauggcggc 900
aagcaagcgc ucgaaacggu gcagcggcug uugccggugc ugugccagga ccauggccug 960
acuccggacc aagugguggc uaucgccagc cacgauggcg gcaagcaagc gcucgaaacg 1020
gugcagcggc uguugccggu gcugugccag gaccauggcc ugaccccgga ccaaguggug 1080
gcuaucgcca gcaacauugg cggcaagcaa gcgcucgaaa cggugcagcg gcuguugccg 1140
gugcugugcc aggaccaugg ccugaccccg gaccaagugg uggcuaucgc cagcaacaau 1200
ggcggcaagc aagcgcucga aacggugcag cggcuguugc cggugcugug ccaggaccau 1260
ggccugaccc cggaccaagu gguggcuauc gccagcaacg guggcggcaa gcaagcgcuc 1320
gaaacggugc agcggcuguu gccggugcug ugccaggacc auggccugac cccggaccaa 1380
gugguggcua ucgccagcaa caauggcggc aagcaagcgc ucgaaacggu gcagcggcug 1440
uugccggugc ugugccagga ccauggccug accccggacc aagugguggc uaucgccagc 1500
aacauuggcg gcaagcaagc gcucgaaagc auuguggccc agcugagccg gccugauccg 1560
gcguuggccg cguugaccaa cgaccaccuc gucgccuugg ccugccucgg cggacguccu 1620
gccauggaug cagugaaaaa gggauugccg cacgcgccgg aauugaucag aagagucaau 1680
cgccguauug gcgaacgcac gucccaucgc guugcgauau cuagaguggg aggaagcucu 1740
cgcagagagu ccaucaaccc auggauucug acugguuucg cugaugccga aggaucauuc 1800
auacuagaca uccgcaaccg aaacaacgaa agcaacagau accgaacuuc gcugagauuc 1860
cagaucaccc ugcacaacaa ggacaaaucg auucuggaga auauccaguc cacuuggaag 1920
gucggcaaga ucacaaacag cagugacaga gccgucaugc ugagggucac ccguuucgaa 1980
gauuugaaag ugauuaucga ccacuucgag aaauauccgc ugauuaccca gaaauugggc 2040
gauuacaagu uguuuaaaca ggcauucagc gucauggaga auaaagaaca ucuuaaggag 2100
aaugggauua aggagcucgu acgaaucaaa gcuaagauga auuggggucu caaugacgaa 2160
uugaaaaaag cauuuccaga gaacauuagc aaagagcgcc cccuuaucaa uaagaacauu 2220
ccgaauuuca aauggcuggc uggauucaca gcuggugaag gcuacuucgg cgugaaucua 2280
aaaaagguaa agggcaccgc aaagguauac gugggacuga gauucucaau cucacagcac 2340
aucagagaca agaaccugau gaauucauug auaacauacc uaggcugugg uuccaucugg 2400
gagaagaaca agucugaguu cagauggcuc gaguucgucg uaaccaaauu cagcgauauc 2460
aacgacaaga ucauuccggu auuccaggaa aauacucuga uuggcgucaa acucgaggac 2520
uuugaagauu ggugcaaggu ugccaaauug aucaaagaga agaaacaccu gaccgaaucc 2580
gguuuggaug agauuaagaa aaucaagcug aacaugaaca aaggucgugg uggaggcggu 2640
agcggaggcg gagggucggc uagcgccgaa uuaccgacau gcgaaaccuu cguguuccuc 2700
gaccuggaag ccacuggccu gcccaaugcu caucccgaga uugccgaaau cucccuguuc 2760
gccauucauc gguucucccu ggaacacccu gagagagacg agagcggaac acuucaacug 2820
ccaagggugc uggacaagcu gacccugugc augugucccg agcagaacuu cacucccaag 2880
gcauccgaaa ucacuggucu gaguaaucag aaccuggcug auaaucacaa ggcuggauuc 2940
aauggugccg ugauucgggc uuugcgggag uuccugaaga ggcagaaaag uccaaucugc 3000
uuggucgccc acaacgguuu ugacuacgac uucccacugc ucaggaccga acuucaacgc 3060
cugggagcag accuuccugg aggaaccgug ugccuggaca cccuacccgc ucugcgugga 3120
cuggacaagg uccaccauca cagcacuagg gcgucucaug gaaagaaguc uuacagccuc 3180
gguaaucucu acaggcggua uuucgaugac gagccucaag cagcccauuc agccgaaggc 3240
gaugucuacg ccuugguuau ggucuuccug caucgggcac aagaacugcu ccaaugcgua 3300
cagacggagg cucagaauug ggaugagauu cagccgaugu auaccccacg uaagcguaga 3360
cuguccggcg guagccugga ugccuga 3387
<210> 22
<211> 1128
<212> PRT
<213> 人工序列
<220>
<223> 在实验室中制备-低活性TCRα megaTAL-Trex2负鼠蛋白质构建体
<400> 22
Met Gly Ser Cys Arg Pro Pro Lys Lys Lys Arg Lys Val Val Asp Leu
1 5 10 15
Arg Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys
20 25 30
Val Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val Gly His Gly
35 40 45
Phe Thr His Ala His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu
50 55 60
Gly Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr Ala Leu Pro Glu
65 70 75 80
Ala Thr His Glu Asp Ile Val Gly Val Gly Lys Gln Trp Ser Gly Ala
85 90 95
Arg Ala Leu Glu Ala Leu Leu Thr Asp Ala Gly Glu Leu Arg Gly Pro
100 105 110
Pro Leu Gln Leu Asp Thr Gly Gln Leu Val Lys Ile Ala Lys Arg Gly
115 120 125
Gly Val Thr Ala Met Glu Ala Val His Ala Ser Arg Asn Ala Leu Thr
130 135 140
Gly Ala Pro Leu Asn Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
145 150 155 160
Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
165 170 175
Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
180 185 190
Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
195 200 205
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val
210 215 220
Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
225 230 235 240
Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln
245 250 255
Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr
260 265 270
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro
275 280 285
Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu
290 295 300
Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu
305 310 315 320
Thr Pro Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln
325 330 335
Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His
340 345 350
Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly
355 360 365
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
370 375 380
Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Asn
385 390 395 400
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu
405 410 415
Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
420 425 430
Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
435 440 445
Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
450 455 460
Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
465 470 475 480
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val
485 490 495
Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Ser Ile Val
500 505 510
Ala Gln Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu Thr Asn Asp
515 520 525
His Leu Val Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala Met Asp Ala
530 535 540
Val Lys Lys Gly Leu Pro His Ala Pro Glu Leu Ile Arg Arg Val Asn
545 550 555 560
Arg Arg Ile Gly Glu Arg Thr Ser His Arg Val Ala Ile Ser Arg Val
565 570 575
Gly Gly Ser Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr Gly
580 585 590
Phe Ala Asp Ala Glu Gly Ser Phe Ile Leu Asp Ile Arg Asn Arg Asn
595 600 605
Asn Glu Ser Asn Arg Tyr Arg Thr Ser Leu Arg Phe Gln Ile Thr Leu
610 615 620
His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp Lys
625 630 635 640
Val Gly Lys Ile Thr Asn Ser Ser Asp Arg Ala Val Met Leu Arg Val
645 650 655
Thr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys Tyr
660 665 670
Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln Ala
675 680 685
Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile Lys
690 695 700
Glu Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly Leu Asn Asp Glu
705 710 715 720
Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Pro Leu Ile
725 730 735
Asn Lys Asn Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe Thr Ala Gly
740 745 750
Glu Gly Tyr Phe Gly Val Asn Leu Lys Lys Val Lys Gly Thr Ala Lys
755 760 765
Val Tyr Val Gly Leu Arg Phe Ser Ile Ser Gln His Ile Arg Asp Lys
770 775 780
Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Ser Ile Trp
785 790 795 800
Glu Lys Asn Lys Ser Glu Phe Arg Trp Leu Glu Phe Val Val Thr Lys
805 810 815
Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn Thr
820 825 830
Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val Ala
835 840 845
Lys Leu Ile Lys Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp Glu
850 855 860
Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Gly Gly Gly Gly
865 870 875 880
Ser Gly Gly Gly Gly Ser Ala Ser Ala Glu Leu Pro Thr Cys Glu Thr
885 890 895
Phe Val Phe Leu Asp Leu Glu Ala Thr Gly Leu Pro Asn Ala His Pro
900 905 910
Glu Ile Ala Glu Ile Ser Leu Phe Ala Ile His Arg Phe Ser Leu Glu
915 920 925
His Pro Glu Arg Asp Glu Ser Gly Thr Leu Gln Leu Pro Arg Val Leu
930 935 940
Asp Lys Leu Thr Leu Cys Met Cys Pro Glu Gln Asn Phe Thr Pro Lys
945 950 955 960
Ala Ser Glu Ile Thr Gly Leu Ser Asn Gln Asn Leu Ala Asp Asn His
965 970 975
Lys Ala Gly Phe Asn Gly Ala Val Ile Arg Ala Leu Arg Glu Phe Leu
980 985 990
Lys Arg Gln Lys Ser Pro Ile Cys Leu Val Ala His Asn Gly Phe Asp
995 1000 1005
Tyr Asp Phe Pro Leu Leu Arg Thr Glu Leu Gln Arg Leu Gly Ala
1010 1015 1020
Asp Leu Pro Gly Gly Thr Val Cys Leu Asp Thr Leu Pro Ala Leu
1025 1030 1035
Arg Gly Leu Asp Lys Val His His His Ser Thr Arg Ala Ser His
1040 1045 1050
Gly Lys Lys Ser Tyr Ser Leu Gly Asn Leu Tyr Arg Arg Tyr Phe
1055 1060 1065
Asp Asp Glu Pro Gln Ala Ala His Ser Ala Glu Gly Asp Val Tyr
1070 1075 1080
Ala Leu Val Met Val Phe Leu His Arg Ala Gln Glu Leu Leu Gln
1085 1090 1095
Cys Val Gln Thr Glu Ala Gln Asn Trp Asp Glu Ile Gln Pro Met
1100 1105 1110
Tyr Thr Pro Arg Lys Arg Arg Leu Ser Gly Gly Ser Leu Asp Ala
1115 1120 1125
<210> 23
<211> 3372
<212> DNA
<213> 人工序列
<220>
<223> 在实验室中制备-低活性TCRα megaTAL-Trex2绵羊DNA构建体
<400> 23
atgggatcct gcaggccacc taagaagaaa cgcaaagtcg tggatctacg cacgctcggc 60
tacagtcagc agcagcaaga gaagatcaaa ccgaaggtgc gttcgacagt ggcgcagcac 120
cacgaggcac tggtgggcca tgggtttaca cacgcgcaca tcgttgcgct cagccaacac 180
ccggcagcgt tagggaccgt cgctgtcacg tatcagcaca taatcacggc gttgccagag 240
gcgacacacg aagacatcgt tggcgtcggc aaacagtggt ccggcgcacg cgccctggag 300
gccttgctca cggatgcggg ggagttgaga ggtccgccgt tacagttgga cacaggccaa 360
cttgtgaaga ttgcaaaacg tggcggcgtg accgcaatgg aggcagtgca tgcatcgcgc 420
aatgcactga cgggtgcccc cctgaacctg accccggacc aagtggtggc tatcgccagc 480
aacattggcg gcaagcaagc gctcgaaacg gtgcagcggc tgttgccggt gctgtgccag 540
gaccatggcc tgaccccgga ccaagtggtg gctatcgcca gcaacattgg cggcaagcaa 600
gcgctcgaaa cggtgcagcg gctgttgccg gtgctgtgcc aggaccatgg cctgaccccg 660
gaccaagtgg tggctatcgc cagcaacatt ggcggcaagc aagcgctcga aacggtgcag 720
cggctgttgc cggtgctgtg ccaggaccat ggcctgaccc cggaccaagt ggtggctatc 780
gccagcaacg gtggcggcaa gcaagcgctc gaaacggtgc agcggctgtt gccggtgctg 840
tgccaggacc atggcctgac tccggaccaa gtggtggcta tcgccagcca cgatggcggc 900
aagcaagcgc tcgaaacggt gcagcggctg ttgccggtgc tgtgccagga ccatggcctg 960
actccggacc aagtggtggc tatcgccagc cacgatggcg gcaagcaagc gctcgaaacg 1020
gtgcagcggc tgttgccggt gctgtgccag gaccatggcc tgaccccgga ccaagtggtg 1080
gctatcgcca gcaacattgg cggcaagcaa gcgctcgaaa cggtgcagcg gctgttgccg 1140
gtgctgtgcc aggaccatgg cctgaccccg gaccaagtgg tggctatcgc cagcaacaat 1200
ggcggcaagc aagcgctcga aacggtgcag cggctgttgc cggtgctgtg ccaggaccat 1260
ggcctgaccc cggaccaagt ggtggctatc gccagcaacg gtggcggcaa gcaagcgctc 1320
gaaacggtgc agcggctgtt gccggtgctg tgccaggacc atggcctgac cccggaccaa 1380
gtggtggcta tcgccagcaa caatggcggc aagcaagcgc tcgaaacggt gcagcggctg 1440
ttgccggtgc tgtgccagga ccatggcctg accccggacc aagtggtggc tatcgccagc 1500
aacattggcg gcaagcaagc gctcgaaagc attgtggccc agctgagccg gcctgatccg 1560
gcgttggccg cgttgaccaa cgaccacctc gtcgccttgg cctgcctcgg cggacgtcct 1620
gccatggatg cagtgaaaaa gggattgccg cacgcgccgg aattgatcag aagagtcaat 1680
cgccgtattg gcgaacgcac gtcccatcgc gttgcgatat ctagagtggg aggaagctct 1740
cgcagagagt ccatcaaccc atggattctg actggtttcg ctgatgccga aggatcattc 1800
atactagaca tccgcaaccg aaacaacgaa agcaacagat accgaacttc gctgagattc 1860
cagatcaccc tgcacaacaa ggacaaatcg attctggaga atatccagtc cacttggaag 1920
gtcggcaaga tcacaaacag cagtgacaga gccgtcatgc tgagggtcac ccgtttcgaa 1980
gatttgaaag tgattatcga ccacttcgag aaatatccgc tgattaccca gaaattgggc 2040
gattacaagt tgtttaaaca ggcattcagc gtcatggaga ataaagaaca tcttaaggag 2100
aatgggatta aggagctcgt acgaatcaaa gctaagatga attggggtct caatgacgaa 2160
ttgaaaaaag catttccaga gaacattagc aaagagcgcc cccttatcaa taagaacatt 2220
ccgaatttca aatggctggc tggattcaca gctggtgaag gctacttcgg cgtgaatcta 2280
aaaaaggtaa agggcaccgc aaaggtatac gtgggactga gattctcaat ctcacagcac 2340
atcagagaca agaacctgat gaattcattg ataacatacc taggctgtgg ttccatctgg 2400
gagaagaaca agtctgagtt cagatggctc gagttcgtcg taaccaaatt cagcgatatc 2460
aacgacaaga tcattccggt attccaggaa aatactctga ttggcgtcaa actcgaggac 2520
tttgaagatt ggtgcaaggt tgccaaattg atcaaagaga agaaacacct gaccgaatcc 2580
ggtttggatg agattaagaa aatcaagctg aacatgaaca aaggtcgtgg tggaggcggt 2640
agcggaggcg gagggtcggc tagctccaaa ggaccgagag ccaagacctt cgtgttcctc 2700
gacctggaag ccactggcct gcccagtgtt gatcccgaga ttgccgagct gtccctgttc 2760
gccgttcatc ggtcctccct ggaagaccct gagtgcgacg agagcggatc accgctgccg 2820
ccaagggtga tggacaagct gaccctgtgc atgtctccca aatacccctt cactgccaag 2880
gcatccgaaa tcactggtct gagtagggaa gggctgggtc gttgtggaaa ggctggattc 2940
gatgggaccg tggttcggac tttgcaggca ttcctgtcca ggcaggaagg tccagtctgc 3000
ttggtcgccc acaacggttt taactacgac ttcccactgc tctgtaccga actgcgtcgc 3060
ctgggagcac accttcctca agatacaatt tgcctggata ccctagtcgc tcttcaagat 3120
ctggacagag cccacggaca cggcactagg gcgcaaggag gcaagtctta cagcctcggt 3180
tcactgttct gttgctattt ccaggcagag ccttccgcag cccattcagc cgaaggcgat 3240
gtccacacct tgcttatggt cttcctgcat cgtgcagcag aactgctcgc ctgggcagat 3300
gagcaggctc tttcatgggc tcacgttgag ccgatgtacg tgccacctga tgacccaagg 3360
ctggaagact ga 3372
<210> 24
<211> 3372
<212> RNA
<213> 人工序列
<220>
<223> 在实验室中制备-低活性TCRα megaTAL-Trex2绵羊RNA构建体
<400> 24
augggauccu gcaggccacc uaagaagaaa cgcaaagucg uggaucuacg cacgcucggc 60
uacagucagc agcagcaaga gaagaucaaa ccgaaggugc guucgacagu ggcgcagcac 120
cacgaggcac uggugggcca uggguuuaca cacgcgcaca ucguugcgcu cagccaacac 180
ccggcagcgu uagggaccgu cgcugucacg uaucagcaca uaaucacggc guugccagag 240
gcgacacacg aagacaucgu uggcgucggc aaacaguggu ccggcgcacg cgcccuggag 300
gccuugcuca cggaugcggg ggaguugaga gguccgccgu uacaguugga cacaggccaa 360
cuugugaaga uugcaaaacg uggcggcgug accgcaaugg aggcagugca ugcaucgcgc 420
aaugcacuga cgggugcccc ccugaaccug accccggacc aagugguggc uaucgccagc 480
aacauuggcg gcaagcaagc gcucgaaacg gugcagcggc uguugccggu gcugugccag 540
gaccauggcc ugaccccgga ccaaguggug gcuaucgcca gcaacauugg cggcaagcaa 600
gcgcucgaaa cggugcagcg gcuguugccg gugcugugcc aggaccaugg ccugaccccg 660
gaccaagugg uggcuaucgc cagcaacauu ggcggcaagc aagcgcucga aacggugcag 720
cggcuguugc cggugcugug ccaggaccau ggccugaccc cggaccaagu gguggcuauc 780
gccagcaacg guggcggcaa gcaagcgcuc gaaacggugc agcggcuguu gccggugcug 840
ugccaggacc auggccugac uccggaccaa gugguggcua ucgccagcca cgauggcggc 900
aagcaagcgc ucgaaacggu gcagcggcug uugccggugc ugugccagga ccauggccug 960
acuccggacc aagugguggc uaucgccagc cacgauggcg gcaagcaagc gcucgaaacg 1020
gugcagcggc uguugccggu gcugugccag gaccauggcc ugaccccgga ccaaguggug 1080
gcuaucgcca gcaacauugg cggcaagcaa gcgcucgaaa cggugcagcg gcuguugccg 1140
gugcugugcc aggaccaugg ccugaccccg gaccaagugg uggcuaucgc cagcaacaau 1200
ggcggcaagc aagcgcucga aacggugcag cggcuguugc cggugcugug ccaggaccau 1260
ggccugaccc cggaccaagu gguggcuauc gccagcaacg guggcggcaa gcaagcgcuc 1320
gaaacggugc agcggcuguu gccggugcug ugccaggacc auggccugac cccggaccaa 1380
gugguggcua ucgccagcaa caauggcggc aagcaagcgc ucgaaacggu gcagcggcug 1440
uugccggugc ugugccagga ccauggccug accccggacc aagugguggc uaucgccagc 1500
aacauuggcg gcaagcaagc gcucgaaagc auuguggccc agcugagccg gccugauccg 1560
gcguuggccg cguugaccaa cgaccaccuc gucgccuugg ccugccucgg cggacguccu 1620
gccauggaug cagugaaaaa gggauugccg cacgcgccgg aauugaucag aagagucaau 1680
cgccguauug gcgaacgcac gucccaucgc guugcgauau cuagaguggg aggaagcucu 1740
cgcagagagu ccaucaaccc auggauucug acugguuucg cugaugccga aggaucauuc 1800
auacuagaca uccgcaaccg aaacaacgaa agcaacagau accgaacuuc gcugagauuc 1860
cagaucaccc ugcacaacaa ggacaaaucg auucuggaga auauccaguc cacuuggaag 1920
gucggcaaga ucacaaacag cagugacaga gccgucaugc ugagggucac ccguuucgaa 1980
gauuugaaag ugauuaucga ccacuucgag aaauauccgc ugauuaccca gaaauugggc 2040
gauuacaagu uguuuaaaca ggcauucagc gucauggaga auaaagaaca ucuuaaggag 2100
aaugggauua aggagcucgu acgaaucaaa gcuaagauga auuggggucu caaugacgaa 2160
uugaaaaaag cauuuccaga gaacauuagc aaagagcgcc cccuuaucaa uaagaacauu 2220
ccgaauuuca aauggcuggc uggauucaca gcuggugaag gcuacuucgg cgugaaucua 2280
aaaaagguaa agggcaccgc aaagguauac gugggacuga gauucucaau cucacagcac 2340
aucagagaca agaaccugau gaauucauug auaacauacc uaggcugugg uuccaucugg 2400
gagaagaaca agucugaguu cagauggcuc gaguucgucg uaaccaaauu cagcgauauc 2460
aacgacaaga ucauuccggu auuccaggaa aauacucuga uuggcgucaa acucgaggac 2520
uuugaagauu ggugcaaggu ugccaaauug aucaaagaga agaaacaccu gaccgaaucc 2580
gguuuggaug agauuaagaa aaucaagcug aacaugaaca aaggucgugg uggaggcggu 2640
agcggaggcg gagggucggc uagcuccaaa ggaccgagag ccaagaccuu cguguuccuc 2700
gaccuggaag ccacuggccu gcccaguguu gaucccgaga uugccgagcu gucccuguuc 2760
gccguucauc gguccucccu ggaagacccu gagugcgacg agagcggauc accgcugccg 2820
ccaaggguga uggacaagcu gacccugugc augucuccca aauaccccuu cacugccaag 2880
gcauccgaaa ucacuggucu gaguagggaa gggcuggguc guuguggaaa ggcuggauuc 2940
gaugggaccg ugguucggac uuugcaggca uuccugucca ggcaggaagg uccagucugc 3000
uuggucgccc acaacgguuu uaacuacgac uucccacugc ucuguaccga acugcgucgc 3060
cugggagcac accuuccuca agauacaauu ugccuggaua cccuagucgc ucuucaagau 3120
cuggacagag cccacggaca cggcacuagg gcgcaaggag gcaagucuua cagccucggu 3180
ucacuguucu guugcuauuu ccaggcagag ccuuccgcag cccauucagc cgaaggcgau 3240
guccacaccu ugcuuauggu cuuccugcau cgugcagcag aacugcucgc cugggcagau 3300
gagcaggcuc uuucaugggc ucacguugag ccgauguacg ugccaccuga ugacccaagg 3360
cuggaagacu ga 3372
<210> 25
<211> 1123
<212> PRT
<213> 人工序列
<220>
<223> 在实验室中制备-低活性TCRα megaTAL-Trex2绵羊蛋白质构建体
<400> 25
Met Gly Ser Cys Arg Pro Pro Lys Lys Lys Arg Lys Val Val Asp Leu
1 5 10 15
Arg Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys
20 25 30
Val Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val Gly His Gly
35 40 45
Phe Thr His Ala His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu
50 55 60
Gly Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr Ala Leu Pro Glu
65 70 75 80
Ala Thr His Glu Asp Ile Val Gly Val Gly Lys Gln Trp Ser Gly Ala
85 90 95
Arg Ala Leu Glu Ala Leu Leu Thr Asp Ala Gly Glu Leu Arg Gly Pro
100 105 110
Pro Leu Gln Leu Asp Thr Gly Gln Leu Val Lys Ile Ala Lys Arg Gly
115 120 125
Gly Val Thr Ala Met Glu Ala Val His Ala Ser Arg Asn Ala Leu Thr
130 135 140
Gly Ala Pro Leu Asn Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
145 150 155 160
Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
165 170 175
Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
180 185 190
Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
195 200 205
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val
210 215 220
Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
225 230 235 240
Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln
245 250 255
Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr
260 265 270
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro
275 280 285
Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu
290 295 300
Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu
305 310 315 320
Thr Pro Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln
325 330 335
Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His
340 345 350
Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly
355 360 365
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
370 375 380
Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Asn
385 390 395 400
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu
405 410 415
Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
420 425 430
Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
435 440 445
Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
450 455 460
Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
465 470 475 480
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val
485 490 495
Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Ser Ile Val
500 505 510
Ala Gln Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu Thr Asn Asp
515 520 525
His Leu Val Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala Met Asp Ala
530 535 540
Val Lys Lys Gly Leu Pro His Ala Pro Glu Leu Ile Arg Arg Val Asn
545 550 555 560
Arg Arg Ile Gly Glu Arg Thr Ser His Arg Val Ala Ile Ser Arg Val
565 570 575
Gly Gly Ser Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr Gly
580 585 590
Phe Ala Asp Ala Glu Gly Ser Phe Ile Leu Asp Ile Arg Asn Arg Asn
595 600 605
Asn Glu Ser Asn Arg Tyr Arg Thr Ser Leu Arg Phe Gln Ile Thr Leu
610 615 620
His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp Lys
625 630 635 640
Val Gly Lys Ile Thr Asn Ser Ser Asp Arg Ala Val Met Leu Arg Val
645 650 655
Thr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys Tyr
660 665 670
Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln Ala
675 680 685
Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile Lys
690 695 700
Glu Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly Leu Asn Asp Glu
705 710 715 720
Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Pro Leu Ile
725 730 735
Asn Lys Asn Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe Thr Ala Gly
740 745 750
Glu Gly Tyr Phe Gly Val Asn Leu Lys Lys Val Lys Gly Thr Ala Lys
755 760 765
Val Tyr Val Gly Leu Arg Phe Ser Ile Ser Gln His Ile Arg Asp Lys
770 775 780
Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Ser Ile Trp
785 790 795 800
Glu Lys Asn Lys Ser Glu Phe Arg Trp Leu Glu Phe Val Val Thr Lys
805 810 815
Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn Thr
820 825 830
Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val Ala
835 840 845
Lys Leu Ile Lys Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp Glu
850 855 860
Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Gly Gly Gly Gly
865 870 875 880
Ser Gly Gly Gly Gly Ser Ala Ser Ser Lys Gly Pro Arg Ala Lys Thr
885 890 895
Phe Val Phe Leu Asp Leu Glu Ala Thr Gly Leu Pro Ser Val Asp Pro
900 905 910
Glu Ile Ala Glu Leu Ser Leu Phe Ala Val His Arg Ser Ser Leu Glu
915 920 925
Asp Pro Glu Cys Asp Glu Ser Gly Ser Pro Leu Pro Pro Arg Val Met
930 935 940
Asp Lys Leu Thr Leu Cys Met Ser Pro Lys Tyr Pro Phe Thr Ala Lys
945 950 955 960
Ala Ser Glu Ile Thr Gly Leu Ser Arg Glu Gly Leu Gly Arg Cys Gly
965 970 975
Lys Ala Gly Phe Asp Gly Thr Val Val Arg Thr Leu Gln Ala Phe Leu
980 985 990
Ser Arg Gln Glu Gly Pro Val Cys Leu Val Ala His Asn Gly Phe Asn
995 1000 1005
Tyr Asp Phe Pro Leu Leu Cys Thr Glu Leu Arg Arg Leu Gly Ala
1010 1015 1020
His Leu Pro Gln Asp Thr Ile Cys Leu Asp Thr Leu Val Ala Leu
1025 1030 1035
Gln Asp Leu Asp Arg Ala His Gly His Gly Thr Arg Ala Gln Gly
1040 1045 1050
Gly Lys Ser Tyr Ser Leu Gly Ser Leu Phe Cys Cys Tyr Phe Gln
1055 1060 1065
Ala Glu Pro Ser Ala Ala His Ser Ala Glu Gly Asp Val His Thr
1070 1075 1080
Leu Leu Met Val Phe Leu His Arg Ala Ala Glu Leu Leu Ala Trp
1085 1090 1095
Ala Asp Glu Gln Ala Leu Ser Trp Ala His Val Glu Pro Met Tyr
1100 1105 1110
Val Pro Pro Asp Asp Pro Arg Leu Glu Asp
1115 1120
<210> 26
<211> 3372
<212> DNA
<213> 人工序列
<220>
<223> 在实验室中制备-低活性TCRα megaTAL-Trex2犰狳DNA构建体
<400> 26
atgggatcct gcaggccacc taagaagaaa cgcaaagtcg tggatctacg cacgctcggc 60
tacagtcagc agcagcaaga gaagatcaaa ccgaaggtgc gttcgacagt ggcgcagcac 120
cacgaggcac tggtgggcca tgggtttaca cacgcgcaca tcgttgcgct cagccaacac 180
ccggcagcgt tagggaccgt cgctgtcacg tatcagcaca taatcacggc gttgccagag 240
gcgacacacg aagacatcgt tggcgtcggc aaacagtggt ccggcgcacg cgccctggag 300
gccttgctca cggatgcggg ggagttgaga ggtccgccgt tacagttgga cacaggccaa 360
cttgtgaaga ttgcaaaacg tggcggcgtg accgcaatgg aggcagtgca tgcatcgcgc 420
aatgcactga cgggtgcccc cctgaacctg accccggacc aagtggtggc tatcgccagc 480
aacattggcg gcaagcaagc gctcgaaacg gtgcagcggc tgttgccggt gctgtgccag 540
gaccatggcc tgaccccgga ccaagtggtg gctatcgcca gcaacattgg cggcaagcaa 600
gcgctcgaaa cggtgcagcg gctgttgccg gtgctgtgcc aggaccatgg cctgaccccg 660
gaccaagtgg tggctatcgc cagcaacatt ggcggcaagc aagcgctcga aacggtgcag 720
cggctgttgc cggtgctgtg ccaggaccat ggcctgaccc cggaccaagt ggtggctatc 780
gccagcaacg gtggcggcaa gcaagcgctc gaaacggtgc agcggctgtt gccggtgctg 840
tgccaggacc atggcctgac tccggaccaa gtggtggcta tcgccagcca cgatggcggc 900
aagcaagcgc tcgaaacggt gcagcggctg ttgccggtgc tgtgccagga ccatggcctg 960
actccggacc aagtggtggc tatcgccagc cacgatggcg gcaagcaagc gctcgaaacg 1020
gtgcagcggc tgttgccggt gctgtgccag gaccatggcc tgaccccgga ccaagtggtg 1080
gctatcgcca gcaacattgg cggcaagcaa gcgctcgaaa cggtgcagcg gctgttgccg 1140
gtgctgtgcc aggaccatgg cctgaccccg gaccaagtgg tggctatcgc cagcaacaat 1200
ggcggcaagc aagcgctcga aacggtgcag cggctgttgc cggtgctgtg ccaggaccat 1260
ggcctgaccc cggaccaagt ggtggctatc gccagcaacg gtggcggcaa gcaagcgctc 1320
gaaacggtgc agcggctgtt gccggtgctg tgccaggacc atggcctgac cccggaccaa 1380
gtggtggcta tcgccagcaa caatggcggc aagcaagcgc tcgaaacggt gcagcggctg 1440
ttgccggtgc tgtgccagga ccatggcctg accccggacc aagtggtggc tatcgccagc 1500
aacattggcg gcaagcaagc gctcgaaagc attgtggccc agctgagccg gcctgatccg 1560
gcgttggccg cgttgaccaa cgaccacctc gtcgccttgg cctgcctcgg cggacgtcct 1620
gccatggatg cagtgaaaaa gggattgccg cacgcgccgg aattgatcag aagagtcaat 1680
cgccgtattg gcgaacgcac gtcccatcgc gttgcgatat ctagagtggg aggaagctct 1740
cgcagagagt ccatcaaccc atggattctg actggtttcg ctgatgccga aggatcattc 1800
atactagaca tccgcaaccg aaacaacgaa agcaacagat accgaacttc gctgagattc 1860
cagatcaccc tgcacaacaa ggacaaatcg attctggaga atatccagtc cacttggaag 1920
gtcggcaaga tcacaaacag cagtgacaga gccgtcatgc tgagggtcac ccgtttcgaa 1980
gatttgaaag tgattatcga ccacttcgag aaatatccgc tgattaccca gaaattgggc 2040
gattacaagt tgtttaaaca ggcattcagc gtcatggaga ataaagaaca tcttaaggag 2100
aatgggatta aggagctcgt acgaatcaaa gctaagatga attggggtct caatgacgaa 2160
ttgaaaaaag catttccaga gaacattagc aaagagcgcc cccttatcaa taagaacatt 2220
ccgaatttca aatggctggc tggattcaca gctggtgaag gctacttcgg cgtgaatcta 2280
aaaaaggtaa agggcaccgc aaaggtatac gtgggactga gattctcaat ctcacagcac 2340
atcagagaca agaacctgat gaattcattg ataacatacc taggctgtgg ttccatctgg 2400
gagaagaaca agtctgagtt cagatggctc gagttcgtcg taaccaaatt cagcgatatc 2460
aacgacaaga tcattccggt attccaggaa aatactctga ttggcgtcaa actcgaggac 2520
tttgaagatt ggtgcaaggt tgccaaattg atcaaagaga agaaacacct gaccgaatcc 2580
ggtttggatg agattaagaa aatcaagctg aacatgaaca aaggtcgtgg tggaggcggt 2640
agcggaggcg gagggtcggc tagctcccaa gcaccgcaag ccgaaacctt cgtgttcctc 2700
gacctggaag ccactggcct gcccaatgtt gatcccgaga ttgccgaaat ctccctgttc 2760
gccgttcatc ggtcctccct ggaacaccct gagagagacg aggccggagt acctgtgctg 2820
ccaagggtgc tggacaagct gaccctgtgc atgtctccca ctcgtccctt cactgccaag 2880
gcatccgaaa tcactggtct gagttcggaa gggctggctc gttgtggaaa ggctggattc 2940
gatgatactg cggctcagac tttgcgggca ttcctgggca ggcaggcagg tccactctgc 3000
ttggtcgccc acaacggttt tgactacgac ttcccactgc tcaggaccga acttcaacgc 3060
ctgggagcac gccttccagc gcataccgtg tgcctggaca ccctacccgc tctgcgtgga 3120
ctggacagag cccactcaca cgctactagg gcgcaaggac gcaagggtta cagcctcggt 3180
tcactgttca ggcggtattt ccaggcagag ccttccgcag cccattcagc cgaaggcgat 3240
gtccgcacct tgcttctggt cttcctgcat cgtgcagcag aactgctcgc ctgggcagat 3300
gggcgggctc gttcatgggc tcacattgcg ccgatgtacg cgccaccgga ggcccctagc 3360
ccggaagcct ga 3372
<210> 27
<211> 3372
<212> RNA
<213> 人工序列
<220>
<223> 在实验室中制备-低活性TCRα megaTAL-Trex2犰狳RNA构建体
<400> 27
augggauccu gcaggccacc uaagaagaaa cgcaaagucg uggaucuacg cacgcucggc 60
uacagucagc agcagcaaga gaagaucaaa ccgaaggugc guucgacagu ggcgcagcac 120
cacgaggcac uggugggcca uggguuuaca cacgcgcaca ucguugcgcu cagccaacac 180
ccggcagcgu uagggaccgu cgcugucacg uaucagcaca uaaucacggc guugccagag 240
gcgacacacg aagacaucgu uggcgucggc aaacaguggu ccggcgcacg cgcccuggag 300
gccuugcuca cggaugcggg ggaguugaga gguccgccgu uacaguugga cacaggccaa 360
cuugugaaga uugcaaaacg uggcggcgug accgcaaugg aggcagugca ugcaucgcgc 420
aaugcacuga cgggugcccc ccugaaccug accccggacc aagugguggc uaucgccagc 480
aacauuggcg gcaagcaagc gcucgaaacg gugcagcggc uguugccggu gcugugccag 540
gaccauggcc ugaccccgga ccaaguggug gcuaucgcca gcaacauugg cggcaagcaa 600
gcgcucgaaa cggugcagcg gcuguugccg gugcugugcc aggaccaugg ccugaccccg 660
gaccaagugg uggcuaucgc cagcaacauu ggcggcaagc aagcgcucga aacggugcag 720
cggcuguugc cggugcugug ccaggaccau ggccugaccc cggaccaagu gguggcuauc 780
gccagcaacg guggcggcaa gcaagcgcuc gaaacggugc agcggcuguu gccggugcug 840
ugccaggacc auggccugac uccggaccaa gugguggcua ucgccagcca cgauggcggc 900
aagcaagcgc ucgaaacggu gcagcggcug uugccggugc ugugccagga ccauggccug 960
acuccggacc aagugguggc uaucgccagc cacgauggcg gcaagcaagc gcucgaaacg 1020
gugcagcggc uguugccggu gcugugccag gaccauggcc ugaccccgga ccaaguggug 1080
gcuaucgcca gcaacauugg cggcaagcaa gcgcucgaaa cggugcagcg gcuguugccg 1140
gugcugugcc aggaccaugg ccugaccccg gaccaagugg uggcuaucgc cagcaacaau 1200
ggcggcaagc aagcgcucga aacggugcag cggcuguugc cggugcugug ccaggaccau 1260
ggccugaccc cggaccaagu gguggcuauc gccagcaacg guggcggcaa gcaagcgcuc 1320
gaaacggugc agcggcuguu gccggugcug ugccaggacc auggccugac cccggaccaa 1380
gugguggcua ucgccagcaa caauggcggc aagcaagcgc ucgaaacggu gcagcggcug 1440
uugccggugc ugugccagga ccauggccug accccggacc aagugguggc uaucgccagc 1500
aacauuggcg gcaagcaagc gcucgaaagc auuguggccc agcugagccg gccugauccg 1560
gcguuggccg cguugaccaa cgaccaccuc gucgccuugg ccugccucgg cggacguccu 1620
gccauggaug cagugaaaaa gggauugccg cacgcgccgg aauugaucag aagagucaau 1680
cgccguauug gcgaacgcac gucccaucgc guugcgauau cuagaguggg aggaagcucu 1740
cgcagagagu ccaucaaccc auggauucug acugguuucg cugaugccga aggaucauuc 1800
auacuagaca uccgcaaccg aaacaacgaa agcaacagau accgaacuuc gcugagauuc 1860
cagaucaccc ugcacaacaa ggacaaaucg auucuggaga auauccaguc cacuuggaag 1920
gucggcaaga ucacaaacag cagugacaga gccgucaugc ugagggucac ccguuucgaa 1980
gauuugaaag ugauuaucga ccacuucgag aaauauccgc ugauuaccca gaaauugggc 2040
gauuacaagu uguuuaaaca ggcauucagc gucauggaga auaaagaaca ucuuaaggag 2100
aaugggauua aggagcucgu acgaaucaaa gcuaagauga auuggggucu caaugacgaa 2160
uugaaaaaag cauuuccaga gaacauuagc aaagagcgcc cccuuaucaa uaagaacauu 2220
ccgaauuuca aauggcuggc uggauucaca gcuggugaag gcuacuucgg cgugaaucua 2280
aaaaagguaa agggcaccgc aaagguauac gugggacuga gauucucaau cucacagcac 2340
aucagagaca agaaccugau gaauucauug auaacauacc uaggcugugg uuccaucugg 2400
gagaagaaca agucugaguu cagauggcuc gaguucgucg uaaccaaauu cagcgauauc 2460
aacgacaaga ucauuccggu auuccaggaa aauacucuga uuggcgucaa acucgaggac 2520
uuugaagauu ggugcaaggu ugccaaauug aucaaagaga agaaacaccu gaccgaaucc 2580
gguuuggaug agauuaagaa aaucaagcug aacaugaaca aaggucgugg uggaggcggu 2640
agcggaggcg gagggucggc uagcucccaa gcaccgcaag ccgaaaccuu cguguuccuc 2700
gaccuggaag ccacuggccu gcccaauguu gaucccgaga uugccgaaau cucccuguuc 2760
gccguucauc gguccucccu ggaacacccu gagagagacg aggccggagu accugugcug 2820
ccaagggugc uggacaagcu gacccugugc augucuccca cucgucccuu cacugccaag 2880
gcauccgaaa ucacuggucu gaguucggaa gggcuggcuc guuguggaaa ggcuggauuc 2940
gaugauacug cggcucagac uuugcgggca uuccugggca ggcaggcagg uccacucugc 3000
uuggucgccc acaacgguuu ugacuacgac uucccacugc ucaggaccga acuucaacgc 3060
cugggagcac gccuuccagc gcauaccgug ugccuggaca cccuacccgc ucugcgugga 3120
cuggacagag cccacucaca cgcuacuagg gcgcaaggac gcaaggguua cagccucggu 3180
ucacuguuca ggcgguauuu ccaggcagag ccuuccgcag cccauucagc cgaaggcgau 3240
guccgcaccu ugcuucuggu cuuccugcau cgugcagcag aacugcucgc cugggcagau 3300
gggcgggcuc guucaugggc ucacauugcg ccgauguacg cgccaccgga ggccccuagc 3360
ccggaagccu ga 3372
<210> 28
<211> 1123
<212> PRT
<213> 人工序列
<220>
<223> 在实验室中制备-低活性TCRα megaTAL-Trex2犰狳蛋白质构建体
<400> 28
Met Gly Ser Cys Arg Pro Pro Lys Lys Lys Arg Lys Val Val Asp Leu
1 5 10 15
Arg Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys
20 25 30
Val Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val Gly His Gly
35 40 45
Phe Thr His Ala His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu
50 55 60
Gly Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr Ala Leu Pro Glu
65 70 75 80
Ala Thr His Glu Asp Ile Val Gly Val Gly Lys Gln Trp Ser Gly Ala
85 90 95
Arg Ala Leu Glu Ala Leu Leu Thr Asp Ala Gly Glu Leu Arg Gly Pro
100 105 110
Pro Leu Gln Leu Asp Thr Gly Gln Leu Val Lys Ile Ala Lys Arg Gly
115 120 125
Gly Val Thr Ala Met Glu Ala Val His Ala Ser Arg Asn Ala Leu Thr
130 135 140
Gly Ala Pro Leu Asn Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
145 150 155 160
Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
165 170 175
Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
180 185 190
Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
195 200 205
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val
210 215 220
Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
225 230 235 240
Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln
245 250 255
Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr
260 265 270
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro
275 280 285
Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu
290 295 300
Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu
305 310 315 320
Thr Pro Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln
325 330 335
Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His
340 345 350
Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly
355 360 365
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
370 375 380
Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Asn
385 390 395 400
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu
405 410 415
Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
420 425 430
Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
435 440 445
Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
450 455 460
Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
465 470 475 480
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val
485 490 495
Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Ser Ile Val
500 505 510
Ala Gln Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu Thr Asn Asp
515 520 525
His Leu Val Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala Met Asp Ala
530 535 540
Val Lys Lys Gly Leu Pro His Ala Pro Glu Leu Ile Arg Arg Val Asn
545 550 555 560
Arg Arg Ile Gly Glu Arg Thr Ser His Arg Val Ala Ile Ser Arg Val
565 570 575
Gly Gly Ser Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr Gly
580 585 590
Phe Ala Asp Ala Glu Gly Ser Phe Ile Leu Asp Ile Arg Asn Arg Asn
595 600 605
Asn Glu Ser Asn Arg Tyr Arg Thr Ser Leu Arg Phe Gln Ile Thr Leu
610 615 620
His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp Lys
625 630 635 640
Val Gly Lys Ile Thr Asn Ser Ser Asp Arg Ala Val Met Leu Arg Val
645 650 655
Thr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys Tyr
660 665 670
Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln Ala
675 680 685
Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile Lys
690 695 700
Glu Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly Leu Asn Asp Glu
705 710 715 720
Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Pro Leu Ile
725 730 735
Asn Lys Asn Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe Thr Ala Gly
740 745 750
Glu Gly Tyr Phe Gly Val Asn Leu Lys Lys Val Lys Gly Thr Ala Lys
755 760 765
Val Tyr Val Gly Leu Arg Phe Ser Ile Ser Gln His Ile Arg Asp Lys
770 775 780
Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Ser Ile Trp
785 790 795 800
Glu Lys Asn Lys Ser Glu Phe Arg Trp Leu Glu Phe Val Val Thr Lys
805 810 815
Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn Thr
820 825 830
Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val Ala
835 840 845
Lys Leu Ile Lys Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp Glu
850 855 860
Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Gly Gly Gly Gly
865 870 875 880
Ser Gly Gly Gly Gly Ser Ala Ser Ser Gln Ala Pro Gln Ala Glu Thr
885 890 895
Phe Val Phe Leu Asp Leu Glu Ala Thr Gly Leu Pro Asn Val Asp Pro
900 905 910
Glu Ile Ala Glu Ile Ser Leu Phe Ala Val His Arg Ser Ser Leu Glu
915 920 925
His Pro Glu Arg Asp Glu Ala Gly Val Pro Val Leu Pro Arg Val Leu
930 935 940
Asp Lys Leu Thr Leu Cys Met Ser Pro Thr Arg Pro Phe Thr Ala Lys
945 950 955 960
Ala Ser Glu Ile Thr Gly Leu Ser Ser Glu Gly Leu Ala Arg Cys Gly
965 970 975
Lys Ala Gly Phe Asp Asp Thr Ala Ala Gln Thr Leu Arg Ala Phe Leu
980 985 990
Gly Arg Gln Ala Gly Pro Leu Cys Leu Val Ala His Asn Gly Phe Asp
995 1000 1005
Tyr Asp Phe Pro Leu Leu Arg Thr Glu Leu Gln Arg Leu Gly Ala
1010 1015 1020
Arg Leu Pro Ala His Thr Val Cys Leu Asp Thr Leu Pro Ala Leu
1025 1030 1035
Arg Gly Leu Asp Arg Ala His Ser His Ala Thr Arg Ala Gln Gly
1040 1045 1050
Arg Lys Gly Tyr Ser Leu Gly Ser Leu Phe Arg Arg Tyr Phe Gln
1055 1060 1065
Ala Glu Pro Ser Ala Ala His Ser Ala Glu Gly Asp Val Arg Thr
1070 1075 1080
Leu Leu Leu Val Phe Leu His Arg Ala Ala Glu Leu Leu Ala Trp
1085 1090 1095
Ala Asp Gly Arg Ala Arg Ser Trp Ala His Ile Ala Pro Met Tyr
1100 1105 1110
Ala Pro Pro Glu Ala Pro Ser Pro Glu Ala
1115 1120
<210> 29
<211> 3372
<212> DNA
<213> 人工序列
<220>
<223> 在实验室中制备-低活性TCRα megaTAL-Trex2美洲豹DNA构建体
<400> 29
atgggatcct gcaggccacc taagaagaaa cgcaaagtcg tggatctacg cacgctcggc 60
tacagtcagc agcagcaaga gaagatcaaa ccgaaggtgc gttcgacagt ggcgcagcac 120
cacgaggcac tggtgggcca tgggtttaca cacgcgcaca tcgttgcgct cagccaacac 180
ccggcagcgt tagggaccgt cgctgtcacg tatcagcaca taatcacggc gttgccagag 240
gcgacacacg aagacatcgt tggcgtcggc aaacagtggt ccggcgcacg cgccctggag 300
gccttgctca cggatgcggg ggagttgaga ggtccgccgt tacagttgga cacaggccaa 360
cttgtgaaga ttgcaaaacg tggcggcgtg accgcaatgg aggcagtgca tgcatcgcgc 420
aatgcactga cgggtgcccc cctgaacctg accccggacc aagtggtggc tatcgccagc 480
aacattggcg gcaagcaagc gctcgaaacg gtgcagcggc tgttgccggt gctgtgccag 540
gaccatggcc tgaccccgga ccaagtggtg gctatcgcca gcaacattgg cggcaagcaa 600
gcgctcgaaa cggtgcagcg gctgttgccg gtgctgtgcc aggaccatgg cctgaccccg 660
gaccaagtgg tggctatcgc cagcaacatt ggcggcaagc aagcgctcga aacggtgcag 720
cggctgttgc cggtgctgtg ccaggaccat ggcctgaccc cggaccaagt ggtggctatc 780
gccagcaacg gtggcggcaa gcaagcgctc gaaacggtgc agcggctgtt gccggtgctg 840
tgccaggacc atggcctgac tccggaccaa gtggtggcta tcgccagcca cgatggcggc 900
aagcaagcgc tcgaaacggt gcagcggctg ttgccggtgc tgtgccagga ccatggcctg 960
actccggacc aagtggtggc tatcgccagc cacgatggcg gcaagcaagc gctcgaaacg 1020
gtgcagcggc tgttgccggt gctgtgccag gaccatggcc tgaccccgga ccaagtggtg 1080
gctatcgcca gcaacattgg cggcaagcaa gcgctcgaaa cggtgcagcg gctgttgccg 1140
gtgctgtgcc aggaccatgg cctgaccccg gaccaagtgg tggctatcgc cagcaacaat 1200
ggcggcaagc aagcgctcga aacggtgcag cggctgttgc cggtgctgtg ccaggaccat 1260
ggcctgaccc cggaccaagt ggtggctatc gccagcaacg gtggcggcaa gcaagcgctc 1320
gaaacggtgc agcggctgtt gccggtgctg tgccaggacc atggcctgac cccggaccaa 1380
gtggtggcta tcgccagcaa caatggcggc aagcaagcgc tcgaaacggt gcagcggctg 1440
ttgccggtgc tgtgccagga ccatggcctg accccggacc aagtggtggc tatcgccagc 1500
aacattggcg gcaagcaagc gctcgaaagc attgtggccc agctgagccg gcctgatccg 1560
gcgttggccg cgttgaccaa cgaccacctc gtcgccttgg cctgcctcgg cggacgtcct 1620
gccatggatg cagtgaaaaa gggattgccg cacgcgccgg aattgatcag aagagtcaat 1680
cgccgtattg gcgaacgcac gtcccatcgc gttgcgatat ctagagtggg aggaagctct 1740
cgcagagagt ccatcaaccc atggattctg actggtttcg ctgatgccga aggatcattc 1800
atactagaca tccgcaaccg aaacaacgaa agcaacagat accgaacttc gctgagattc 1860
cagatcaccc tgcacaacaa ggacaaatcg attctggaga atatccagtc cacttggaag 1920
gtcggcaaga tcacaaacag cagtgacaga gccgtcatgc tgagggtcac ccgtttcgaa 1980
gatttgaaag tgattatcga ccacttcgag aaatatccgc tgattaccca gaaattgggc 2040
gattacaagt tgtttaaaca ggcattcagc gtcatggaga ataaagaaca tcttaaggag 2100
aatgggatta aggagctcgt acgaatcaaa gctaagatga attggggtct caatgacgaa 2160
ttgaaaaaag catttccaga gaacattagc aaagagcgcc cccttatcaa taagaacatt 2220
ccgaatttca aatggctggc tggattcaca gctggtgaag gctacttcgg cgtgaatcta 2280
aaaaaggtaa agggcaccgc aaaggtatac gtgggactga gattctcaat ctcacagcac 2340
atcagagaca agaacctgat gaattcattg ataacatacc taggctgtgg ttccatctgg 2400
gagaagaaca agtctgagtt cagatggctc gagttcgtcg taaccaaatt cagcgatatc 2460
aacgacaaga tcattccggt attccaggaa aatactctga ttggcgtcaa actcgaggac 2520
tttgaagatt ggtgcaaggt tgccaaattg atcaaagaga agaaacacct gaccgaatcc 2580
ggtttggatg agattaagaa aatcaagctg aacatgaaca aaggtcgtgg tggaggcggt 2640
agcggaggcg gagggtcggc tagctccgaa gtaccgagag ccgaaacctt cgtgttcctc 2700
gacctggaag ccactggcct gcccagtatt gatcccgagg ttgccgaaat ctccctgttc 2760
gccgttcatc ggtcctccct ggaaaaccct gacagagacg agagcggagt acctgtgctg 2820
ccaagggtgc tggacaagct gaccctgtgc atgtctcccg agcgtccctt cactgccaag 2880
gcatccgaaa tcactggtct gagttcggat tccctggctc gttgtagaaa ggctggattc 2940
gattcttccg tggttcggac tttgcaggca ttcctgtcca ggcaggaagg tccagtctgc 3000
ttggtcgccc acaacggttt tgactacgac ttcccactgc tctgtaccga actgcgtcgc 3060
ctgggagcac accttccacc ggatacaatt tgcctggaca ccctacccgc tctgcgtgga 3120
ctggacagag cccactcaca cggcactagg gcgcaaggat tcaagggtta cagcctcggt 3180
tcactgttca ggcggtattt ccatgcagag ccttccgcag cccattcagc cgaaggcgat 3240
gtccacacct tgcttatggt cttcctgcat cgtgcagcag aactgctctc ctgggcagat 3300
gagcaggctc gttcatgggc tcgcattaag ccgatgtacg agccaccgga tggccctagc 3360
gtggaagcct ga 3372
<210> 30
<211> 3372
<212> RNA
<213> 人工序列
<220>
<223> 在实验室中制备-低活性TCRα megaTAL-Trex2美洲豹RNA构建体
<400> 30
augggauccu gcaggccacc uaagaagaaa cgcaaagucg uggaucuacg cacgcucggc 60
uacagucagc agcagcaaga gaagaucaaa ccgaaggugc guucgacagu ggcgcagcac 120
cacgaggcac uggugggcca uggguuuaca cacgcgcaca ucguugcgcu cagccaacac 180
ccggcagcgu uagggaccgu cgcugucacg uaucagcaca uaaucacggc guugccagag 240
gcgacacacg aagacaucgu uggcgucggc aaacaguggu ccggcgcacg cgcccuggag 300
gccuugcuca cggaugcggg ggaguugaga gguccgccgu uacaguugga cacaggccaa 360
cuugugaaga uugcaaaacg uggcggcgug accgcaaugg aggcagugca ugcaucgcgc 420
aaugcacuga cgggugcccc ccugaaccug accccggacc aagugguggc uaucgccagc 480
aacauuggcg gcaagcaagc gcucgaaacg gugcagcggc uguugccggu gcugugccag 540
gaccauggcc ugaccccgga ccaaguggug gcuaucgcca gcaacauugg cggcaagcaa 600
gcgcucgaaa cggugcagcg gcuguugccg gugcugugcc aggaccaugg ccugaccccg 660
gaccaagugg uggcuaucgc cagcaacauu ggcggcaagc aagcgcucga aacggugcag 720
cggcuguugc cggugcugug ccaggaccau ggccugaccc cggaccaagu gguggcuauc 780
gccagcaacg guggcggcaa gcaagcgcuc gaaacggugc agcggcuguu gccggugcug 840
ugccaggacc auggccugac uccggaccaa gugguggcua ucgccagcca cgauggcggc 900
aagcaagcgc ucgaaacggu gcagcggcug uugccggugc ugugccagga ccauggccug 960
acuccggacc aagugguggc uaucgccagc cacgauggcg gcaagcaagc gcucgaaacg 1020
gugcagcggc uguugccggu gcugugccag gaccauggcc ugaccccgga ccaaguggug 1080
gcuaucgcca gcaacauugg cggcaagcaa gcgcucgaaa cggugcagcg gcuguugccg 1140
gugcugugcc aggaccaugg ccugaccccg gaccaagugg uggcuaucgc cagcaacaau 1200
ggcggcaagc aagcgcucga aacggugcag cggcuguugc cggugcugug ccaggaccau 1260
ggccugaccc cggaccaagu gguggcuauc gccagcaacg guggcggcaa gcaagcgcuc 1320
gaaacggugc agcggcuguu gccggugcug ugccaggacc auggccugac cccggaccaa 1380
gugguggcua ucgccagcaa caauggcggc aagcaagcgc ucgaaacggu gcagcggcug 1440
uugccggugc ugugccagga ccauggccug accccggacc aagugguggc uaucgccagc 1500
aacauuggcg gcaagcaagc gcucgaaagc auuguggccc agcugagccg gccugauccg 1560
gcguuggccg cguugaccaa cgaccaccuc gucgccuugg ccugccucgg cggacguccu 1620
gccauggaug cagugaaaaa gggauugccg cacgcgccgg aauugaucag aagagucaau 1680
cgccguauug gcgaacgcac gucccaucgc guugcgauau cuagaguggg aggaagcucu 1740
cgcagagagu ccaucaaccc auggauucug acugguuucg cugaugccga aggaucauuc 1800
auacuagaca uccgcaaccg aaacaacgaa agcaacagau accgaacuuc gcugagauuc 1860
cagaucaccc ugcacaacaa ggacaaaucg auucuggaga auauccaguc cacuuggaag 1920
gucggcaaga ucacaaacag cagugacaga gccgucaugc ugagggucac ccguuucgaa 1980
gauuugaaag ugauuaucga ccacuucgag aaauauccgc ugauuaccca gaaauugggc 2040
gauuacaagu uguuuaaaca ggcauucagc gucauggaga auaaagaaca ucuuaaggag 2100
aaugggauua aggagcucgu acgaaucaaa gcuaagauga auuggggucu caaugacgaa 2160
uugaaaaaag cauuuccaga gaacauuagc aaagagcgcc cccuuaucaa uaagaacauu 2220
ccgaauuuca aauggcuggc uggauucaca gcuggugaag gcuacuucgg cgugaaucua 2280
aaaaagguaa agggcaccgc aaagguauac gugggacuga gauucucaau cucacagcac 2340
aucagagaca agaaccugau gaauucauug auaacauacc uaggcugugg uuccaucugg 2400
gagaagaaca agucugaguu cagauggcuc gaguucgucg uaaccaaauu cagcgauauc 2460
aacgacaaga ucauuccggu auuccaggaa aauacucuga uuggcgucaa acucgaggac 2520
uuugaagauu ggugcaaggu ugccaaauug aucaaagaga agaaacaccu gaccgaaucc 2580
gguuuggaug agauuaagaa aaucaagcug aacaugaaca aaggucgugg uggaggcggu 2640
agcggaggcg gagggucggc uagcuccgaa guaccgagag ccgaaaccuu cguguuccuc 2700
gaccuggaag ccacuggccu gcccaguauu gaucccgagg uugccgaaau cucccuguuc 2760
gccguucauc gguccucccu ggaaaacccu gacagagacg agagcggagu accugugcug 2820
ccaagggugc uggacaagcu gacccugugc augucucccg agcgucccuu cacugccaag 2880
gcauccgaaa ucacuggucu gaguucggau ucccuggcuc guuguagaaa ggcuggauuc 2940
gauucuuccg ugguucggac uuugcaggca uuccugucca ggcaggaagg uccagucugc 3000
uuggucgccc acaacgguuu ugacuacgac uucccacugc ucuguaccga acugcgucgc 3060
cugggagcac accuuccacc ggauacaauu ugccuggaca cccuacccgc ucugcgugga 3120
cuggacagag cccacucaca cggcacuagg gcgcaaggau ucaaggguua cagccucggu 3180
ucacuguuca ggcgguauuu ccaugcagag ccuuccgcag cccauucagc cgaaggcgau 3240
guccacaccu ugcuuauggu cuuccugcau cgugcagcag aacugcucuc cugggcagau 3300
gagcaggcuc guucaugggc ucgcauuaag ccgauguacg agccaccgga uggcccuagc 3360
guggaagccu ga 3372
<210> 31
<211> 1123
<212> PRT
<213> 人工序列
<220>
<223> 在实验室中制备-低活性TCRα megaTAL-Trex2美洲豹蛋白质构建体
<400> 31
Met Gly Ser Cys Arg Pro Pro Lys Lys Lys Arg Lys Val Val Asp Leu
1 5 10 15
Arg Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys
20 25 30
Val Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val Gly His Gly
35 40 45
Phe Thr His Ala His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu
50 55 60
Gly Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr Ala Leu Pro Glu
65 70 75 80
Ala Thr His Glu Asp Ile Val Gly Val Gly Lys Gln Trp Ser Gly Ala
85 90 95
Arg Ala Leu Glu Ala Leu Leu Thr Asp Ala Gly Glu Leu Arg Gly Pro
100 105 110
Pro Leu Gln Leu Asp Thr Gly Gln Leu Val Lys Ile Ala Lys Arg Gly
115 120 125
Gly Val Thr Ala Met Glu Ala Val His Ala Ser Arg Asn Ala Leu Thr
130 135 140
Gly Ala Pro Leu Asn Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
145 150 155 160
Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
165 170 175
Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
180 185 190
Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
195 200 205
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val
210 215 220
Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
225 230 235 240
Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln
245 250 255
Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr
260 265 270
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro
275 280 285
Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu
290 295 300
Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu
305 310 315 320
Thr Pro Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln
325 330 335
Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His
340 345 350
Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly
355 360 365
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
370 375 380
Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Asn
385 390 395 400
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu
405 410 415
Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
420 425 430
Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
435 440 445
Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
450 455 460
Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
465 470 475 480
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val
485 490 495
Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Ser Ile Val
500 505 510
Ala Gln Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu Thr Asn Asp
515 520 525
His Leu Val Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala Met Asp Ala
530 535 540
Val Lys Lys Gly Leu Pro His Ala Pro Glu Leu Ile Arg Arg Val Asn
545 550 555 560
Arg Arg Ile Gly Glu Arg Thr Ser His Arg Val Ala Ile Ser Arg Val
565 570 575
Gly Gly Ser Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr Gly
580 585 590
Phe Ala Asp Ala Glu Gly Ser Phe Ile Leu Asp Ile Arg Asn Arg Asn
595 600 605
Asn Glu Ser Asn Arg Tyr Arg Thr Ser Leu Arg Phe Gln Ile Thr Leu
610 615 620
His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp Lys
625 630 635 640
Val Gly Lys Ile Thr Asn Ser Ser Asp Arg Ala Val Met Leu Arg Val
645 650 655
Thr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys Tyr
660 665 670
Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln Ala
675 680 685
Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile Lys
690 695 700
Glu Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly Leu Asn Asp Glu
705 710 715 720
Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Pro Leu Ile
725 730 735
Asn Lys Asn Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe Thr Ala Gly
740 745 750
Glu Gly Tyr Phe Gly Val Asn Leu Lys Lys Val Lys Gly Thr Ala Lys
755 760 765
Val Tyr Val Gly Leu Arg Phe Ser Ile Ser Gln His Ile Arg Asp Lys
770 775 780
Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Ser Ile Trp
785 790 795 800
Glu Lys Asn Lys Ser Glu Phe Arg Trp Leu Glu Phe Val Val Thr Lys
805 810 815
Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn Thr
820 825 830
Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val Ala
835 840 845
Lys Leu Ile Lys Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp Glu
850 855 860
Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Gly Gly Gly Gly
865 870 875 880
Ser Gly Gly Gly Gly Ser Ala Ser Ser Glu Val Pro Arg Ala Glu Thr
885 890 895
Phe Val Phe Leu Asp Leu Glu Ala Thr Gly Leu Pro Ser Ile Asp Pro
900 905 910
Glu Val Ala Glu Ile Ser Leu Phe Ala Val His Arg Ser Ser Leu Glu
915 920 925
Asn Pro Asp Arg Asp Glu Ser Gly Val Pro Val Leu Pro Arg Val Leu
930 935 940
Asp Lys Leu Thr Leu Cys Met Ser Pro Glu Arg Pro Phe Thr Ala Lys
945 950 955 960
Ala Ser Glu Ile Thr Gly Leu Ser Ser Asp Ser Leu Ala Arg Cys Arg
965 970 975
Lys Ala Gly Phe Asp Ser Ser Val Val Arg Thr Leu Gln Ala Phe Leu
980 985 990
Ser Arg Gln Glu Gly Pro Val Cys Leu Val Ala His Asn Gly Phe Asp
995 1000 1005
Tyr Asp Phe Pro Leu Leu Cys Thr Glu Leu Arg Arg Leu Gly Ala
1010 1015 1020
His Leu Pro Pro Asp Thr Ile Cys Leu Asp Thr Leu Pro Ala Leu
1025 1030 1035
Arg Gly Leu Asp Arg Ala His Ser His Gly Thr Arg Ala Gln Gly
1040 1045 1050
Phe Lys Gly Tyr Ser Leu Gly Ser Leu Phe Arg Arg Tyr Phe His
1055 1060 1065
Ala Glu Pro Ser Ala Ala His Ser Ala Glu Gly Asp Val His Thr
1070 1075 1080
Leu Leu Met Val Phe Leu His Arg Ala Ala Glu Leu Leu Ser Trp
1085 1090 1095
Ala Asp Glu Gln Ala Arg Ser Trp Ala Arg Ile Lys Pro Met Tyr
1100 1105 1110
Glu Pro Pro Asp Gly Pro Ser Val Glu Ala
1115 1120
<210> 32
<211> 3372
<212> DNA
<213> 人工序列
<220>
<223> 在实验室中制备-低活性TCRα megaTAL-Trex2小鼠DNA构建体
<400> 32
atgggatcct gcaggccacc taagaagaaa cgcaaagtcg tggatctacg cacgctcggc 60
tacagtcagc agcagcaaga gaagatcaaa ccgaaggtgc gttcgacagt ggcgcagcac 120
cacgaggcac tggtgggcca tgggtttaca cacgcgcaca tcgttgcgct cagccaacac 180
ccggcagcgt tagggaccgt cgctgtcacg tatcagcaca taatcacggc gttgccagag 240
gcgacacacg aagacatcgt tggcgtcggc aaacagtggt ccggcgcacg cgccctggag 300
gccttgctca cggatgcggg ggagttgaga ggtccgccgt tacagttgga cacaggccaa 360
cttgtgaaga ttgcaaaacg tggcggcgtg accgcaatgg aggcagtgca tgcatcgcgc 420
aatgcactga cgggtgcccc cctgaacctg accccggacc aagtggtggc tatcgccagc 480
aacattggcg gcaagcaagc gctcgaaacg gtgcagcggc tgttgccggt gctgtgccag 540
gaccatggcc tgaccccgga ccaagtggtg gctatcgcca gcaacattgg cggcaagcaa 600
gcgctcgaaa cggtgcagcg gctgttgccg gtgctgtgcc aggaccatgg cctgaccccg 660
gaccaagtgg tggctatcgc cagcaacatt ggcggcaagc aagcgctcga aacggtgcag 720
cggctgttgc cggtgctgtg ccaggaccat ggcctgaccc cggaccaagt ggtggctatc 780
gccagcaacg gtggcggcaa gcaagcgctc gaaacggtgc agcggctgtt gccggtgctg 840
tgccaggacc atggcctgac tccggaccaa gtggtggcta tcgccagcca cgatggcggc 900
aagcaagcgc tcgaaacggt gcagcggctg ttgccggtgc tgtgccagga ccatggcctg 960
actccggacc aagtggtggc tatcgccagc cacgatggcg gcaagcaagc gctcgaaacg 1020
gtgcagcggc tgttgccggt gctgtgccag gaccatggcc tgaccccgga ccaagtggtg 1080
gctatcgcca gcaacattgg cggcaagcaa gcgctcgaaa cggtgcagcg gctgttgccg 1140
gtgctgtgcc aggaccatgg cctgaccccg gaccaagtgg tggctatcgc cagcaacaat 1200
ggcggcaagc aagcgctcga aacggtgcag cggctgttgc cggtgctgtg ccaggaccat 1260
ggcctgaccc cggaccaagt ggtggctatc gccagcaacg gtggcggcaa gcaagcgctc 1320
gaaacggtgc agcggctgtt gccggtgctg tgccaggacc atggcctgac cccggaccaa 1380
gtggtggcta tcgccagcaa caatggcggc aagcaagcgc tcgaaacggt gcagcggctg 1440
ttgccggtgc tgtgccagga ccatggcctg accccggacc aagtggtggc tatcgccagc 1500
aacattggcg gcaagcaagc gctcgaaagc attgtggccc agctgagccg gcctgatccg 1560
gcgttggccg cgttgaccaa cgaccacctc gtcgccttgg cctgcctcgg cggacgtcct 1620
gccatggatg cagtgaaaaa gggattgccg cacgcgccgg aattgatcag aagagtcaat 1680
cgccgtattg gcgaacgcac gtcccatcgc gttgcgatat ctagagtggg aggaagctct 1740
cgcagagagt ccatcaaccc atggattctg actggtttcg ctgatgccga aggatcattc 1800
atactagaca tccgcaaccg aaacaacgaa agcaacagat accgaacttc gctgagattc 1860
cagatcaccc tgcacaacaa ggacaaatcg attctggaga atatccagtc cacttggaag 1920
gtcggcaaga tcacaaacag cagtgacaga gccgtcatgc tgagggtcac ccgtttcgaa 1980
gatttgaaag tgattatcga ccacttcgag aaatatccgc tgattaccca gaaattgggc 2040
gattacaagt tgtttaaaca ggcattcagc gtcatggaga ataaagaaca tcttaaggag 2100
aatgggatta aggagctcgt acgaatcaaa gctaagatga attggggtct caatgacgaa 2160
ttgaaaaaag catttccaga gaacattagc aaagagcgcc cccttatcaa taagaacatt 2220
ccgaatttca aatggctggc tggattcaca gctggtgaag gctacttcgg cgtgaatcta 2280
aaaaaggtaa agggcaccgc aaaggtatac gtgggactga gattctcaat ctcacagcac 2340
atcagagaca agaacctgat gaattcattg ataacatacc taggctgtgg ttccatctgg 2400
gagaagaaca agtctgagtt cagatggctc gagttcgtcg taaccaaatt cagcgatatc 2460
aacgacaaga tcattccggt attccaggaa aatactctga ttggcgtcaa actcgaggac 2520
tttgaagatt ggtgcaaggt tgccaaattg atcaaagaga agaaacacct gaccgaatcc 2580
ggtttggatg agattaagaa aatcaagctg aacatgaaca aaggtcgtgg tggaggcggt 2640
agcggaggcg gagggtcggc tagctccgaa ccgccgagag ccgaaacctt cgtgttcctc 2700
gacctggagg ccactggcct gccgaacatg gacccggaaa ttgccgaaat ctccctgttc 2760
gccgtgcacc ggtcctccct ggaaaaccct gagcgggacg atagcggttc cctggtgctg 2820
cccagggtgc tggacaagct gaccctgtgc atgtgccccg agcgcccctt cactgccaag 2880
gcatccgaaa tcactggtct gagctcggaa tccctgatgc actgcggcaa ggccggattc 2940
aacggcgccg tggtccggac tttgcaggga ttcctgtcca gacaggaggg ccccatctgc 3000
ttggtcgccc acaacggttt tgactacgac ttccccctgc tctgcaccga actgcagcgc 3060
ctgggagccc acctccctca ggacaccgtg tgcctggaca ccctgcccgc actgcgcgga 3120
ctggacagag cccactcaca cggcaccagg gcgcagggac gcaagtccta cagcctcgca 3180
tcactgttcc atcggtattt ccaggccgag ccttccgccg cccattccgc cgaaggggat 3240
gtgcacacct tgcttctgat cttcctgcat cgcgctcccg aactgctggc ctgggcggac 3300
gagcaggccc ggtcctgggc ccacattgag ccgatgtacg tgccgcccga tggacctagc 3360
ctggaagcct ga 3372
<210> 33
<211> 3372
<212> RNA
<213> 人工序列
<220>
<223> 在实验室中制备-低活性TCRα megaTAL-Trex2小鼠RNA构建体
<400> 33
augggauccu gcaggccacc uaagaagaaa cgcaaagucg uggaucuacg cacgcucggc 60
uacagucagc agcagcaaga gaagaucaaa ccgaaggugc guucgacagu ggcgcagcac 120
cacgaggcac uggugggcca uggguuuaca cacgcgcaca ucguugcgcu cagccaacac 180
ccggcagcgu uagggaccgu cgcugucacg uaucagcaca uaaucacggc guugccagag 240
gcgacacacg aagacaucgu uggcgucggc aaacaguggu ccggcgcacg cgcccuggag 300
gccuugcuca cggaugcggg ggaguugaga gguccgccgu uacaguugga cacaggccaa 360
cuugugaaga uugcaaaacg uggcggcgug accgcaaugg aggcagugca ugcaucgcgc 420
aaugcacuga cgggugcccc ccugaaccug accccggacc aagugguggc uaucgccagc 480
aacauuggcg gcaagcaagc gcucgaaacg gugcagcggc uguugccggu gcugugccag 540
gaccauggcc ugaccccgga ccaaguggug gcuaucgcca gcaacauugg cggcaagcaa 600
gcgcucgaaa cggugcagcg gcuguugccg gugcugugcc aggaccaugg ccugaccccg 660
gaccaagugg uggcuaucgc cagcaacauu ggcggcaagc aagcgcucga aacggugcag 720
cggcuguugc cggugcugug ccaggaccau ggccugaccc cggaccaagu gguggcuauc 780
gccagcaacg guggcggcaa gcaagcgcuc gaaacggugc agcggcuguu gccggugcug 840
ugccaggacc auggccugac uccggaccaa gugguggcua ucgccagcca cgauggcggc 900
aagcaagcgc ucgaaacggu gcagcggcug uugccggugc ugugccagga ccauggccug 960
acuccggacc aagugguggc uaucgccagc cacgauggcg gcaagcaagc gcucgaaacg 1020
gugcagcggc uguugccggu gcugugccag gaccauggcc ugaccccgga ccaaguggug 1080
gcuaucgcca gcaacauugg cggcaagcaa gcgcucgaaa cggugcagcg gcuguugccg 1140
gugcugugcc aggaccaugg ccugaccccg gaccaagugg uggcuaucgc cagcaacaau 1200
ggcggcaagc aagcgcucga aacggugcag cggcuguugc cggugcugug ccaggaccau 1260
ggccugaccc cggaccaagu gguggcuauc gccagcaacg guggcggcaa gcaagcgcuc 1320
gaaacggugc agcggcuguu gccggugcug ugccaggacc auggccugac cccggaccaa 1380
gugguggcua ucgccagcaa caauggcggc aagcaagcgc ucgaaacggu gcagcggcug 1440
uugccggugc ugugccagga ccauggccug accccggacc aagugguggc uaucgccagc 1500
aacauuggcg gcaagcaagc gcucgaaagc auuguggccc agcugagccg gccugauccg 1560
gcguuggccg cguugaccaa cgaccaccuc gucgccuugg ccugccucgg cggacguccu 1620
gccauggaug cagugaaaaa gggauugccg cacgcgccgg aauugaucag aagagucaau 1680
cgccguauug gcgaacgcac gucccaucgc guugcgauau cuagaguggg aggaagcucu 1740
cgcagagagu ccaucaaccc auggauucug acugguuucg cugaugccga aggaucauuc 1800
auacuagaca uccgcaaccg aaacaacgaa agcaacagau accgaacuuc gcugagauuc 1860
cagaucaccc ugcacaacaa ggacaaaucg auucuggaga auauccaguc cacuuggaag 1920
gucggcaaga ucacaaacag cagugacaga gccgucaugc ugagggucac ccguuucgaa 1980
gauuugaaag ugauuaucga ccacuucgag aaauauccgc ugauuaccca gaaauugggc 2040
gauuacaagu uguuuaaaca ggcauucagc gucauggaga auaaagaaca ucuuaaggag 2100
aaugggauua aggagcucgu acgaaucaaa gcuaagauga auuggggucu caaugacgaa 2160
uugaaaaaag cauuuccaga gaacauuagc aaagagcgcc cccuuaucaa uaagaacauu 2220
ccgaauuuca aauggcuggc uggauucaca gcuggugaag gcuacuucgg cgugaaucua 2280
aaaaagguaa agggcaccgc aaagguauac gugggacuga gauucucaau cucacagcac 2340
aucagagaca agaaccugau gaauucauug auaacauacc uaggcugugg uuccaucugg 2400
gagaagaaca agucugaguu cagauggcuc gaguucgucg uaaccaaauu cagcgauauc 2460
aacgacaaga ucauuccggu auuccaggaa aauacucuga uuggcgucaa acucgaggac 2520
uuugaagauu ggugcaaggu ugccaaauug aucaaagaga agaaacaccu gaccgaaucc 2580
gguuuggaug agauuaagaa aaucaagcug aacaugaaca aaggucgugg uggaggcggu 2640
agcggaggcg gagggucggc uagcuccgaa ccgccgagag ccgaaaccuu cguguuccuc 2700
gaccuggagg ccacuggccu gccgaacaug gacccggaaa uugccgaaau cucccuguuc 2760
gccgugcacc gguccucccu ggaaaacccu gagcgggacg auagcgguuc ccuggugcug 2820
cccagggugc uggacaagcu gacccugugc augugccccg agcgccccuu cacugccaag 2880
gcauccgaaa ucacuggucu gagcucggaa ucccugaugc acugcggcaa ggccggauuc 2940
aacggcgccg ugguccggac uuugcaggga uuccugucca gacaggaggg ccccaucugc 3000
uuggucgccc acaacgguuu ugacuacgac uucccccugc ucugcaccga acugcagcgc 3060
cugggagccc accucccuca ggacaccgug ugccuggaca cccugcccgc acugcgcgga 3120
cuggacagag cccacucaca cggcaccagg gcgcagggac gcaaguccua cagccucgca 3180
ucacuguucc aucgguauuu ccaggccgag ccuuccgccg cccauuccgc cgaaggggau 3240
gugcacaccu ugcuucugau cuuccugcau cgcgcucccg aacugcuggc cugggcggac 3300
gagcaggccc gguccugggc ccacauugag ccgauguacg ugccgcccga uggaccuagc 3360
cuggaagccu ga 3372
<210> 34
<211> 1123
<212> PRT
<213> 人工序列
<220>
<223> 在实验室中制备-低活性TCRα megaTAL-Trex2小鼠蛋白质构建体
<400> 34
Met Gly Ser Cys Arg Pro Pro Lys Lys Lys Arg Lys Val Val Asp Leu
1 5 10 15
Arg Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys
20 25 30
Val Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val Gly His Gly
35 40 45
Phe Thr His Ala His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu
50 55 60
Gly Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr Ala Leu Pro Glu
65 70 75 80
Ala Thr His Glu Asp Ile Val Gly Val Gly Lys Gln Trp Ser Gly Ala
85 90 95
Arg Ala Leu Glu Ala Leu Leu Thr Asp Ala Gly Glu Leu Arg Gly Pro
100 105 110
Pro Leu Gln Leu Asp Thr Gly Gln Leu Val Lys Ile Ala Lys Arg Gly
115 120 125
Gly Val Thr Ala Met Glu Ala Val His Ala Ser Arg Asn Ala Leu Thr
130 135 140
Gly Ala Pro Leu Asn Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
145 150 155 160
Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
165 170 175
Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
180 185 190
Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
195 200 205
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val
210 215 220
Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
225 230 235 240
Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln
245 250 255
Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr
260 265 270
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro
275 280 285
Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu
290 295 300
Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu
305 310 315 320
Thr Pro Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln
325 330 335
Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His
340 345 350
Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly
355 360 365
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
370 375 380
Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Asn
385 390 395 400
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu
405 410 415
Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
420 425 430
Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
435 440 445
Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
450 455 460
Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
465 470 475 480
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val
485 490 495
Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Ser Ile Val
500 505 510
Ala Gln Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu Thr Asn Asp
515 520 525
His Leu Val Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala Met Asp Ala
530 535 540
Val Lys Lys Gly Leu Pro His Ala Pro Glu Leu Ile Arg Arg Val Asn
545 550 555 560
Arg Arg Ile Gly Glu Arg Thr Ser His Arg Val Ala Ile Ser Arg Val
565 570 575
Gly Gly Ser Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr Gly
580 585 590
Phe Ala Asp Ala Glu Gly Ser Phe Ile Leu Asp Ile Arg Asn Arg Asn
595 600 605
Asn Glu Ser Asn Arg Tyr Arg Thr Ser Leu Arg Phe Gln Ile Thr Leu
610 615 620
His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp Lys
625 630 635 640
Val Gly Lys Ile Thr Asn Ser Ser Asp Arg Ala Val Met Leu Arg Val
645 650 655
Thr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys Tyr
660 665 670
Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln Ala
675 680 685
Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile Lys
690 695 700
Glu Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly Leu Asn Asp Glu
705 710 715 720
Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Pro Leu Ile
725 730 735
Asn Lys Asn Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe Thr Ala Gly
740 745 750
Glu Gly Tyr Phe Gly Val Asn Leu Lys Lys Val Lys Gly Thr Ala Lys
755 760 765
Val Tyr Val Gly Leu Arg Phe Ser Ile Ser Gln His Ile Arg Asp Lys
770 775 780
Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Ser Ile Trp
785 790 795 800
Glu Lys Asn Lys Ser Glu Phe Arg Trp Leu Glu Phe Val Val Thr Lys
805 810 815
Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn Thr
820 825 830
Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val Ala
835 840 845
Lys Leu Ile Lys Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp Glu
850 855 860
Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Gly Gly Gly Gly
865 870 875 880
Ser Gly Gly Gly Gly Ser Ala Ser Ser Glu Pro Pro Arg Ala Glu Thr
885 890 895
Phe Val Phe Leu Asp Leu Glu Ala Thr Gly Leu Pro Asn Met Asp Pro
900 905 910
Glu Ile Ala Glu Ile Ser Leu Phe Ala Val His Arg Ser Ser Leu Glu
915 920 925
Asn Pro Glu Arg Asp Asp Ser Gly Ser Leu Val Leu Pro Arg Val Leu
930 935 940
Asp Lys Leu Thr Leu Cys Met Cys Pro Glu Arg Pro Phe Thr Ala Lys
945 950 955 960
Ala Ser Glu Ile Thr Gly Leu Ser Ser Glu Ser Leu Met His Cys Gly
965 970 975
Lys Ala Gly Phe Asn Gly Ala Val Val Arg Thr Leu Gln Gly Phe Leu
980 985 990
Ser Arg Gln Glu Gly Pro Ile Cys Leu Val Ala His Asn Gly Phe Asp
995 1000 1005
Tyr Asp Phe Pro Leu Leu Cys Thr Glu Leu Gln Arg Leu Gly Ala
1010 1015 1020
His Leu Pro Gln Asp Thr Val Cys Leu Asp Thr Leu Pro Ala Leu
1025 1030 1035
Arg Gly Leu Asp Arg Ala His Ser His Gly Thr Arg Ala Gln Gly
1040 1045 1050
Arg Lys Ser Tyr Ser Leu Ala Ser Leu Phe His Arg Tyr Phe Gln
1055 1060 1065
Ala Glu Pro Ser Ala Ala His Ser Ala Glu Gly Asp Val His Thr
1070 1075 1080
Leu Leu Leu Ile Phe Leu His Arg Ala Pro Glu Leu Leu Ala Trp
1085 1090 1095
Ala Asp Glu Gln Ala Arg Ser Trp Ala His Ile Glu Pro Met Tyr
1100 1105 1110
Val Pro Pro Asp Gly Pro Ser Leu Glu Ala
1115 1120
<210> 35
<211> 3504
<212> DNA
<213> 人工序列
<220>
<223> 在实验室中制备-低活性TCRα megaTAL-hsRad1 DNA构建体
<400> 35
atgggatcct gcaggccacc taagaagaaa cgcaaagtcg tggatctacg cacgctcggc 60
tacagtcagc agcagcaaga gaagatcaaa ccgaaggtgc gttcgacagt ggcgcagcac 120
cacgaggcac tggtgggcca tgggtttaca cacgcgcaca tcgttgcgct cagccaacac 180
ccggcagcgt tagggaccgt cgctgtcacg tatcagcaca taatcacggc gttgccagag 240
gcgacacacg aagacatcgt tggcgtcggc aaacagtggt ccggcgcacg cgccctggag 300
gccttgctca cggatgcggg ggagttgaga ggtccgccgt tacagttgga cacaggccaa 360
cttgtgaaga ttgcaaaacg tggcggcgtg accgcaatgg aggcagtgca tgcatcgcgc 420
aatgcactga cgggtgcccc cctgaacctg accccggacc aagtggtggc tatcgccagc 480
aacattggcg gcaagcaagc gctcgaaacg gtgcagcggc tgttgccggt gctgtgccag 540
gaccatggcc tgaccccgga ccaagtggtg gctatcgcca gcaacattgg cggcaagcaa 600
gcgctcgaaa cggtgcagcg gctgttgccg gtgctgtgcc aggaccatgg cctgaccccg 660
gaccaagtgg tggctatcgc cagcaacatt ggcggcaagc aagcgctcga aacggtgcag 720
cggctgttgc cggtgctgtg ccaggaccat ggcctgaccc cggaccaagt ggtggctatc 780
gccagcaacg gtggcggcaa gcaagcgctc gaaacggtgc agcggctgtt gccggtgctg 840
tgccaggacc atggcctgac tccggaccaa gtggtggcta tcgccagcca cgatggcggc 900
aagcaagcgc tcgaaacggt gcagcggctg ttgccggtgc tgtgccagga ccatggcctg 960
actccggacc aagtggtggc tatcgccagc cacgatggcg gcaagcaagc gctcgaaacg 1020
gtgcagcggc tgttgccggt gctgtgccag gaccatggcc tgaccccgga ccaagtggtg 1080
gctatcgcca gcaacattgg cggcaagcaa gcgctcgaaa cggtgcagcg gctgttgccg 1140
gtgctgtgcc aggaccatgg cctgaccccg gaccaagtgg tggctatcgc cagcaacaat 1200
ggcggcaagc aagcgctcga aacggtgcag cggctgttgc cggtgctgtg ccaggaccat 1260
ggcctgaccc cggaccaagt ggtggctatc gccagcaacg gtggcggcaa gcaagcgctc 1320
gaaacggtgc agcggctgtt gccggtgctg tgccaggacc atggcctgac cccggaccaa 1380
gtggtggcta tcgccagcaa caatggcggc aagcaagcgc tcgaaacggt gcagcggctg 1440
ttgccggtgc tgtgccagga ccatggcctg accccggacc aagtggtggc tatcgccagc 1500
aacattggcg gcaagcaagc gctcgaaagc attgtggccc agctgagccg gcctgatccg 1560
gcgttggccg cgttgaccaa cgaccacctc gtcgccttgg cctgcctcgg cggacgtcct 1620
gccatggatg cagtgaaaaa gggattgccg cacgcgccgg aattgatcag aagagtcaat 1680
cgccgtattg gcgaacgcac gtcccatcgc gttgcgatat ctagagtggg aggaagctct 1740
cgcagagagt ccatcaaccc atggattctg actggtttcg ctgatgccga aggatcattc 1800
atactagaca tccgcaaccg aaacaacgaa agcaacagat accgaacttc gctgagattc 1860
cagatcaccc tgcacaacaa ggacaaatcg attctggaga atatccagtc cacttggaag 1920
gtcggcaaga tcacaaacag cagtgacaga gccgtcatgc tgagggtcac ccgtttcgaa 1980
gatttgaaag tgattatcga ccacttcgag aaatatccgc tgattaccca gaaattgggc 2040
gattacaagt tgtttaaaca ggcattcagc gtcatggaga ataaagaaca tcttaaggag 2100
aatgggatta aggagctcgt acgaatcaaa gctaagatga attggggtct caatgacgaa 2160
ttgaaaaaag catttccaga gaacattagc aaagagcgcc cccttatcaa taagaacatt 2220
ccgaatttca aatggctggc tggattcaca gctggtgaag gctacttcgg cgtgaatcta 2280
aaaaaggtaa agggcaccgc aaaggtatac gtgggactga gattctcaat ctcacagcac 2340
atcagagaca agaacctgat gaattcattg ataacatacc taggctgtgg ttccatctgg 2400
gagaagaaca agtctgagtt cagatggctc gagttcgtcg taaccaaatt cagcgatatc 2460
aacgacaaga tcattccggt attccaggaa aatactctga ttggcgtcaa actcgaggac 2520
tttgaagatt ggtgcaaggt tgccaaattg atcaaagaga agaaacacct gaccgaatcc 2580
ggtttggatg agattaagaa aatcaagctg aacatgaaca aaggtcgtgg tggaggcggt 2640
agcggaggcg gagggtcgcc gcttctgacc caacagatcc aagacgagga cgatcagtac 2700
agccttgttg ccagccttga caacgtgagg aatctctcca ctatcttgaa agctattcat 2760
ttccgagaac atgccacctg tttcgcaact aagaacggta tcaaagtaac agtggagaat 2820
gcaaagtgtg tgcaagcaaa tgccttcatt caggctggca tcttccaaga gttcaaagtt 2880
caggaagagt ctgttacttt ccgaatcaac ctaactgtcc tcttagactg tttatctatc 2940
ttcggatcaa gtccaatgcc agggacttta actgcactcc gaatgtgtta ccaaggttac 3000
ggataccctt tgatgctgtt cctcgaagaa ggaggagtgg tgacagtctg caagatcaat 3060
acacaggaac cagaggagac cctggacttc gatttctgct ccaccaatgt tatcaacaag 3120
atcattctcc agtcagaagg gctccgtgaa gcattctctg aattggatat gacgagtgaa 3180
gtcctacaga tcaccatgtc tcctgacaag ccttacttca ggttatctac tttcggaaat 3240
gcaggaagtt cccaccttga ctatcccaaa gattctgatt tgatggaggc atttcattgt 3300
aatcagactc aagtgaacag atacaagata tccctactga aaccctctac aaaggcatta 3360
gtcctatctt gtaaggtatc tattcggaca gataacagag gcttcctttc attacagtat 3420
atgattagaa atgaagatgg acaaatctgc tttgtggagt attactgctg ccctgatgag 3480
gaagttcctg aatctgagtc ttga 3504
<210> 36
<211> 3504
<212> RNA
<213> 人工序列
<220>
<223> 在实验室中制备-低活性TCRα megaTAL-hsRad1 RNA构建体
<400> 36
augggauccu gcaggccacc uaagaagaaa cgcaaagucg uggaucuacg cacgcucggc 60
uacagucagc agcagcaaga gaagaucaaa ccgaaggugc guucgacagu ggcgcagcac 120
cacgaggcac uggugggcca uggguuuaca cacgcgcaca ucguugcgcu cagccaacac 180
ccggcagcgu uagggaccgu cgcugucacg uaucagcaca uaaucacggc guugccagag 240
gcgacacacg aagacaucgu uggcgucggc aaacaguggu ccggcgcacg cgcccuggag 300
gccuugcuca cggaugcggg ggaguugaga gguccgccgu uacaguugga cacaggccaa 360
cuugugaaga uugcaaaacg uggcggcgug accgcaaugg aggcagugca ugcaucgcgc 420
aaugcacuga cgggugcccc ccugaaccug accccggacc aagugguggc uaucgccagc 480
aacauuggcg gcaagcaagc gcucgaaacg gugcagcggc uguugccggu gcugugccag 540
gaccauggcc ugaccccgga ccaaguggug gcuaucgcca gcaacauugg cggcaagcaa 600
gcgcucgaaa cggugcagcg gcuguugccg gugcugugcc aggaccaugg ccugaccccg 660
gaccaagugg uggcuaucgc cagcaacauu ggcggcaagc aagcgcucga aacggugcag 720
cggcuguugc cggugcugug ccaggaccau ggccugaccc cggaccaagu gguggcuauc 780
gccagcaacg guggcggcaa gcaagcgcuc gaaacggugc agcggcuguu gccggugcug 840
ugccaggacc auggccugac uccggaccaa gugguggcua ucgccagcca cgauggcggc 900
aagcaagcgc ucgaaacggu gcagcggcug uugccggugc ugugccagga ccauggccug 960
acuccggacc aagugguggc uaucgccagc cacgauggcg gcaagcaagc gcucgaaacg 1020
gugcagcggc uguugccggu gcugugccag gaccauggcc ugaccccgga ccaaguggug 1080
gcuaucgcca gcaacauugg cggcaagcaa gcgcucgaaa cggugcagcg gcuguugccg 1140
gugcugugcc aggaccaugg ccugaccccg gaccaagugg uggcuaucgc cagcaacaau 1200
ggcggcaagc aagcgcucga aacggugcag cggcuguugc cggugcugug ccaggaccau 1260
ggccugaccc cggaccaagu gguggcuauc gccagcaacg guggcggcaa gcaagcgcuc 1320
gaaacggugc agcggcuguu gccggugcug ugccaggacc auggccugac cccggaccaa 1380
gugguggcua ucgccagcaa caauggcggc aagcaagcgc ucgaaacggu gcagcggcug 1440
uugccggugc ugugccagga ccauggccug accccggacc aagugguggc uaucgccagc 1500
aacauuggcg gcaagcaagc gcucgaaagc auuguggccc agcugagccg gccugauccg 1560
gcguuggccg cguugaccaa cgaccaccuc gucgccuugg ccugccucgg cggacguccu 1620
gccauggaug cagugaaaaa gggauugccg cacgcgccgg aauugaucag aagagucaau 1680
cgccguauug gcgaacgcac gucccaucgc guugcgauau cuagaguggg aggaagcucu 1740
cgcagagagu ccaucaaccc auggauucug acugguuucg cugaugccga aggaucauuc 1800
auacuagaca uccgcaaccg aaacaacgaa agcaacagau accgaacuuc gcugagauuc 1860
cagaucaccc ugcacaacaa ggacaaaucg auucuggaga auauccaguc cacuuggaag 1920
gucggcaaga ucacaaacag cagugacaga gccgucaugc ugagggucac ccguuucgaa 1980
gauuugaaag ugauuaucga ccacuucgag aaauauccgc ugauuaccca gaaauugggc 2040
gauuacaagu uguuuaaaca ggcauucagc gucauggaga auaaagaaca ucuuaaggag 2100
aaugggauua aggagcucgu acgaaucaaa gcuaagauga auuggggucu caaugacgaa 2160
uugaaaaaag cauuuccaga gaacauuagc aaagagcgcc cccuuaucaa uaagaacauu 2220
ccgaauuuca aauggcuggc uggauucaca gcuggugaag gcuacuucgg cgugaaucua 2280
aaaaagguaa agggcaccgc aaagguauac gugggacuga gauucucaau cucacagcac 2340
aucagagaca agaaccugau gaauucauug auaacauacc uaggcugugg uuccaucugg 2400
gagaagaaca agucugaguu cagauggcuc gaguucgucg uaaccaaauu cagcgauauc 2460
aacgacaaga ucauuccggu auuccaggaa aauacucuga uuggcgucaa acucgaggac 2520
uuugaagauu ggugcaaggu ugccaaauug aucaaagaga agaaacaccu gaccgaaucc 2580
gguuuggaug agauuaagaa aaucaagcug aacaugaaca aaggucgugg uggaggcggu 2640
agcggaggcg gagggucgcc gcuucugacc caacagaucc aagacgagga cgaucaguac 2700
agccuuguug ccagccuuga caacgugagg aaucucucca cuaucuugaa agcuauucau 2760
uuccgagaac augccaccug uuucgcaacu aagaacggua ucaaaguaac aguggagaau 2820
gcaaagugug ugcaagcaaa ugccuucauu caggcuggca ucuuccaaga guucaaaguu 2880
caggaagagu cuguuacuuu ccgaaucaac cuaacugucc ucuuagacug uuuaucuauc 2940
uucggaucaa guccaaugcc agggacuuua acugcacucc gaauguguua ccaagguuac 3000
ggauacccuu ugaugcuguu ccucgaagaa ggaggagugg ugacagucug caagaucaau 3060
acacaggaac cagaggagac ccuggacuuc gauuucugcu ccaccaaugu uaucaacaag 3120
aucauucucc agucagaagg gcuccgugaa gcauucucug aauuggauau gacgagugaa 3180
guccuacaga ucaccauguc uccugacaag ccuuacuuca gguuaucuac uuucggaaau 3240
gcaggaaguu cccaccuuga cuaucccaaa gauucugauu ugauggaggc auuucauugu 3300
aaucagacuc aagugaacag auacaagaua ucccuacuga aacccucuac aaaggcauua 3360
guccuaucuu guaagguauc uauucggaca gauaacagag gcuuccuuuc auuacaguau 3420
augauuagaa augaagaugg acaaaucugc uuuguggagu auuacugcug cccugaugag 3480
gaaguuccug aaucugaguc uuga 3504
<210> 37
<211> 1167
<212> PRT
<213> 人工序列
<220>
<223> 在实验室中制备-低活性TCRα megaTAL-hsRad1蛋白质构建体
<400> 37
Met Gly Ser Cys Arg Pro Pro Lys Lys Lys Arg Lys Val Val Asp Leu
1 5 10 15
Arg Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys
20 25 30
Val Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val Gly His Gly
35 40 45
Phe Thr His Ala His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu
50 55 60
Gly Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr Ala Leu Pro Glu
65 70 75 80
Ala Thr His Glu Asp Ile Val Gly Val Gly Lys Gln Trp Ser Gly Ala
85 90 95
Arg Ala Leu Glu Ala Leu Leu Thr Asp Ala Gly Glu Leu Arg Gly Pro
100 105 110
Pro Leu Gln Leu Asp Thr Gly Gln Leu Val Lys Ile Ala Lys Arg Gly
115 120 125
Gly Val Thr Ala Met Glu Ala Val His Ala Ser Arg Asn Ala Leu Thr
130 135 140
Gly Ala Pro Leu Asn Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
145 150 155 160
Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
165 170 175
Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
180 185 190
Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
195 200 205
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val
210 215 220
Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
225 230 235 240
Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln
245 250 255
Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr
260 265 270
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro
275 280 285
Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu
290 295 300
Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu
305 310 315 320
Thr Pro Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln
325 330 335
Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His
340 345 350
Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly
355 360 365
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
370 375 380
Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Asn
385 390 395 400
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu
405 410 415
Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
420 425 430
Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
435 440 445
Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
450 455 460
Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
465 470 475 480
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val
485 490 495
Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Ser Ile Val
500 505 510
Ala Gln Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu Thr Asn Asp
515 520 525
His Leu Val Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala Met Asp Ala
530 535 540
Val Lys Lys Gly Leu Pro His Ala Pro Glu Leu Ile Arg Arg Val Asn
545 550 555 560
Arg Arg Ile Gly Glu Arg Thr Ser His Arg Val Ala Ile Ser Arg Val
565 570 575
Gly Gly Ser Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr Gly
580 585 590
Phe Ala Asp Ala Glu Gly Ser Phe Ile Leu Asp Ile Arg Asn Arg Asn
595 600 605
Asn Glu Ser Asn Arg Tyr Arg Thr Ser Leu Arg Phe Gln Ile Thr Leu
610 615 620
His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp Lys
625 630 635 640
Val Gly Lys Ile Thr Asn Ser Ser Asp Arg Ala Val Met Leu Arg Val
645 650 655
Thr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys Tyr
660 665 670
Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln Ala
675 680 685
Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile Lys
690 695 700
Glu Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly Leu Asn Asp Glu
705 710 715 720
Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Pro Leu Ile
725 730 735
Asn Lys Asn Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe Thr Ala Gly
740 745 750
Glu Gly Tyr Phe Gly Val Asn Leu Lys Lys Val Lys Gly Thr Ala Lys
755 760 765
Val Tyr Val Gly Leu Arg Phe Ser Ile Ser Gln His Ile Arg Asp Lys
770 775 780
Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Ser Ile Trp
785 790 795 800
Glu Lys Asn Lys Ser Glu Phe Arg Trp Leu Glu Phe Val Val Thr Lys
805 810 815
Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn Thr
820 825 830
Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val Ala
835 840 845
Lys Leu Ile Lys Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp Glu
850 855 860
Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Gly Gly Gly Gly
865 870 875 880
Ser Gly Gly Gly Gly Ser Pro Leu Leu Thr Gln Gln Ile Gln Asp Glu
885 890 895
Asp Asp Gln Tyr Ser Leu Val Ala Ser Leu Asp Asn Val Arg Asn Leu
900 905 910
Ser Thr Ile Leu Lys Ala Ile His Phe Arg Glu His Ala Thr Cys Phe
915 920 925
Ala Thr Lys Asn Gly Ile Lys Val Thr Val Glu Asn Ala Lys Cys Val
930 935 940
Gln Ala Asn Ala Phe Ile Gln Ala Gly Ile Phe Gln Glu Phe Lys Val
945 950 955 960
Gln Glu Glu Ser Val Thr Phe Arg Ile Asn Leu Thr Val Leu Leu Asp
965 970 975
Cys Leu Ser Ile Phe Gly Ser Ser Pro Met Pro Gly Thr Leu Thr Ala
980 985 990
Leu Arg Met Cys Tyr Gln Gly Tyr Gly Tyr Pro Leu Met Leu Phe Leu
995 1000 1005
Glu Glu Gly Gly Val Val Thr Val Cys Lys Ile Asn Thr Gln Glu
1010 1015 1020
Pro Glu Glu Thr Leu Asp Phe Asp Phe Cys Ser Thr Asn Val Ile
1025 1030 1035
Asn Lys Ile Ile Leu Gln Ser Glu Gly Leu Arg Glu Ala Phe Ser
1040 1045 1050
Glu Leu Asp Met Thr Ser Glu Val Leu Gln Ile Thr Met Ser Pro
1055 1060 1065
Asp Lys Pro Tyr Phe Arg Leu Ser Thr Phe Gly Asn Ala Gly Ser
1070 1075 1080
Ser His Leu Asp Tyr Pro Lys Asp Ser Asp Leu Met Glu Ala Phe
1085 1090 1095
His Cys Asn Gln Thr Gln Val Asn Arg Tyr Lys Ile Ser Leu Leu
1100 1105 1110
Lys Pro Ser Thr Lys Ala Leu Val Leu Ser Cys Lys Val Ser Ile
1115 1120 1125
Arg Thr Asp Asn Arg Gly Phe Leu Ser Leu Gln Tyr Met Ile Arg
1130 1135 1140
Asn Glu Asp Gly Gln Ile Cys Phe Val Glu Tyr Tyr Cys Cys Pro
1145 1150 1155
Asp Glu Glu Val Pro Glu Ser Glu Ser
1160 1165
<210> 38
<211> 3870
<212> DNA
<213> 人工序列
<220>
<223> 在实验室中制备-低活性TCRα megaTAL-hsRad9a DNA构建体
<400> 38
atgggatcct gcaggccacc taagaagaaa cgcaaagtcg tggatctacg cacgctcggc 60
tacagtcagc agcagcaaga gaagatcaaa ccgaaggtgc gttcgacagt ggcgcagcac 120
cacgaggcac tggtgggcca tgggtttaca cacgcgcaca tcgttgcgct cagccaacac 180
ccggcagcgt tagggaccgt cgctgtcacg tatcagcaca taatcacggc gttgccagag 240
gcgacacacg aagacatcgt tggcgtcggc aaacagtggt ccggcgcacg cgccctggag 300
gccttgctca cggatgcggg ggagttgaga ggtccgccgt tacagttgga cacaggccaa 360
cttgtgaaga ttgcaaaacg tggcggcgtg accgcaatgg aggcagtgca tgcatcgcgc 420
aatgcactga cgggtgcccc cctgaacctg accccggacc aagtggtggc tatcgccagc 480
aacattggcg gcaagcaagc gctcgaaacg gtgcagcggc tgttgccggt gctgtgccag 540
gaccatggcc tgaccccgga ccaagtggtg gctatcgcca gcaacattgg cggcaagcaa 600
gcgctcgaaa cggtgcagcg gctgttgccg gtgctgtgcc aggaccatgg cctgaccccg 660
gaccaagtgg tggctatcgc cagcaacatt ggcggcaagc aagcgctcga aacggtgcag 720
cggctgttgc cggtgctgtg ccaggaccat ggcctgaccc cggaccaagt ggtggctatc 780
gccagcaacg gtggcggcaa gcaagcgctc gaaacggtgc agcggctgtt gccggtgctg 840
tgccaggacc atggcctgac tccggaccaa gtggtggcta tcgccagcca cgatggcggc 900
aagcaagcgc tcgaaacggt gcagcggctg ttgccggtgc tgtgccagga ccatggcctg 960
actccggacc aagtggtggc tatcgccagc cacgatggcg gcaagcaagc gctcgaaacg 1020
gtgcagcggc tgttgccggt gctgtgccag gaccatggcc tgaccccgga ccaagtggtg 1080
gctatcgcca gcaacattgg cggcaagcaa gcgctcgaaa cggtgcagcg gctgttgccg 1140
gtgctgtgcc aggaccatgg cctgaccccg gaccaagtgg tggctatcgc cagcaacaat 1200
ggcggcaagc aagcgctcga aacggtgcag cggctgttgc cggtgctgtg ccaggaccat 1260
ggcctgaccc cggaccaagt ggtggctatc gccagcaacg gtggcggcaa gcaagcgctc 1320
gaaacggtgc agcggctgtt gccggtgctg tgccaggacc atggcctgac cccggaccaa 1380
gtggtggcta tcgccagcaa caatggcggc aagcaagcgc tcgaaacggt gcagcggctg 1440
ttgccggtgc tgtgccagga ccatggcctg accccggacc aagtggtggc tatcgccagc 1500
aacattggcg gcaagcaagc gctcgaaagc attgtggccc agctgagccg gcctgatccg 1560
gcgttggccg cgttgaccaa cgaccacctc gtcgccttgg cctgcctcgg cggacgtcct 1620
gccatggatg cagtgaaaaa gggattgccg cacgcgccgg aattgatcag aagagtcaat 1680
cgccgtattg gcgaacgcac gtcccatcgc gttgcgatat ctagagtggg aggaagctct 1740
cgcagagagt ccatcaaccc atggattctg actggtttcg ctgatgccga aggatcattc 1800
atactagaca tccgcaaccg aaacaacgaa agcaacagat accgaacttc gctgagattc 1860
cagatcaccc tgcacaacaa ggacaaatcg attctggaga atatccagtc cacttggaag 1920
gtcggcaaga tcacaaacag cagtgacaga gccgtcatgc tgagggtcac ccgtttcgaa 1980
gatttgaaag tgattatcga ccacttcgag aaatatccgc tgattaccca gaaattgggc 2040
gattacaagt tgtttaaaca ggcattcagc gtcatggaga ataaagaaca tcttaaggag 2100
aatgggatta aggagctcgt acgaatcaaa gctaagatga attggggtct caatgacgaa 2160
ttgaaaaaag catttccaga gaacattagc aaagagcgcc cccttatcaa taagaacatt 2220
ccgaatttca aatggctggc tggattcaca gctggtgaag gctacttcgg cgtgaatcta 2280
aaaaaggtaa agggcaccgc aaaggtatac gtgggactga gattctcaat ctcacagcac 2340
atcagagaca agaacctgat gaattcattg ataacatacc taggctgtgg ttccatctgg 2400
gagaagaaca agtctgagtt cagatggctc gagttcgtcg taaccaaatt cagcgatatc 2460
aacgacaaga tcattccggt attccaggaa aatactctga ttggcgtcaa actcgaggac 2520
tttgaagatt ggtgcaaggt tgccaaattg atcaaagaga agaaacacct gaccgaatcc 2580
ggtttggatg agattaagaa aatcaagctg aacatgaaca aaggtcgtgg tggaggcggt 2640
agcggaggcg gagggtcggc tagcggcaaa ggagggttct tcagcaggtg gtggcggagc 2700
gggaggacga tagggcaagt gtgtgagcag aagcagccag agggctgggt ctgtggctgc 2760
cctctgacca cgtccctctc cctgctcttc gtggcccaac tctccctccg aacggtgaac 2820
tcctcccgct ctgcctatgc ctgctttctc tttgctccgc tcttcttcca gcaataccag 2880
gcagccactc ctggtcaaga cctgctgcga tgtaagattc tgatgaagtc tttcctgtct 2940
gtcttccgct cactggcgat gctggagaag acggtggaga agtgctgcat ctccctgaat 3000
ggtcggagca gtcgcctggt agtccaactg cattgcaagt tcggcgtgcg gaagactcac 3060
aacctgtcct tccaggactg tgagtccctt caggccgtct ttgacccagc ctcgtgccct 3120
cacatgctcc gagcaccagc acgggttctg ggagaggctg ttctgccctt ctctcctgca 3180
ctggctgaag tgacgctggg cattggccgt ggacgcaggg tcatcctgcg tagttaccac 3240
gaggaggagg cagacagcac tgccaaagcg atggtgactg agatgtgcct tggagaggag 3300
gatttccagc aactacaggc ccaggaagga gttgccatca ctttctgcct caaggagttc 3360
cgagggctcc tgagtttcgc agagtcagca aacttgaatc ttagcattca tttcgatgct 3420
ccaggcaggc cagccatctt caccatcaag gactctttgc tggacggcca ctttgtctta 3480
gccacactct cagacaccga ctcgcactcc caagacctgg gctcaccaga gcgtcaccag 3540
ccagtgcctc aactccaggc tcacagcaca cctcaccctg acgactttgc caatgacgac 3600
attgactctt acatgatagc gatggaaacc actataggca atgagggctc gcgtgtgctg 3660
ccctccattt ccctttcacc tggccctcag cctcccaaga gcccaggacc tcactccgag 3720
gaggaagatg aggctgagcc cagcacagtg cctgggactc ctccacccaa gaagttccgc 3780
tcactgttct tcggctccat cctggctcct gttcgctcac cacagggtcc tagccctgtg 3840
ctggcggaag acagtgaggg tgaaggctga 3870
<210> 39
<211> 3870
<212> RNA
<213> 人工序列
<220>
<223> 在实验室中制备-低活性TCRα megaTAL-hsRad9a RNA构建体
<400> 39
augggauccu gcaggccacc uaagaagaaa cgcaaagucg uggaucuacg cacgcucggc 60
uacagucagc agcagcaaga gaagaucaaa ccgaaggugc guucgacagu ggcgcagcac 120
cacgaggcac uggugggcca uggguuuaca cacgcgcaca ucguugcgcu cagccaacac 180
ccggcagcgu uagggaccgu cgcugucacg uaucagcaca uaaucacggc guugccagag 240
gcgacacacg aagacaucgu uggcgucggc aaacaguggu ccggcgcacg cgcccuggag 300
gccuugcuca cggaugcggg ggaguugaga gguccgccgu uacaguugga cacaggccaa 360
cuugugaaga uugcaaaacg uggcggcgug accgcaaugg aggcagugca ugcaucgcgc 420
aaugcacuga cgggugcccc ccugaaccug accccggacc aagugguggc uaucgccagc 480
aacauuggcg gcaagcaagc gcucgaaacg gugcagcggc uguugccggu gcugugccag 540
gaccauggcc ugaccccgga ccaaguggug gcuaucgcca gcaacauugg cggcaagcaa 600
gcgcucgaaa cggugcagcg gcuguugccg gugcugugcc aggaccaugg ccugaccccg 660
gaccaagugg uggcuaucgc cagcaacauu ggcggcaagc aagcgcucga aacggugcag 720
cggcuguugc cggugcugug ccaggaccau ggccugaccc cggaccaagu gguggcuauc 780
gccagcaacg guggcggcaa gcaagcgcuc gaaacggugc agcggcuguu gccggugcug 840
ugccaggacc auggccugac uccggaccaa gugguggcua ucgccagcca cgauggcggc 900
aagcaagcgc ucgaaacggu gcagcggcug uugccggugc ugugccagga ccauggccug 960
acuccggacc aagugguggc uaucgccagc cacgauggcg gcaagcaagc gcucgaaacg 1020
gugcagcggc uguugccggu gcugugccag gaccauggcc ugaccccgga ccaaguggug 1080
gcuaucgcca gcaacauugg cggcaagcaa gcgcucgaaa cggugcagcg gcuguugccg 1140
gugcugugcc aggaccaugg ccugaccccg gaccaagugg uggcuaucgc cagcaacaau 1200
ggcggcaagc aagcgcucga aacggugcag cggcuguugc cggugcugug ccaggaccau 1260
ggccugaccc cggaccaagu gguggcuauc gccagcaacg guggcggcaa gcaagcgcuc 1320
gaaacggugc agcggcuguu gccggugcug ugccaggacc auggccugac cccggaccaa 1380
gugguggcua ucgccagcaa caauggcggc aagcaagcgc ucgaaacggu gcagcggcug 1440
uugccggugc ugugccagga ccauggccug accccggacc aagugguggc uaucgccagc 1500
aacauuggcg gcaagcaagc gcucgaaagc auuguggccc agcugagccg gccugauccg 1560
gcguuggccg cguugaccaa cgaccaccuc gucgccuugg ccugccucgg cggacguccu 1620
gccauggaug cagugaaaaa gggauugccg cacgcgccgg aauugaucag aagagucaau 1680
cgccguauug gcgaacgcac gucccaucgc guugcgauau cuagaguggg aggaagcucu 1740
cgcagagagu ccaucaaccc auggauucug acugguuucg cugaugccga aggaucauuc 1800
auacuagaca uccgcaaccg aaacaacgaa agcaacagau accgaacuuc gcugagauuc 1860
cagaucaccc ugcacaacaa ggacaaaucg auucuggaga auauccaguc cacuuggaag 1920
gucggcaaga ucacaaacag cagugacaga gccgucaugc ugagggucac ccguuucgaa 1980
gauuugaaag ugauuaucga ccacuucgag aaauauccgc ugauuaccca gaaauugggc 2040
gauuacaagu uguuuaaaca ggcauucagc gucauggaga auaaagaaca ucuuaaggag 2100
aaugggauua aggagcucgu acgaaucaaa gcuaagauga auuggggucu caaugacgaa 2160
uugaaaaaag cauuuccaga gaacauuagc aaagagcgcc cccuuaucaa uaagaacauu 2220
ccgaauuuca aauggcuggc uggauucaca gcuggugaag gcuacuucgg cgugaaucua 2280
aaaaagguaa agggcaccgc aaagguauac gugggacuga gauucucaau cucacagcac 2340
aucagagaca agaaccugau gaauucauug auaacauacc uaggcugugg uuccaucugg 2400
gagaagaaca agucugaguu cagauggcuc gaguucgucg uaaccaaauu cagcgauauc 2460
aacgacaaga ucauuccggu auuccaggaa aauacucuga uuggcgucaa acucgaggac 2520
uuugaagauu ggugcaaggu ugccaaauug aucaaagaga agaaacaccu gaccgaaucc 2580
gguuuggaug agauuaagaa aaucaagcug aacaugaaca aaggucgugg uggaggcggu 2640
agcggaggcg gagggucggc uagcggcaaa ggaggguucu ucagcaggug guggcggagc 2700
gggaggacga uagggcaagu gugugagcag aagcagccag agggcugggu cuguggcugc 2760
ccucugacca cgucccucuc ccugcucuuc guggcccaac ucucccuccg aacggugaac 2820
uccucccgcu cugccuaugc cugcuuucuc uuugcuccgc ucuucuucca gcaauaccag 2880
gcagccacuc cuggucaaga ccugcugcga uguaagauuc ugaugaaguc uuuccugucu 2940
gucuuccgcu cacuggcgau gcuggagaag acgguggaga agugcugcau cucccugaau 3000
ggucggagca gucgccuggu aguccaacug cauugcaagu ucggcgugcg gaagacucac 3060
aaccuguccu uccaggacug ugagucccuu caggccgucu uugacccagc cucgugcccu 3120
cacaugcucc gagcaccagc acggguucug ggagaggcug uucugcccuu cucuccugca 3180
cuggcugaag ugacgcuggg cauuggccgu ggacgcaggg ucauccugcg uaguuaccac 3240
gaggaggagg cagacagcac ugccaaagcg auggugacug agaugugccu uggagaggag 3300
gauuuccagc aacuacaggc ccaggaagga guugccauca cuuucugccu caaggaguuc 3360
cgagggcucc ugaguuucgc agagucagca aacuugaauc uuagcauuca uuucgaugcu 3420
ccaggcaggc cagccaucuu caccaucaag gacucuuugc uggacggcca cuuugucuua 3480
gccacacucu cagacaccga cucgcacucc caagaccugg gcucaccaga gcgucaccag 3540
ccagugccuc aacuccaggc ucacagcaca ccucacccug acgacuuugc caaugacgac 3600
auugacucuu acaugauagc gauggaaacc acuauaggca augagggcuc gcgugugcug 3660
cccuccauuu cccuuucacc uggcccucag ccucccaaga gcccaggacc ucacuccgag 3720
gaggaagaug aggcugagcc cagcacagug ccugggacuc cuccacccaa gaaguuccgc 3780
ucacuguucu ucggcuccau ccuggcuccu guucgcucac cacagggucc uagcccugug 3840
cuggcggaag acagugaggg ugaaggcuga 3870
<210> 40
<211> 1289
<212> PRT
<213> 人工序列
<220>
<223> 在实验室中制备-低活性TCRα megaTAL-hsRad9a蛋白质构建体
<400> 40
Met Gly Ser Cys Arg Pro Pro Lys Lys Lys Arg Lys Val Val Asp Leu
1 5 10 15
Arg Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys
20 25 30
Val Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val Gly His Gly
35 40 45
Phe Thr His Ala His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu
50 55 60
Gly Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr Ala Leu Pro Glu
65 70 75 80
Ala Thr His Glu Asp Ile Val Gly Val Gly Lys Gln Trp Ser Gly Ala
85 90 95
Arg Ala Leu Glu Ala Leu Leu Thr Asp Ala Gly Glu Leu Arg Gly Pro
100 105 110
Pro Leu Gln Leu Asp Thr Gly Gln Leu Val Lys Ile Ala Lys Arg Gly
115 120 125
Gly Val Thr Ala Met Glu Ala Val His Ala Ser Arg Asn Ala Leu Thr
130 135 140
Gly Ala Pro Leu Asn Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
145 150 155 160
Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
165 170 175
Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
180 185 190
Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
195 200 205
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val
210 215 220
Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
225 230 235 240
Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln
245 250 255
Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr
260 265 270
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro
275 280 285
Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu
290 295 300
Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu
305 310 315 320
Thr Pro Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln
325 330 335
Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His
340 345 350
Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly
355 360 365
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
370 375 380
Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Asn
385 390 395 400
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu
405 410 415
Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
420 425 430
Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
435 440 445
Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
450 455 460
Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
465 470 475 480
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val
485 490 495
Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Ser Ile Val
500 505 510
Ala Gln Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu Thr Asn Asp
515 520 525
His Leu Val Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala Met Asp Ala
530 535 540
Val Lys Lys Gly Leu Pro His Ala Pro Glu Leu Ile Arg Arg Val Asn
545 550 555 560
Arg Arg Ile Gly Glu Arg Thr Ser His Arg Val Ala Ile Ser Arg Val
565 570 575
Gly Gly Ser Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr Gly
580 585 590
Phe Ala Asp Ala Glu Gly Ser Phe Ile Leu Asp Ile Arg Asn Arg Asn
595 600 605
Asn Glu Ser Asn Arg Tyr Arg Thr Ser Leu Arg Phe Gln Ile Thr Leu
610 615 620
His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp Lys
625 630 635 640
Val Gly Lys Ile Thr Asn Ser Ser Asp Arg Ala Val Met Leu Arg Val
645 650 655
Thr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys Tyr
660 665 670
Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln Ala
675 680 685
Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile Lys
690 695 700
Glu Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly Leu Asn Asp Glu
705 710 715 720
Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Pro Leu Ile
725 730 735
Asn Lys Asn Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe Thr Ala Gly
740 745 750
Glu Gly Tyr Phe Gly Val Asn Leu Lys Lys Val Lys Gly Thr Ala Lys
755 760 765
Val Tyr Val Gly Leu Arg Phe Ser Ile Ser Gln His Ile Arg Asp Lys
770 775 780
Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Ser Ile Trp
785 790 795 800
Glu Lys Asn Lys Ser Glu Phe Arg Trp Leu Glu Phe Val Val Thr Lys
805 810 815
Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn Thr
820 825 830
Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val Ala
835 840 845
Lys Leu Ile Lys Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp Glu
850 855 860
Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Gly Gly Gly Gly
865 870 875 880
Ser Gly Gly Gly Gly Ser Ala Ser Gly Lys Gly Gly Phe Phe Ser Arg
885 890 895
Trp Trp Arg Ser Gly Arg Thr Ile Gly Gln Val Cys Glu Gln Lys Gln
900 905 910
Pro Glu Gly Trp Val Cys Gly Cys Pro Leu Thr Thr Ser Leu Ser Leu
915 920 925
Leu Phe Val Ala Gln Leu Ser Leu Arg Thr Val Asn Ser Ser Arg Ser
930 935 940
Ala Tyr Ala Cys Phe Leu Phe Ala Pro Leu Phe Phe Gln Gln Tyr Gln
945 950 955 960
Ala Ala Thr Pro Gly Gln Asp Leu Leu Arg Cys Lys Ile Leu Met Lys
965 970 975
Ser Phe Leu Ser Val Phe Arg Ser Leu Ala Met Leu Glu Lys Thr Val
980 985 990
Glu Lys Cys Cys Ile Ser Leu Asn Gly Arg Ser Ser Arg Leu Val Val
995 1000 1005
Gln Leu His Cys Lys Phe Gly Val Arg Lys Thr His Asn Leu Ser
1010 1015 1020
Phe Gln Asp Cys Glu Ser Leu Gln Ala Val Phe Asp Pro Ala Ser
1025 1030 1035
Cys Pro His Met Leu Arg Ala Pro Ala Arg Val Leu Gly Glu Ala
1040 1045 1050
Val Leu Pro Phe Ser Pro Ala Leu Ala Glu Val Thr Leu Gly Ile
1055 1060 1065
Gly Arg Gly Arg Arg Val Ile Leu Arg Ser Tyr His Glu Glu Glu
1070 1075 1080
Ala Asp Ser Thr Ala Lys Ala Met Val Thr Glu Met Cys Leu Gly
1085 1090 1095
Glu Glu Asp Phe Gln Gln Leu Gln Ala Gln Glu Gly Val Ala Ile
1100 1105 1110
Thr Phe Cys Leu Lys Glu Phe Arg Gly Leu Leu Ser Phe Ala Glu
1115 1120 1125
Ser Ala Asn Leu Asn Leu Ser Ile His Phe Asp Ala Pro Gly Arg
1130 1135 1140
Pro Ala Ile Phe Thr Ile Lys Asp Ser Leu Leu Asp Gly His Phe
1145 1150 1155
Val Leu Ala Thr Leu Ser Asp Thr Asp Ser His Ser Gln Asp Leu
1160 1165 1170
Gly Ser Pro Glu Arg His Gln Pro Val Pro Gln Leu Gln Ala His
1175 1180 1185
Ser Thr Pro His Pro Asp Asp Phe Ala Asn Asp Asp Ile Asp Ser
1190 1195 1200
Tyr Met Ile Ala Met Glu Thr Thr Ile Gly Asn Glu Gly Ser Arg
1205 1210 1215
Val Leu Pro Ser Ile Ser Leu Ser Pro Gly Pro Gln Pro Pro Lys
1220 1225 1230
Ser Pro Gly Pro His Ser Glu Glu Glu Asp Glu Ala Glu Pro Ser
1235 1240 1245
Thr Val Pro Gly Thr Pro Pro Pro Lys Lys Phe Arg Ser Leu Phe
1250 1255 1260
Phe Gly Ser Ile Leu Ala Pro Val Arg Ser Pro Gln Gly Pro Ser
1265 1270 1275
Pro Val Leu Ala Glu Asp Ser Glu Gly Glu Gly
1280 1285
<210> 41
<211> 4086
<212> DNA
<213> 人工序列
<220>
<223> 在实验室中制备-低活性TCRα megaTAL-ExoI DNA构建体
<400> 41
atgggatcct gcaggccacc taagaagaaa cgcaaagtcg tggatctacg cacgctcggc 60
tacagtcagc agcagcaaga gaagatcaaa ccgaaggtgc gttcgacagt ggcgcagcac 120
cacgaggcac tggtgggcca tgggtttaca cacgcgcaca tcgttgcgct cagccaacac 180
ccggcagcgt tagggaccgt cgctgtcacg tatcagcaca taatcacggc gttgccagag 240
gcgacacacg aagacatcgt tggcgtcggc aaacagtggt ccggcgcacg cgccctggag 300
gccttgctca cggatgcggg ggagttgaga ggtccgccgt tacagttgga cacaggccaa 360
cttgtgaaga ttgcaaaacg tggcggcgtg accgcaatgg aggcagtgca tgcatcgcgc 420
aatgcactga cgggtgcccc cctgaacctg accccggacc aagtggtggc tatcgccagc 480
aacattggcg gcaagcaagc gctcgaaacg gtgcagcggc tgttgccggt gctgtgccag 540
gaccatggcc tgaccccgga ccaagtggtg gctatcgcca gcaacattgg cggcaagcaa 600
gcgctcgaaa cggtgcagcg gctgttgccg gtgctgtgcc aggaccatgg cctgaccccg 660
gaccaagtgg tggctatcgc cagcaacatt ggcggcaagc aagcgctcga aacggtgcag 720
cggctgttgc cggtgctgtg ccaggaccat ggcctgaccc cggaccaagt ggtggctatc 780
gccagcaacg gtggcggcaa gcaagcgctc gaaacggtgc agcggctgtt gccggtgctg 840
tgccaggacc atggcctgac tccggaccaa gtggtggcta tcgccagcca cgatggcggc 900
aagcaagcgc tcgaaacggt gcagcggctg ttgccggtgc tgtgccagga ccatggcctg 960
actccggacc aagtggtggc tatcgccagc cacgatggcg gcaagcaagc gctcgaaacg 1020
gtgcagcggc tgttgccggt gctgtgccag gaccatggcc tgaccccgga ccaagtggtg 1080
gctatcgcca gcaacattgg cggcaagcaa gcgctcgaaa cggtgcagcg gctgttgccg 1140
gtgctgtgcc aggaccatgg cctgaccccg gaccaagtgg tggctatcgc cagcaacaat 1200
ggcggcaagc aagcgctcga aacggtgcag cggctgttgc cggtgctgtg ccaggaccat 1260
ggcctgaccc cggaccaagt ggtggctatc gccagcaacg gtggcggcaa gcaagcgctc 1320
gaaacggtgc agcggctgtt gccggtgctg tgccaggacc atggcctgac cccggaccaa 1380
gtggtggcta tcgccagcaa caatggcggc aagcaagcgc tcgaaacggt gcagcggctg 1440
ttgccggtgc tgtgccagga ccatggcctg accccggacc aagtggtggc tatcgccagc 1500
aacattggcg gcaagcaagc gctcgaaagc attgtggccc agctgagccg gcctgatccg 1560
gcgttggccg cgttgaccaa cgaccacctc gtcgccttgg cctgcctcgg cggacgtcct 1620
gccatggatg cagtgaaaaa gggattgccg cacgcgccgg aattgatcag aagagtcaat 1680
cgccgtattg gcgaacgcac gtcccatcgc gttgcgatat ctagagtggg aggaagctct 1740
cgcagagagt ccatcaaccc atggattctg actggtttcg ctgatgccga aggatcattc 1800
atactagaca tccgcaaccg aaacaacgaa agcaacagat accgaacttc gctgagattc 1860
cagatcaccc tgcacaacaa ggacaaatcg attctggaga atatccagtc cacttggaag 1920
gtcggcaaga tcacaaacag cagtgacaga gccgtcatgc tgagggtcac ccgtttcgaa 1980
gatttgaaag tgattatcga ccacttcgag aaatatccgc tgattaccca gaaattgggc 2040
gattacaagt tgtttaaaca ggcattcagc gtcatggaga ataaagaaca tcttaaggag 2100
aatgggatta aggagctcgt acgaatcaaa gctaagatga attggggtct caatgacgaa 2160
ttgaaaaaag catttccaga gaacattagc aaagagcgcc cccttatcaa taagaacatt 2220
ccgaatttca aatggctggc tggattcaca gctggtgaag gctacttcgg cgtgaatcta 2280
aaaaaggtaa agggcaccgc aaaggtatac gtgggactga gattctcaat ctcacagcac 2340
atcagagaca agaacctgat gaattcattg ataacatacc taggctgtgg ttccatctgg 2400
gagaagaaca agtctgagtt cagatggctc gagttcgtcg taaccaaatt cagcgatatc 2460
aacgacaaga tcattccggt attccaggaa aatactctga ttggcgtcaa actcgaggac 2520
tttgaagatt ggtgcaaggt tgccaaattg atcaaagaga agaaacacct gaccgaatcc 2580
ggtttggatg agattaagaa aatcaagctg aacatgaaca aaggtcgtgg tggaggcggt 2640
agcggaggcg gagggtcggc tagcaacgat ggcaagcagc aaagtacatt ccttttccac 2700
gactacgaaa cctttggaac ccaccccgct ctggatcggc cagcacagtt cgctgccata 2760
agaaccgaca acgagttcaa tgtgatcggt gaacccgagg tgttttactg caaacctgcg 2820
gacgattatc tgcctcaacc gggagccgtt ctgattacgg gaattacccc ccaagaagcg 2880
cgtgccaagg gcgaaaatga agctgcattt gccgctagaa tccatagcct gtttactgtt 2940
ccgaagacgt gtatcttggg atacaacaat gtacgctttg atgacgaagt aactcgtaac 3000
gtgttctatc ggaacttcta tgacccttac gcctggagtt ggcagcacga caattctcga 3060
tgggatcttc tggatgtgat gagagcctgc tacgctctca ggccagaggg cattaactgg 3120
cctgagaacg acgatgggct tccgtcattt cgccttgagc atcttaccaa agcgaatggc 3180
atcgaacact ccaatgcaca cgacgctatg gctgatgtct acgcgactat tgcaatggcg 3240
aaattggtca agacaagaca gccgaggttg tttgactacc tgtttacgca tcgaaacaaa 3300
cataagctca tggcactgat agatgtccct caaatgaagc ccctcgttca cgtcagcggt 3360
atgttcggag cgtggagagg aaacacgtcc tgggttgctc ctctggcatg gcatccagag 3420
aacagaaacg cggtaatcat ggtcgatctc gcaggagaca ttagcccact tctcgaattg 3480
gattccgaca ccttgcgaga acgcctttac acagcgaaaa ctgatttggg tgataacgct 3540
gccgtacctg tcaaattggt acatatcaac aaatgtccgg tgctcgcgca agccaacacc 3600
ctgagacctg aagacgcgga tagactgggg ataaaccggc agcactgttt ggataatctc 3660
aagatactca gggagaatcc gcaggtgcgc gaaaaagtcg tagccatctt tgcggaggca 3720
gaaccattta cgccgtctga caatgtagat gctcagttat acaatggctt ctttagtgac 3780
gctgaccgag ctgcgatgaa gattgtgcta gagactgaac cgagaaacct ctctgcattg 3840
gacatcactt ttgtcgataa gcgaatagag aagttgctgt tcaactatcg tgcccgaaac 3900
tttccaggga cactcgatta cgcagagcaa caaaggtggc ttgagcatcg caggcaggtg 3960
tttaccccag agttcttgca gggttacgct gaggagattc aaatgttggc acagcagtat 4020
gcagatgata aggagaaagt cgcgttgctg aaggctctgt ggcaatacgc cgaagagata 4080
gtgtga 4086
<210> 42
<211> 4086
<212> RNA
<213> 人工序列
<220>
<223> 在实验室中制备-低活性TCRα megaTAL-ExoI RNA构建体
<400> 42
augggauccu gcaggccacc uaagaagaaa cgcaaagucg uggaucuacg cacgcucggc 60
uacagucagc agcagcaaga gaagaucaaa ccgaaggugc guucgacagu ggcgcagcac 120
cacgaggcac uggugggcca uggguuuaca cacgcgcaca ucguugcgcu cagccaacac 180
ccggcagcgu uagggaccgu cgcugucacg uaucagcaca uaaucacggc guugccagag 240
gcgacacacg aagacaucgu uggcgucggc aaacaguggu ccggcgcacg cgcccuggag 300
gccuugcuca cggaugcggg ggaguugaga gguccgccgu uacaguugga cacaggccaa 360
cuugugaaga uugcaaaacg uggcggcgug accgcaaugg aggcagugca ugcaucgcgc 420
aaugcacuga cgggugcccc ccugaaccug accccggacc aagugguggc uaucgccagc 480
aacauuggcg gcaagcaagc gcucgaaacg gugcagcggc uguugccggu gcugugccag 540
gaccauggcc ugaccccgga ccaaguggug gcuaucgcca gcaacauugg cggcaagcaa 600
gcgcucgaaa cggugcagcg gcuguugccg gugcugugcc aggaccaugg ccugaccccg 660
gaccaagugg uggcuaucgc cagcaacauu ggcggcaagc aagcgcucga aacggugcag 720
cggcuguugc cggugcugug ccaggaccau ggccugaccc cggaccaagu gguggcuauc 780
gccagcaacg guggcggcaa gcaagcgcuc gaaacggugc agcggcuguu gccggugcug 840
ugccaggacc auggccugac uccggaccaa gugguggcua ucgccagcca cgauggcggc 900
aagcaagcgc ucgaaacggu gcagcggcug uugccggugc ugugccagga ccauggccug 960
acuccggacc aagugguggc uaucgccagc cacgauggcg gcaagcaagc gcucgaaacg 1020
gugcagcggc uguugccggu gcugugccag gaccauggcc ugaccccgga ccaaguggug 1080
gcuaucgcca gcaacauugg cggcaagcaa gcgcucgaaa cggugcagcg gcuguugccg 1140
gugcugugcc aggaccaugg ccugaccccg gaccaagugg uggcuaucgc cagcaacaau 1200
ggcggcaagc aagcgcucga aacggugcag cggcuguugc cggugcugug ccaggaccau 1260
ggccugaccc cggaccaagu gguggcuauc gccagcaacg guggcggcaa gcaagcgcuc 1320
gaaacggugc agcggcuguu gccggugcug ugccaggacc auggccugac cccggaccaa 1380
gugguggcua ucgccagcaa caauggcggc aagcaagcgc ucgaaacggu gcagcggcug 1440
uugccggugc ugugccagga ccauggccug accccggacc aagugguggc uaucgccagc 1500
aacauuggcg gcaagcaagc gcucgaaagc auuguggccc agcugagccg gccugauccg 1560
gcguuggccg cguugaccaa cgaccaccuc gucgccuugg ccugccucgg cggacguccu 1620
gccauggaug cagugaaaaa gggauugccg cacgcgccgg aauugaucag aagagucaau 1680
cgccguauug gcgaacgcac gucccaucgc guugcgauau cuagaguggg aggaagcucu 1740
cgcagagagu ccaucaaccc auggauucug acugguuucg cugaugccga aggaucauuc 1800
auacuagaca uccgcaaccg aaacaacgaa agcaacagau accgaacuuc gcugagauuc 1860
cagaucaccc ugcacaacaa ggacaaaucg auucuggaga auauccaguc cacuuggaag 1920
gucggcaaga ucacaaacag cagugacaga gccgucaugc ugagggucac ccguuucgaa 1980
gauuugaaag ugauuaucga ccacuucgag aaauauccgc ugauuaccca gaaauugggc 2040
gauuacaagu uguuuaaaca ggcauucagc gucauggaga auaaagaaca ucuuaaggag 2100
aaugggauua aggagcucgu acgaaucaaa gcuaagauga auuggggucu caaugacgaa 2160
uugaaaaaag cauuuccaga gaacauuagc aaagagcgcc cccuuaucaa uaagaacauu 2220
ccgaauuuca aauggcuggc uggauucaca gcuggugaag gcuacuucgg cgugaaucua 2280
aaaaagguaa agggcaccgc aaagguauac gugggacuga gauucucaau cucacagcac 2340
aucagagaca agaaccugau gaauucauug auaacauacc uaggcugugg uuccaucugg 2400
gagaagaaca agucugaguu cagauggcuc gaguucgucg uaaccaaauu cagcgauauc 2460
aacgacaaga ucauuccggu auuccaggaa aauacucuga uuggcgucaa acucgaggac 2520
uuugaagauu ggugcaaggu ugccaaauug aucaaagaga agaaacaccu gaccgaaucc 2580
gguuuggaug agauuaagaa aaucaagcug aacaugaaca aaggucgugg uggaggcggu 2640
agcggaggcg gagggucggc uagcaacgau ggcaagcagc aaaguacauu ccuuuuccac 2700
gacuacgaaa ccuuuggaac ccaccccgcu cuggaucggc cagcacaguu cgcugccaua 2760
agaaccgaca acgaguucaa ugugaucggu gaacccgagg uguuuuacug caaaccugcg 2820
gacgauuauc ugccucaacc gggagccguu cugauuacgg gaauuacccc ccaagaagcg 2880
cgugccaagg gcgaaaauga agcugcauuu gccgcuagaa uccauagccu guuuacuguu 2940
ccgaagacgu guaucuuggg auacaacaau guacgcuuug augacgaagu aacucguaac 3000
guguucuauc ggaacuucua ugacccuuac gccuggaguu ggcagcacga caauucucga 3060
ugggaucuuc uggaugugau gagagccugc uacgcucuca ggccagaggg cauuaacugg 3120
ccugagaacg acgaugggcu uccgucauuu cgccuugagc aucuuaccaa agcgaauggc 3180
aucgaacacu ccaaugcaca cgacgcuaug gcugaugucu acgcgacuau ugcaauggcg 3240
aaauugguca agacaagaca gccgagguug uuugacuacc uguuuacgca ucgaaacaaa 3300
cauaagcuca uggcacugau agaugucccu caaaugaagc cccucguuca cgucagcggu 3360
auguucggag cguggagagg aaacacgucc uggguugcuc cucuggcaug gcauccagag 3420
aacagaaacg cgguaaucau ggucgaucuc gcaggagaca uuagcccacu ucucgaauug 3480
gauuccgaca ccuugcgaga acgccuuuac acagcgaaaa cugauuuggg ugauaacgcu 3540
gccguaccug ucaaauuggu acauaucaac aaauguccgg ugcucgcgca agccaacacc 3600
cugagaccug aagacgcgga uagacugggg auaaaccggc agcacuguuu ggauaaucuc 3660
aagauacuca gggagaaucc gcaggugcgc gaaaaagucg uagccaucuu ugcggaggca 3720
gaaccauuua cgccgucuga caauguagau gcucaguuau acaauggcuu cuuuagugac 3780
gcugaccgag cugcgaugaa gauugugcua gagacugaac cgagaaaccu cucugcauug 3840
gacaucacuu uugucgauaa gcgaauagag aaguugcugu ucaacuaucg ugcccgaaac 3900
uuuccaggga cacucgauua cgcagagcaa caaagguggc uugagcaucg caggcaggug 3960
uuuaccccag aguucuugca ggguuacgcu gaggagauuc aaauguuggc acagcaguau 4020
gcagaugaua aggagaaagu cgcguugcug aaggcucugu ggcaauacgc cgaagagaua 4080
guguga 4086
<210> 43
<211> 1361
<212> PRT
<213> 人工序列
<220>
<223> 在实验室中制备-低活性TCRα megaTAL-ExoI蛋白质构建体
<400> 43
Met Gly Ser Cys Arg Pro Pro Lys Lys Lys Arg Lys Val Val Asp Leu
1 5 10 15
Arg Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys
20 25 30
Val Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val Gly His Gly
35 40 45
Phe Thr His Ala His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu
50 55 60
Gly Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr Ala Leu Pro Glu
65 70 75 80
Ala Thr His Glu Asp Ile Val Gly Val Gly Lys Gln Trp Ser Gly Ala
85 90 95
Arg Ala Leu Glu Ala Leu Leu Thr Asp Ala Gly Glu Leu Arg Gly Pro
100 105 110
Pro Leu Gln Leu Asp Thr Gly Gln Leu Val Lys Ile Ala Lys Arg Gly
115 120 125
Gly Val Thr Ala Met Glu Ala Val His Ala Ser Arg Asn Ala Leu Thr
130 135 140
Gly Ala Pro Leu Asn Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
145 150 155 160
Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
165 170 175
Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
180 185 190
Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
195 200 205
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val
210 215 220
Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
225 230 235 240
Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln
245 250 255
Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr
260 265 270
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro
275 280 285
Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu
290 295 300
Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu
305 310 315 320
Thr Pro Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln
325 330 335
Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His
340 345 350
Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly
355 360 365
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
370 375 380
Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Asn
385 390 395 400
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu
405 410 415
Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
420 425 430
Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
435 440 445
Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
450 455 460
Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
465 470 475 480
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val
485 490 495
Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Ser Ile Val
500 505 510
Ala Gln Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu Thr Asn Asp
515 520 525
His Leu Val Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala Met Asp Ala
530 535 540
Val Lys Lys Gly Leu Pro His Ala Pro Glu Leu Ile Arg Arg Val Asn
545 550 555 560
Arg Arg Ile Gly Glu Arg Thr Ser His Arg Val Ala Ile Ser Arg Val
565 570 575
Gly Gly Ser Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr Gly
580 585 590
Phe Ala Asp Ala Glu Gly Ser Phe Ile Leu Asp Ile Arg Asn Arg Asn
595 600 605
Asn Glu Ser Asn Arg Tyr Arg Thr Ser Leu Arg Phe Gln Ile Thr Leu
610 615 620
His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp Lys
625 630 635 640
Val Gly Lys Ile Thr Asn Ser Ser Asp Arg Ala Val Met Leu Arg Val
645 650 655
Thr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys Tyr
660 665 670
Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln Ala
675 680 685
Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile Lys
690 695 700
Glu Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly Leu Asn Asp Glu
705 710 715 720
Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Pro Leu Ile
725 730 735
Asn Lys Asn Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe Thr Ala Gly
740 745 750
Glu Gly Tyr Phe Gly Val Asn Leu Lys Lys Val Lys Gly Thr Ala Lys
755 760 765
Val Tyr Val Gly Leu Arg Phe Ser Ile Ser Gln His Ile Arg Asp Lys
770 775 780
Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Ser Ile Trp
785 790 795 800
Glu Lys Asn Lys Ser Glu Phe Arg Trp Leu Glu Phe Val Val Thr Lys
805 810 815
Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn Thr
820 825 830
Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val Ala
835 840 845
Lys Leu Ile Lys Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp Glu
850 855 860
Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Gly Gly Gly Gly
865 870 875 880
Ser Gly Gly Gly Gly Ser Ala Ser Asn Asp Gly Lys Gln Gln Ser Thr
885 890 895
Phe Leu Phe His Asp Tyr Glu Thr Phe Gly Thr His Pro Ala Leu Asp
900 905 910
Arg Pro Ala Gln Phe Ala Ala Ile Arg Thr Asp Asn Glu Phe Asn Val
915 920 925
Ile Gly Glu Pro Glu Val Phe Tyr Cys Lys Pro Ala Asp Asp Tyr Leu
930 935 940
Pro Gln Pro Gly Ala Val Leu Ile Thr Gly Ile Thr Pro Gln Glu Ala
945 950 955 960
Arg Ala Lys Gly Glu Asn Glu Ala Ala Phe Ala Ala Arg Ile His Ser
965 970 975
Leu Phe Thr Val Pro Lys Thr Cys Ile Leu Gly Tyr Asn Asn Val Arg
980 985 990
Phe Asp Asp Glu Val Thr Arg Asn Val Phe Tyr Arg Asn Phe Tyr Asp
995 1000 1005
Pro Tyr Ala Trp Ser Trp Gln His Asp Asn Ser Arg Trp Asp Leu
1010 1015 1020
Leu Asp Val Met Arg Ala Cys Tyr Ala Leu Arg Pro Glu Gly Ile
1025 1030 1035
Asn Trp Pro Glu Asn Asp Asp Gly Leu Pro Ser Phe Arg Leu Glu
1040 1045 1050
His Leu Thr Lys Ala Asn Gly Ile Glu His Ser Asn Ala His Asp
1055 1060 1065
Ala Met Ala Asp Val Tyr Ala Thr Ile Ala Met Ala Lys Leu Val
1070 1075 1080
Lys Thr Arg Gln Pro Arg Leu Phe Asp Tyr Leu Phe Thr His Arg
1085 1090 1095
Asn Lys His Lys Leu Met Ala Leu Ile Asp Val Pro Gln Met Lys
1100 1105 1110
Pro Leu Val His Val Ser Gly Met Phe Gly Ala Trp Arg Gly Asn
1115 1120 1125
Thr Ser Trp Val Ala Pro Leu Ala Trp His Pro Glu Asn Arg Asn
1130 1135 1140
Ala Val Ile Met Val Asp Leu Ala Gly Asp Ile Ser Pro Leu Leu
1145 1150 1155
Glu Leu Asp Ser Asp Thr Leu Arg Glu Arg Leu Tyr Thr Ala Lys
1160 1165 1170
Thr Asp Leu Gly Asp Asn Ala Ala Val Pro Val Lys Leu Val His
1175 1180 1185
Ile Asn Lys Cys Pro Val Leu Ala Gln Ala Asn Thr Leu Arg Pro
1190 1195 1200
Glu Asp Ala Asp Arg Leu Gly Ile Asn Arg Gln His Cys Leu Asp
1205 1210 1215
Asn Leu Lys Ile Leu Arg Glu Asn Pro Gln Val Arg Glu Lys Val
1220 1225 1230
Val Ala Ile Phe Ala Glu Ala Glu Pro Phe Thr Pro Ser Asp Asn
1235 1240 1245
Val Asp Ala Gln Leu Tyr Asn Gly Phe Phe Ser Asp Ala Asp Arg
1250 1255 1260
Ala Ala Met Lys Ile Val Leu Glu Thr Glu Pro Arg Asn Leu Ser
1265 1270 1275
Ala Leu Asp Ile Thr Phe Val Asp Lys Arg Ile Glu Lys Leu Leu
1280 1285 1290
Phe Asn Tyr Arg Ala Arg Asn Phe Pro Gly Thr Leu Asp Tyr Ala
1295 1300 1305
Glu Gln Gln Arg Trp Leu Glu His Arg Arg Gln Val Phe Thr Pro
1310 1315 1320
Glu Phe Leu Gln Gly Tyr Ala Glu Glu Ile Gln Met Leu Ala Gln
1325 1330 1335
Gln Tyr Ala Asp Asp Lys Glu Lys Val Ala Leu Leu Lys Ala Leu
1340 1345 1350
Trp Gln Tyr Ala Glu Glu Ile Val
1355 1360
<210> 44
<211> 3324
<212> DNA
<213> 人工序列
<220>
<223> 在实验室中制备-低活性TCRα megaTAL-ExoX DNA构建体
<400> 44
atgggatcct gcaggccacc taagaagaaa cgcaaagtcg tggatctacg cacgctcggc 60
tacagtcagc agcagcaaga gaagatcaaa ccgaaggtgc gttcgacagt ggcgcagcac 120
cacgaggcac tggtgggcca tgggtttaca cacgcgcaca tcgttgcgct cagccaacac 180
ccggcagcgt tagggaccgt cgctgtcacg tatcagcaca taatcacggc gttgccagag 240
gcgacacacg aagacatcgt tggcgtcggc aaacagtggt ccggcgcacg cgccctggag 300
gccttgctca cggatgcggg ggagttgaga ggtccgccgt tacagttgga cacaggccaa 360
cttgtgaaga ttgcaaaacg tggcggcgtg accgcaatgg aggcagtgca tgcatcgcgc 420
aatgcactga cgggtgcccc cctgaacctg accccggacc aagtggtggc tatcgccagc 480
aacattggcg gcaagcaagc gctcgaaacg gtgcagcggc tgttgccggt gctgtgccag 540
gaccatggcc tgaccccgga ccaagtggtg gctatcgcca gcaacattgg cggcaagcaa 600
gcgctcgaaa cggtgcagcg gctgttgccg gtgctgtgcc aggaccatgg cctgaccccg 660
gaccaagtgg tggctatcgc cagcaacatt ggcggcaagc aagcgctcga aacggtgcag 720
cggctgttgc cggtgctgtg ccaggaccat ggcctgaccc cggaccaagt ggtggctatc 780
gccagcaacg gtggcggcaa gcaagcgctc gaaacggtgc agcggctgtt gccggtgctg 840
tgccaggacc atggcctgac tccggaccaa gtggtggcta tcgccagcca cgatggcggc 900
aagcaagcgc tcgaaacggt gcagcggctg ttgccggtgc tgtgccagga ccatggcctg 960
actccggacc aagtggtggc tatcgccagc cacgatggcg gcaagcaagc gctcgaaacg 1020
gtgcagcggc tgttgccggt gctgtgccag gaccatggcc tgaccccgga ccaagtggtg 1080
gctatcgcca gcaacattgg cggcaagcaa gcgctcgaaa cggtgcagcg gctgttgccg 1140
gtgctgtgcc aggaccatgg cctgaccccg gaccaagtgg tggctatcgc cagcaacaat 1200
ggcggcaagc aagcgctcga aacggtgcag cggctgttgc cggtgctgtg ccaggaccat 1260
ggcctgaccc cggaccaagt ggtggctatc gccagcaacg gtggcggcaa gcaagcgctc 1320
gaaacggtgc agcggctgtt gccggtgctg tgccaggacc atggcctgac cccggaccaa 1380
gtggtggcta tcgccagcaa caatggcggc aagcaagcgc tcgaaacggt gcagcggctg 1440
ttgccggtgc tgtgccagga ccatggcctg accccggacc aagtggtggc tatcgccagc 1500
aacattggcg gcaagcaagc gctcgaaagc attgtggccc agctgagccg gcctgatccg 1560
gcgttggccg cgttgaccaa cgaccacctc gtcgccttgg cctgcctcgg cggacgtcct 1620
gccatggatg cagtgaaaaa gggattgccg cacgcgccgg aattgatcag aagagtcaat 1680
cgccgtattg gcgaacgcac gtcccatcgc gttgcgatat ctagagtggg aggaagctct 1740
cgcagagagt ccatcaaccc atggattctg actggtttcg ctgatgccga aggatcattc 1800
atactagaca tccgcaaccg aaacaacgaa agcaacagat accgaacttc gctgagattc 1860
cagatcaccc tgcacaacaa ggacaaatcg attctggaga atatccagtc cacttggaag 1920
gtcggcaaga tcacaaacag cagtgacaga gccgtcatgc tgagggtcac ccgtttcgaa 1980
gatttgaaag tgattatcga ccacttcgag aaatatccgc tgattaccca gaaattgggc 2040
gattacaagt tgtttaaaca ggcattcagc gtcatggaga ataaagaaca tcttaaggag 2100
aatgggatta aggagctcgt acgaatcaaa gctaagatga attggggtct caatgacgaa 2160
ttgaaaaaag catttccaga gaacattagc aaagagcgcc cccttatcaa taagaacatt 2220
ccgaatttca aatggctggc tggattcaca gctggtgaag gctacttcgg cgtgaatcta 2280
aaaaaggtaa agggcaccgc aaaggtatac gtgggactga gattctcaat ctcacagcac 2340
atcagagaca agaacctgat gaattcattg ataacatacc taggctgtgg ttccatctgg 2400
gagaagaaca agtctgagtt cagatggctc gagttcgtcg taaccaaatt cagcgatatc 2460
aacgacaaga tcattccggt attccaggaa aatactctga ttggcgtcaa actcgaggac 2520
tttgaagatt ggtgcaaggt tgccaaattg atcaaagaga agaaacacct gaccgaatcc 2580
ggtttggatg agattaagaa aatcaagctg aacatgaaca aaggtcgtgg tggaggcggt 2640
agcggaggcg gagggtcggc tagcctgcgg atcatagaca cggaaacctg tggactccag 2700
ggtggtatcg tggagatagc cagtgtcgat gtaattgatg ggaaaatcgt aaacccgatg 2760
agccacctcg tccgaccgga taggccgata agtccgcagg ctatggctat acatagaatc 2820
acggaggcta tggtagctga caagccttgg atcgaggacg ttatcccaca ctactatggg 2880
agcgaatggt atgtagcgca caacgcatca tttgataggc gcgtgcttcc cgaaatgcct 2940
ggggagtgga tttgcacaat gaaactcgct cgccgactgt ggcctggtat caagtatagt 3000
aacatggcac tttacaagac ccgcaaactc aacgtacaga cacctccggg tctccaccac 3060
catagggcac tctatgattg ttacataacc gctgctctcc ttatagacat aatgaatacc 3120
tccgggtgga ccgccgaaca aatggcagac ataacgggaa gacctagttt gatgacaacc 3180
tttacatttg gaaagtatcg gggcaaggcg gtgagcgatg tggccgaaag agatcccggt 3240
tatcttagat ggctcttcaa taacttggat tctatgtcac ctgaacttcg ccttaccctg 3300
aagcattacc ttgagaatac ctga 3324
<210> 45
<211> 3324
<212> RNA
<213> 人工序列
<220>
<223> 在实验室中制备-低活性TCRα megaTAL-ExoX RNA构建体
<400> 45
augggauccu gcaggccacc uaagaagaaa cgcaaagucg uggaucuacg cacgcucggc 60
uacagucagc agcagcaaga gaagaucaaa ccgaaggugc guucgacagu ggcgcagcac 120
cacgaggcac uggugggcca uggguuuaca cacgcgcaca ucguugcgcu cagccaacac 180
ccggcagcgu uagggaccgu cgcugucacg uaucagcaca uaaucacggc guugccagag 240
gcgacacacg aagacaucgu uggcgucggc aaacaguggu ccggcgcacg cgcccuggag 300
gccuugcuca cggaugcggg ggaguugaga gguccgccgu uacaguugga cacaggccaa 360
cuugugaaga uugcaaaacg uggcggcgug accgcaaugg aggcagugca ugcaucgcgc 420
aaugcacuga cgggugcccc ccugaaccug accccggacc aagugguggc uaucgccagc 480
aacauuggcg gcaagcaagc gcucgaaacg gugcagcggc uguugccggu gcugugccag 540
gaccauggcc ugaccccgga ccaaguggug gcuaucgcca gcaacauugg cggcaagcaa 600
gcgcucgaaa cggugcagcg gcuguugccg gugcugugcc aggaccaugg ccugaccccg 660
gaccaagugg uggcuaucgc cagcaacauu ggcggcaagc aagcgcucga aacggugcag 720
cggcuguugc cggugcugug ccaggaccau ggccugaccc cggaccaagu gguggcuauc 780
gccagcaacg guggcggcaa gcaagcgcuc gaaacggugc agcggcuguu gccggugcug 840
ugccaggacc auggccugac uccggaccaa gugguggcua ucgccagcca cgauggcggc 900
aagcaagcgc ucgaaacggu gcagcggcug uugccggugc ugugccagga ccauggccug 960
acuccggacc aagugguggc uaucgccagc cacgauggcg gcaagcaagc gcucgaaacg 1020
gugcagcggc uguugccggu gcugugccag gaccauggcc ugaccccgga ccaaguggug 1080
gcuaucgcca gcaacauugg cggcaagcaa gcgcucgaaa cggugcagcg gcuguugccg 1140
gugcugugcc aggaccaugg ccugaccccg gaccaagugg uggcuaucgc cagcaacaau 1200
ggcggcaagc aagcgcucga aacggugcag cggcuguugc cggugcugug ccaggaccau 1260
ggccugaccc cggaccaagu gguggcuauc gccagcaacg guggcggcaa gcaagcgcuc 1320
gaaacggugc agcggcuguu gccggugcug ugccaggacc auggccugac cccggaccaa 1380
gugguggcua ucgccagcaa caauggcggc aagcaagcgc ucgaaacggu gcagcggcug 1440
uugccggugc ugugccagga ccauggccug accccggacc aagugguggc uaucgccagc 1500
aacauuggcg gcaagcaagc gcucgaaagc auuguggccc agcugagccg gccugauccg 1560
gcguuggccg cguugaccaa cgaccaccuc gucgccuugg ccugccucgg cggacguccu 1620
gccauggaug cagugaaaaa gggauugccg cacgcgccgg aauugaucag aagagucaau 1680
cgccguauug gcgaacgcac gucccaucgc guugcgauau cuagaguggg aggaagcucu 1740
cgcagagagu ccaucaaccc auggauucug acugguuucg cugaugccga aggaucauuc 1800
auacuagaca uccgcaaccg aaacaacgaa agcaacagau accgaacuuc gcugagauuc 1860
cagaucaccc ugcacaacaa ggacaaaucg auucuggaga auauccaguc cacuuggaag 1920
gucggcaaga ucacaaacag cagugacaga gccgucaugc ugagggucac ccguuucgaa 1980
gauuugaaag ugauuaucga ccacuucgag aaauauccgc ugauuaccca gaaauugggc 2040
gauuacaagu uguuuaaaca ggcauucagc gucauggaga auaaagaaca ucuuaaggag 2100
aaugggauua aggagcucgu acgaaucaaa gcuaagauga auuggggucu caaugacgaa 2160
uugaaaaaag cauuuccaga gaacauuagc aaagagcgcc cccuuaucaa uaagaacauu 2220
ccgaauuuca aauggcuggc uggauucaca gcuggugaag gcuacuucgg cgugaaucua 2280
aaaaagguaa agggcaccgc aaagguauac gugggacuga gauucucaau cucacagcac 2340
aucagagaca agaaccugau gaauucauug auaacauacc uaggcugugg uuccaucugg 2400
gagaagaaca agucugaguu cagauggcuc gaguucgucg uaaccaaauu cagcgauauc 2460
aacgacaaga ucauuccggu auuccaggaa aauacucuga uuggcgucaa acucgaggac 2520
uuugaagauu ggugcaaggu ugccaaauug aucaaagaga agaaacaccu gaccgaaucc 2580
gguuuggaug agauuaagaa aaucaagcug aacaugaaca aaggucgugg uggaggcggu 2640
agcggaggcg gagggucggc uagccugcgg aucauagaca cggaaaccug uggacuccag 2700
ggugguaucg uggagauagc cagugucgau guaauugaug ggaaaaucgu aaacccgaug 2760
agccaccucg uccgaccgga uaggccgaua aguccgcagg cuauggcuau acauagaauc 2820
acggaggcua ugguagcuga caagccuugg aucgaggacg uuaucccaca cuacuauggg 2880
agcgaauggu auguagcgca caacgcauca uuugauaggc gcgugcuucc cgaaaugccu 2940
ggggagugga uuugcacaau gaaacucgcu cgccgacugu ggccugguau caaguauagu 3000
aacauggcac uuuacaagac ccgcaaacuc aacguacaga caccuccggg ucuccaccac 3060
cauagggcac ucuaugauug uuacauaacc gcugcucucc uuauagacau aaugaauacc 3120
uccgggugga ccgccgaaca aauggcagac auaacgggaa gaccuaguuu gaugacaacc 3180
uuuacauuug gaaaguaucg gggcaaggcg gugagcgaug uggccgaaag agaucccggu 3240
uaucuuagau ggcucuucaa uaacuuggau ucuaugucac cugaacuucg ccuuacccug 3300
aagcauuacc uugagaauac cuga 3324
<210> 46
<211> 1107
<212> PRT
<213> 人工序列
<220>
<223> 在实验室中制备-低活性TCRα megaTAL-ExoX蛋白质构建体
<400> 46
Met Gly Ser Cys Arg Pro Pro Lys Lys Lys Arg Lys Val Val Asp Leu
1 5 10 15
Arg Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys
20 25 30
Val Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val Gly His Gly
35 40 45
Phe Thr His Ala His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu
50 55 60
Gly Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr Ala Leu Pro Glu
65 70 75 80
Ala Thr His Glu Asp Ile Val Gly Val Gly Lys Gln Trp Ser Gly Ala
85 90 95
Arg Ala Leu Glu Ala Leu Leu Thr Asp Ala Gly Glu Leu Arg Gly Pro
100 105 110
Pro Leu Gln Leu Asp Thr Gly Gln Leu Val Lys Ile Ala Lys Arg Gly
115 120 125
Gly Val Thr Ala Met Glu Ala Val His Ala Ser Arg Asn Ala Leu Thr
130 135 140
Gly Ala Pro Leu Asn Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
145 150 155 160
Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
165 170 175
Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
180 185 190
Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
195 200 205
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val
210 215 220
Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
225 230 235 240
Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln
245 250 255
Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr
260 265 270
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro
275 280 285
Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu
290 295 300
Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu
305 310 315 320
Thr Pro Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln
325 330 335
Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His
340 345 350
Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly
355 360 365
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
370 375 380
Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Asn
385 390 395 400
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu
405 410 415
Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
420 425 430
Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
435 440 445
Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
450 455 460
Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
465 470 475 480
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val
485 490 495
Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Ser Ile Val
500 505 510
Ala Gln Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu Thr Asn Asp
515 520 525
His Leu Val Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala Met Asp Ala
530 535 540
Val Lys Lys Gly Leu Pro His Ala Pro Glu Leu Ile Arg Arg Val Asn
545 550 555 560
Arg Arg Ile Gly Glu Arg Thr Ser His Arg Val Ala Ile Ser Arg Val
565 570 575
Gly Gly Ser Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr Gly
580 585 590
Phe Ala Asp Ala Glu Gly Ser Phe Ile Leu Asp Ile Arg Asn Arg Asn
595 600 605
Asn Glu Ser Asn Arg Tyr Arg Thr Ser Leu Arg Phe Gln Ile Thr Leu
610 615 620
His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp Lys
625 630 635 640
Val Gly Lys Ile Thr Asn Ser Ser Asp Arg Ala Val Met Leu Arg Val
645 650 655
Thr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys Tyr
660 665 670
Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln Ala
675 680 685
Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile Lys
690 695 700
Glu Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly Leu Asn Asp Glu
705 710 715 720
Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Pro Leu Ile
725 730 735
Asn Lys Asn Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe Thr Ala Gly
740 745 750
Glu Gly Tyr Phe Gly Val Asn Leu Lys Lys Val Lys Gly Thr Ala Lys
755 760 765
Val Tyr Val Gly Leu Arg Phe Ser Ile Ser Gln His Ile Arg Asp Lys
770 775 780
Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Ser Ile Trp
785 790 795 800
Glu Lys Asn Lys Ser Glu Phe Arg Trp Leu Glu Phe Val Val Thr Lys
805 810 815
Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn Thr
820 825 830
Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val Ala
835 840 845
Lys Leu Ile Lys Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp Glu
850 855 860
Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Gly Gly Gly Gly
865 870 875 880
Ser Gly Gly Gly Gly Ser Ala Ser Leu Arg Ile Ile Asp Thr Glu Thr
885 890 895
Cys Gly Leu Gln Gly Gly Ile Val Glu Ile Ala Ser Val Asp Val Ile
900 905 910
Asp Gly Lys Ile Val Asn Pro Met Ser His Leu Val Arg Pro Asp Arg
915 920 925
Pro Ile Ser Pro Gln Ala Met Ala Ile His Arg Ile Thr Glu Ala Met
930 935 940
Val Ala Asp Lys Pro Trp Ile Glu Asp Val Ile Pro His Tyr Tyr Gly
945 950 955 960
Ser Glu Trp Tyr Val Ala His Asn Ala Ser Phe Asp Arg Arg Val Leu
965 970 975
Pro Glu Met Pro Gly Glu Trp Ile Cys Thr Met Lys Leu Ala Arg Arg
980 985 990
Leu Trp Pro Gly Ile Lys Tyr Ser Asn Met Ala Leu Tyr Lys Thr Arg
995 1000 1005
Lys Leu Asn Val Gln Thr Pro Pro Gly Leu His His His Arg Ala
1010 1015 1020
Leu Tyr Asp Cys Tyr Ile Thr Ala Ala Leu Leu Ile Asp Ile Met
1025 1030 1035
Asn Thr Ser Gly Trp Thr Ala Glu Gln Met Ala Asp Ile Thr Gly
1040 1045 1050
Arg Pro Ser Leu Met Thr Thr Phe Thr Phe Gly Lys Tyr Arg Gly
1055 1060 1065
Lys Ala Val Ser Asp Val Ala Glu Arg Asp Pro Gly Tyr Leu Arg
1070 1075 1080
Trp Leu Phe Asn Asn Leu Asp Ser Met Ser Pro Glu Leu Arg Leu
1085 1090 1095
Thr Leu Lys His Tyr Leu Glu Asn Thr
1100 1105
<210> 47
<211> 3537
<212> DNA
<213> 人工序列
<220>
<223> 在实验室中制备-低活性TCRα megaTAL-T5FEN DNA构建体
<400> 47
atgggatcct gcaggccacc taagaagaaa cgcaaagtcg tggatctacg cacgctcggc 60
tacagtcagc agcagcaaga gaagatcaaa ccgaaggtgc gttcgacagt ggcgcagcac 120
cacgaggcac tggtgggcca tgggtttaca cacgcgcaca tcgttgcgct cagccaacac 180
ccggcagcgt tagggaccgt cgctgtcacg tatcagcaca taatcacggc gttgccagag 240
gcgacacacg aagacatcgt tggcgtcggc aaacagtggt ccggcgcacg cgccctggag 300
gccttgctca cggatgcggg ggagttgaga ggtccgccgt tacagttgga cacaggccaa 360
cttgtgaaga ttgcaaaacg tggcggcgtg accgcaatgg aggcagtgca tgcatcgcgc 420
aatgcactga cgggtgcccc cctgaacctg accccggacc aagtggtggc tatcgccagc 480
aacattggcg gcaagcaagc gctcgaaacg gtgcagcggc tgttgccggt gctgtgccag 540
gaccatggcc tgaccccgga ccaagtggtg gctatcgcca gcaacattgg cggcaagcaa 600
gcgctcgaaa cggtgcagcg gctgttgccg gtgctgtgcc aggaccatgg cctgaccccg 660
gaccaagtgg tggctatcgc cagcaacatt ggcggcaagc aagcgctcga aacggtgcag 720
cggctgttgc cggtgctgtg ccaggaccat ggcctgaccc cggaccaagt ggtggctatc 780
gccagcaacg gtggcggcaa gcaagcgctc gaaacggtgc agcggctgtt gccggtgctg 840
tgccaggacc atggcctgac tccggaccaa gtggtggcta tcgccagcca cgatggcggc 900
aagcaagcgc tcgaaacggt gcagcggctg ttgccggtgc tgtgccagga ccatggcctg 960
actccggacc aagtggtggc tatcgccagc cacgatggcg gcaagcaagc gctcgaaacg 1020
gtgcagcggc tgttgccggt gctgtgccag gaccatggcc tgaccccgga ccaagtggtg 1080
gctatcgcca gcaacattgg cggcaagcaa gcgctcgaaa cggtgcagcg gctgttgccg 1140
gtgctgtgcc aggaccatgg cctgaccccg gaccaagtgg tggctatcgc cagcaacaat 1200
ggcggcaagc aagcgctcga aacggtgcag cggctgttgc cggtgctgtg ccaggaccat 1260
ggcctgaccc cggaccaagt ggtggctatc gccagcaacg gtggcggcaa gcaagcgctc 1320
gaaacggtgc agcggctgtt gccggtgctg tgccaggacc atggcctgac cccggaccaa 1380
gtggtggcta tcgccagcaa caatggcggc aagcaagcgc tcgaaacggt gcagcggctg 1440
ttgccggtgc tgtgccagga ccatggcctg accccggacc aagtggtggc tatcgccagc 1500
aacattggcg gcaagcaagc gctcgaaagc attgtggccc agctgagccg gcctgatccg 1560
gcgttggccg cgttgaccaa cgaccacctc gtcgccttgg cctgcctcgg cggacgtcct 1620
gccatggatg cagtgaaaaa gggattgccg cacgcgccgg aattgatcag aagagtcaat 1680
cgccgtattg gcgaacgcac gtcccatcgc gttgcgatat ctagagtggg aggaagctct 1740
cgcagagagt ccatcaaccc atggattctg actggtttcg ctgatgccga aggatcattc 1800
atactagaca tccgcaaccg aaacaacgaa agcaacagat accgaacttc gctgagattc 1860
cagatcaccc tgcacaacaa ggacaaatcg attctggaga atatccagtc cacttggaag 1920
gtcggcaaga tcacaaacag cagtgacaga gccgtcatgc tgagggtcac ccgtttcgaa 1980
gatttgaaag tgattatcga ccacttcgag aaatatccgc tgattaccca gaaattgggc 2040
gattacaagt tgtttaaaca ggcattcagc gtcatggaga ataaagaaca tcttaaggag 2100
aatgggatta aggagctcgt acgaatcaaa gctaagatga attggggtct caatgacgaa 2160
ttgaaaaaag catttccaga gaacattagc aaagagcgcc cccttatcaa taagaacatt 2220
ccgaatttca aatggctggc tggattcaca gctggtgaag gctacttcgg cgtgaatcta 2280
aaaaaggtaa agggcaccgc aaaggtatac gtgggactga gattctcaat ctcacagcac 2340
atcagagaca agaacctgat gaattcattg ataacatacc taggctgtgg ttccatctgg 2400
gagaagaaca agtctgagtt cagatggctc gagttcgtcg taaccaaatt cagcgatatc 2460
aacgacaaga tcattccggt attccaggaa aatactctga ttggcgtcaa actcgaggac 2520
tttgaagatt ggtgcaaggt tgccaaattg atcaaagaga agaaacacct gaccgaatcc 2580
ggtttggatg agattaagaa aatcaagctg aacatgaaca aaggtcgtgg tggaggcggt 2640
agcggaggcg gagggtcggc tagcagtaag agctggggga agttcataga ggaggaggaa 2700
gctgaaatgg caagcagacg caatttgatg attgtggacg gcactaatct tggtttcagg 2760
ttcaaacata acaatagtaa gaagccattt gccagcagct atgtttctac gatacaatca 2820
ctggctaaaa gttacagtgc gaggactaca atagtccttg gcgacaaagg caaatcagtg 2880
ttcaggctcg aacatctccc tgaatacaag ggcaaccgcg atgagaagta tgcacagcga 2940
acggaagaag agaaagccct ggacgagcaa ttctttgaat atctcaaaga tgcctttgaa 3000
ctgtgcaaaa ctacatttcc aacctttacc atccggggag ttgaggcaga tgacatggca 3060
gcctacattg taaaactgat agggcacctc tacgaccatg tttggctcat atccactgac 3120
ggagactggg acactttgtt gaccgataaa gtgtcaaggt tctcattcac tactcgtcgg 3180
gaatatcatt tgcgcgatat gtatgaacac cataacgtcg atgacgtgga acaattcatc 3240
tctcttaaag caatcatggg cgatctgggg gacaacatta ggggggttga ggggatcgga 3300
gcgaaaaggg gatacaatat catacgggaa tttggcaacg tccttgacat catcgaccag 3360
ctccccttgc ccggaaagca gaagtacatt caaaacctca acgcttcaga agaactgctc 3420
tttaggaact tgatactggt agacctccca acttactgcg tggatgcaat cgcggcggta 3480
ggacaggacg tgcttgacaa attcacgaag gacatactgg agattgccga acagtga 3537
<210> 48
<211> 3537
<212> RNA
<213> 人工序列
<220>
<223> 在实验室中制备-低活性TCRα megaTAL-T5FEN RNA构建体
<400> 48
augggauccu gcaggccacc uaagaagaaa cgcaaagucg uggaucuacg cacgcucggc 60
uacagucagc agcagcaaga gaagaucaaa ccgaaggugc guucgacagu ggcgcagcac 120
cacgaggcac uggugggcca uggguuuaca cacgcgcaca ucguugcgcu cagccaacac 180
ccggcagcgu uagggaccgu cgcugucacg uaucagcaca uaaucacggc guugccagag 240
gcgacacacg aagacaucgu uggcgucggc aaacaguggu ccggcgcacg cgcccuggag 300
gccuugcuca cggaugcggg ggaguugaga gguccgccgu uacaguugga cacaggccaa 360
cuugugaaga uugcaaaacg uggcggcgug accgcaaugg aggcagugca ugcaucgcgc 420
aaugcacuga cgggugcccc ccugaaccug accccggacc aagugguggc uaucgccagc 480
aacauuggcg gcaagcaagc gcucgaaacg gugcagcggc uguugccggu gcugugccag 540
gaccauggcc ugaccccgga ccaaguggug gcuaucgcca gcaacauugg cggcaagcaa 600
gcgcucgaaa cggugcagcg gcuguugccg gugcugugcc aggaccaugg ccugaccccg 660
gaccaagugg uggcuaucgc cagcaacauu ggcggcaagc aagcgcucga aacggugcag 720
cggcuguugc cggugcugug ccaggaccau ggccugaccc cggaccaagu gguggcuauc 780
gccagcaacg guggcggcaa gcaagcgcuc gaaacggugc agcggcuguu gccggugcug 840
ugccaggacc auggccugac uccggaccaa gugguggcua ucgccagcca cgauggcggc 900
aagcaagcgc ucgaaacggu gcagcggcug uugccggugc ugugccagga ccauggccug 960
acuccggacc aagugguggc uaucgccagc cacgauggcg gcaagcaagc gcucgaaacg 1020
gugcagcggc uguugccggu gcugugccag gaccauggcc ugaccccgga ccaaguggug 1080
gcuaucgcca gcaacauugg cggcaagcaa gcgcucgaaa cggugcagcg gcuguugccg 1140
gugcugugcc aggaccaugg ccugaccccg gaccaagugg uggcuaucgc cagcaacaau 1200
ggcggcaagc aagcgcucga aacggugcag cggcuguugc cggugcugug ccaggaccau 1260
ggccugaccc cggaccaagu gguggcuauc gccagcaacg guggcggcaa gcaagcgcuc 1320
gaaacggugc agcggcuguu gccggugcug ugccaggacc auggccugac cccggaccaa 1380
gugguggcua ucgccagcaa caauggcggc aagcaagcgc ucgaaacggu gcagcggcug 1440
uugccggugc ugugccagga ccauggccug accccggacc aagugguggc uaucgccagc 1500
aacauuggcg gcaagcaagc gcucgaaagc auuguggccc agcugagccg gccugauccg 1560
gcguuggccg cguugaccaa cgaccaccuc gucgccuugg ccugccucgg cggacguccu 1620
gccauggaug cagugaaaaa gggauugccg cacgcgccgg aauugaucag aagagucaau 1680
cgccguauug gcgaacgcac gucccaucgc guugcgauau cuagaguggg aggaagcucu 1740
cgcagagagu ccaucaaccc auggauucug acugguuucg cugaugccga aggaucauuc 1800
auacuagaca uccgcaaccg aaacaacgaa agcaacagau accgaacuuc gcugagauuc 1860
cagaucaccc ugcacaacaa ggacaaaucg auucuggaga auauccaguc cacuuggaag 1920
gucggcaaga ucacaaacag cagugacaga gccgucaugc ugagggucac ccguuucgaa 1980
gauuugaaag ugauuaucga ccacuucgag aaauauccgc ugauuaccca gaaauugggc 2040
gauuacaagu uguuuaaaca ggcauucagc gucauggaga auaaagaaca ucuuaaggag 2100
aaugggauua aggagcucgu acgaaucaaa gcuaagauga auuggggucu caaugacgaa 2160
uugaaaaaag cauuuccaga gaacauuagc aaagagcgcc cccuuaucaa uaagaacauu 2220
ccgaauuuca aauggcuggc uggauucaca gcuggugaag gcuacuucgg cgugaaucua 2280
aaaaagguaa agggcaccgc aaagguauac gugggacuga gauucucaau cucacagcac 2340
aucagagaca agaaccugau gaauucauug auaacauacc uaggcugugg uuccaucugg 2400
gagaagaaca agucugaguu cagauggcuc gaguucgucg uaaccaaauu cagcgauauc 2460
aacgacaaga ucauuccggu auuccaggaa aauacucuga uuggcgucaa acucgaggac 2520
uuugaagauu ggugcaaggu ugccaaauug aucaaagaga agaaacaccu gaccgaaucc 2580
gguuuggaug agauuaagaa aaucaagcug aacaugaaca aaggucgugg uggaggcggu 2640
agcggaggcg gagggucggc uagcaguaag agcuggggga aguucauaga ggaggaggaa 2700
gcugaaaugg caagcagacg caauuugaug auuguggacg gcacuaaucu ugguuucagg 2760
uucaaacaua acaauaguaa gaagccauuu gccagcagcu auguuucuac gauacaauca 2820
cuggcuaaaa guuacagugc gaggacuaca auaguccuug gcgacaaagg caaaucagug 2880
uucaggcucg aacaucuccc ugaauacaag ggcaaccgcg augagaagua ugcacagcga 2940
acggaagaag agaaagcccu ggacgagcaa uucuuugaau aucucaaaga ugccuuugaa 3000
cugugcaaaa cuacauuucc aaccuuuacc auccggggag uugaggcaga ugacauggca 3060
gccuacauug uaaaacugau agggcaccuc uacgaccaug uuuggcucau auccacugac 3120
ggagacuggg acacuuuguu gaccgauaaa gugucaaggu ucucauucac uacucgucgg 3180
gaauaucauu ugcgcgauau guaugaacac cauaacgucg augacgugga acaauucauc 3240
ucucuuaaag caaucauggg cgaucugggg gacaacauua gggggguuga ggggaucgga 3300
gcgaaaaggg gauacaauau cauacgggaa uuuggcaacg uccuugacau caucgaccag 3360
cuccccuugc ccggaaagca gaaguacauu caaaaccuca acgcuucaga agaacugcuc 3420
uuuaggaacu ugauacuggu agaccuccca acuuacugcg uggaugcaau cgcggcggua 3480
ggacaggacg ugcuugacaa auucacgaag gacauacugg agauugccga acaguga 3537
<210> 49
<211> 1178
<212> PRT
<213> 人工序列
<220>
<223> 在实验室中制备-低活性TCRα megaTAL-T5FEN蛋白质构建体
<400> 49
Met Gly Ser Cys Arg Pro Pro Lys Lys Lys Arg Lys Val Val Asp Leu
1 5 10 15
Arg Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys
20 25 30
Val Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val Gly His Gly
35 40 45
Phe Thr His Ala His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu
50 55 60
Gly Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr Ala Leu Pro Glu
65 70 75 80
Ala Thr His Glu Asp Ile Val Gly Val Gly Lys Gln Trp Ser Gly Ala
85 90 95
Arg Ala Leu Glu Ala Leu Leu Thr Asp Ala Gly Glu Leu Arg Gly Pro
100 105 110
Pro Leu Gln Leu Asp Thr Gly Gln Leu Val Lys Ile Ala Lys Arg Gly
115 120 125
Gly Val Thr Ala Met Glu Ala Val His Ala Ser Arg Asn Ala Leu Thr
130 135 140
Gly Ala Pro Leu Asn Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
145 150 155 160
Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
165 170 175
Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
180 185 190
Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
195 200 205
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val
210 215 220
Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
225 230 235 240
Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln
245 250 255
Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr
260 265 270
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro
275 280 285
Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu
290 295 300
Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu
305 310 315 320
Thr Pro Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln
325 330 335
Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His
340 345 350
Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly
355 360 365
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
370 375 380
Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Asn
385 390 395 400
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu
405 410 415
Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
420 425 430
Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
435 440 445
Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
450 455 460
Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
465 470 475 480
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val
485 490 495
Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Ser Ile Val
500 505 510
Ala Gln Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu Thr Asn Asp
515 520 525
His Leu Val Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala Met Asp Ala
530 535 540
Val Lys Lys Gly Leu Pro His Ala Pro Glu Leu Ile Arg Arg Val Asn
545 550 555 560
Arg Arg Ile Gly Glu Arg Thr Ser His Arg Val Ala Ile Ser Arg Val
565 570 575
Gly Gly Ser Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr Gly
580 585 590
Phe Ala Asp Ala Glu Gly Ser Phe Ile Leu Asp Ile Arg Asn Arg Asn
595 600 605
Asn Glu Ser Asn Arg Tyr Arg Thr Ser Leu Arg Phe Gln Ile Thr Leu
610 615 620
His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp Lys
625 630 635 640
Val Gly Lys Ile Thr Asn Ser Ser Asp Arg Ala Val Met Leu Arg Val
645 650 655
Thr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys Tyr
660 665 670
Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln Ala
675 680 685
Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile Lys
690 695 700
Glu Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly Leu Asn Asp Glu
705 710 715 720
Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Pro Leu Ile
725 730 735
Asn Lys Asn Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe Thr Ala Gly
740 745 750
Glu Gly Tyr Phe Gly Val Asn Leu Lys Lys Val Lys Gly Thr Ala Lys
755 760 765
Val Tyr Val Gly Leu Arg Phe Ser Ile Ser Gln His Ile Arg Asp Lys
770 775 780
Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Ser Ile Trp
785 790 795 800
Glu Lys Asn Lys Ser Glu Phe Arg Trp Leu Glu Phe Val Val Thr Lys
805 810 815
Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn Thr
820 825 830
Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val Ala
835 840 845
Lys Leu Ile Lys Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp Glu
850 855 860
Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Gly Gly Gly Gly
865 870 875 880
Ser Gly Gly Gly Gly Ser Ala Ser Ser Lys Ser Trp Gly Lys Phe Ile
885 890 895
Glu Glu Glu Glu Ala Glu Met Ala Ser Arg Arg Asn Leu Met Ile Val
900 905 910
Asp Gly Thr Asn Leu Gly Phe Arg Phe Lys His Asn Asn Ser Lys Lys
915 920 925
Pro Phe Ala Ser Ser Tyr Val Ser Thr Ile Gln Ser Leu Ala Lys Ser
930 935 940
Tyr Ser Ala Arg Thr Thr Ile Val Leu Gly Asp Lys Gly Lys Ser Val
945 950 955 960
Phe Arg Leu Glu His Leu Pro Glu Tyr Lys Gly Asn Arg Asp Glu Lys
965 970 975
Tyr Ala Gln Arg Thr Glu Glu Glu Lys Ala Leu Asp Glu Gln Phe Phe
980 985 990
Glu Tyr Leu Lys Asp Ala Phe Glu Leu Cys Lys Thr Thr Phe Pro Thr
995 1000 1005
Phe Thr Ile Arg Gly Val Glu Ala Asp Asp Met Ala Ala Tyr Ile
1010 1015 1020
Val Lys Leu Ile Gly His Leu Tyr Asp His Val Trp Leu Ile Ser
1025 1030 1035
Thr Asp Gly Asp Trp Asp Thr Leu Leu Thr Asp Lys Val Ser Arg
1040 1045 1050
Phe Ser Phe Thr Thr Arg Arg Glu Tyr His Leu Arg Asp Met Tyr
1055 1060 1065
Glu His His Asn Val Asp Asp Val Glu Gln Phe Ile Ser Leu Lys
1070 1075 1080
Ala Ile Met Gly Asp Leu Gly Asp Asn Ile Arg Gly Val Glu Gly
1085 1090 1095
Ile Gly Ala Lys Arg Gly Tyr Asn Ile Ile Arg Glu Phe Gly Asn
1100 1105 1110
Val Leu Asp Ile Ile Asp Gln Leu Pro Leu Pro Gly Lys Gln Lys
1115 1120 1125
Tyr Ile Gln Asn Leu Asn Ala Ser Glu Glu Leu Leu Phe Arg Asn
1130 1135 1140
Leu Ile Leu Val Asp Leu Pro Thr Tyr Cys Val Asp Ala Ile Ala
1145 1150 1155
Ala Val Gly Gln Asp Val Leu Asp Lys Phe Thr Lys Asp Ile Leu
1160 1165 1170
Glu Ile Ala Glu Gln
1175
<210> 50
<211> 3342
<212> DNA
<213> 人工序列
<220>
<223> 在实验室中制备-低活性TCRα megaTAL-λExo DNA构建体
<400> 50
atgggatcct gcaggccacc taagaagaaa cgcaaagtcg tggatctacg cacgctcggc 60
tacagtcagc agcagcaaga gaagatcaaa ccgaaggtgc gttcgacagt ggcgcagcac 120
cacgaggcac tggtgggcca tgggtttaca cacgcgcaca tcgttgcgct cagccaacac 180
ccggcagcgt tagggaccgt cgctgtcacg tatcagcaca taatcacggc gttgccagag 240
gcgacacacg aagacatcgt tggcgtcggc aaacagtggt ccggcgcacg cgccctggag 300
gccttgctca cggatgcggg ggagttgaga ggtccgccgt tacagttgga cacaggccaa 360
cttgtgaaga ttgcaaaacg tggcggcgtg accgcaatgg aggcagtgca tgcatcgcgc 420
aatgcactga cgggtgcccc cctgaacctg accccggacc aagtggtggc tatcgccagc 480
aacattggcg gcaagcaagc gctcgaaacg gtgcagcggc tgttgccggt gctgtgccag 540
gaccatggcc tgaccccgga ccaagtggtg gctatcgcca gcaacattgg cggcaagcaa 600
gcgctcgaaa cggtgcagcg gctgttgccg gtgctgtgcc aggaccatgg cctgaccccg 660
gaccaagtgg tggctatcgc cagcaacatt ggcggcaagc aagcgctcga aacggtgcag 720
cggctgttgc cggtgctgtg ccaggaccat ggcctgaccc cggaccaagt ggtggctatc 780
gccagcaacg gtggcggcaa gcaagcgctc gaaacggtgc agcggctgtt gccggtgctg 840
tgccaggacc atggcctgac tccggaccaa gtggtggcta tcgccagcca cgatggcggc 900
aagcaagcgc tcgaaacggt gcagcggctg ttgccggtgc tgtgccagga ccatggcctg 960
actccggacc aagtggtggc tatcgccagc cacgatggcg gcaagcaagc gctcgaaacg 1020
gtgcagcggc tgttgccggt gctgtgccag gaccatggcc tgaccccgga ccaagtggtg 1080
gctatcgcca gcaacattgg cggcaagcaa gcgctcgaaa cggtgcagcg gctgttgccg 1140
gtgctgtgcc aggaccatgg cctgaccccg gaccaagtgg tggctatcgc cagcaacaat 1200
ggcggcaagc aagcgctcga aacggtgcag cggctgttgc cggtgctgtg ccaggaccat 1260
ggcctgaccc cggaccaagt ggtggctatc gccagcaacg gtggcggcaa gcaagcgctc 1320
gaaacggtgc agcggctgtt gccggtgctg tgccaggacc atggcctgac cccggaccaa 1380
gtggtggcta tcgccagcaa caatggcggc aagcaagcgc tcgaaacggt gcagcggctg 1440
ttgccggtgc tgtgccagga ccatggcctg accccggacc aagtggtggc tatcgccagc 1500
aacattggcg gcaagcaagc gctcgaaagc attgtggccc agctgagccg gcctgatccg 1560
gcgttggccg cgttgaccaa cgaccacctc gtcgccttgg cctgcctcgg cggacgtcct 1620
gccatggatg cagtgaaaaa gggattgccg cacgcgccgg aattgatcag aagagtcaat 1680
cgccgtattg gcgaacgcac gtcccatcgc gttgcgatat ctagagtggg aggaagctct 1740
cgcagagagt ccatcaaccc atggattctg actggtttcg ctgatgccga aggatcattc 1800
atactagaca tccgcaaccg aaacaacgaa agcaacagat accgaacttc gctgagattc 1860
cagatcaccc tgcacaacaa ggacaaatcg attctggaga atatccagtc cacttggaag 1920
gtcggcaaga tcacaaacag cagtgacaga gccgtcatgc tgagggtcac ccgtttcgaa 1980
gatttgaaag tgattatcga ccacttcgag aaatatccgc tgattaccca gaaattgggc 2040
gattacaagt tgtttaaaca ggcattcagc gtcatggaga ataaagaaca tcttaaggag 2100
aatgggatta aggagctcgt acgaatcaaa gctaagatga attggggtct caatgacgaa 2160
ttgaaaaaag catttccaga gaacattagc aaagagcgcc cccttatcaa taagaacatt 2220
ccgaatttca aatggctggc tggattcaca gctggtgaag gctacttcgg cgtgaatcta 2280
aaaaaggtaa agggcaccgc aaaggtatac gtgggactga gattctcaat ctcacagcac 2340
atcagagaca agaacctgat gaattcattg ataacatacc taggctgtgg ttccatctgg 2400
gagaagaaca agtctgagtt cagatggctc gagttcgtcg taaccaaatt cagcgatatc 2460
aacgacaaga tcattccggt attccaggaa aatactctga ttggcgtcaa actcgaggac 2520
tttgaagatt ggtgcaaggt tgccaaattg atcaaagaga agaaacacct gaccgaatcc 2580
ggtttggatg agattaagaa aatcaagctg aacatgaaca aaggtcgtgg tggaggcggt 2640
agcggaggcg gagggtcggc tagcactccc gacatcatac tgcaacgaac cggaattgac 2700
gttcgagcgg tcgagcaagg ggatgatgcc tggcataagt tgagacttgg tgtgataact 2760
gcatcagagg tacataatgt cattgcgaag ccccggagcg ggaaaaagtg gcccgacatg 2820
aagatgtcat actttcatac actgctggcc gaggtctgca cgggtgttgc tcccgaggtt 2880
aatgccaaag cacttgcatg gggtaaacag tacgaaaacg acgctcggac cctctttgag 2940
ttcacatctg gtgtgaatgt gacagagagc ccaatcattt accgagacga gagcatgaga 3000
acggcttgca gtcctgatgg cctgtgcagc gacggtaacg gactggagct aaagtgcccg 3060
tttactagcc gagactttat gaagtttagg ctcggtgggt ttgaggccat caagagtgct 3120
tacatggcgc aagtacagta ttccatgtgg gtcactcgca aaaacgcttg gtactttgct 3180
aactatgatc cccgaatgaa gagggagggc ctgcattatg tggtcataga acgggacgaa 3240
aagtatatgg cgagcttcga cgaaattgta ccggagttca tagagaagat ggatgaagca 3300
ctcgccgaga ttgggttcgt ctttggggaa caatggaggt ga 3342
<210> 51
<211> 3342
<212> RNA
<213> 人工序列
<220>
<223> 在实验室中制备-低活性TCRα megaTAL-λExo RNA构建体
<400> 51
augggauccu gcaggccacc uaagaagaaa cgcaaagucg uggaucuacg cacgcucggc 60
uacagucagc agcagcaaga gaagaucaaa ccgaaggugc guucgacagu ggcgcagcac 120
cacgaggcac uggugggcca uggguuuaca cacgcgcaca ucguugcgcu cagccaacac 180
ccggcagcgu uagggaccgu cgcugucacg uaucagcaca uaaucacggc guugccagag 240
gcgacacacg aagacaucgu uggcgucggc aaacaguggu ccggcgcacg cgcccuggag 300
gccuugcuca cggaugcggg ggaguugaga gguccgccgu uacaguugga cacaggccaa 360
cuugugaaga uugcaaaacg uggcggcgug accgcaaugg aggcagugca ugcaucgcgc 420
aaugcacuga cgggugcccc ccugaaccug accccggacc aagugguggc uaucgccagc 480
aacauuggcg gcaagcaagc gcucgaaacg gugcagcggc uguugccggu gcugugccag 540
gaccauggcc ugaccccgga ccaaguggug gcuaucgcca gcaacauugg cggcaagcaa 600
gcgcucgaaa cggugcagcg gcuguugccg gugcugugcc aggaccaugg ccugaccccg 660
gaccaagugg uggcuaucgc cagcaacauu ggcggcaagc aagcgcucga aacggugcag 720
cggcuguugc cggugcugug ccaggaccau ggccugaccc cggaccaagu gguggcuauc 780
gccagcaacg guggcggcaa gcaagcgcuc gaaacggugc agcggcuguu gccggugcug 840
ugccaggacc auggccugac uccggaccaa gugguggcua ucgccagcca cgauggcggc 900
aagcaagcgc ucgaaacggu gcagcggcug uugccggugc ugugccagga ccauggccug 960
acuccggacc aagugguggc uaucgccagc cacgauggcg gcaagcaagc gcucgaaacg 1020
gugcagcggc uguugccggu gcugugccag gaccauggcc ugaccccgga ccaaguggug 1080
gcuaucgcca gcaacauugg cggcaagcaa gcgcucgaaa cggugcagcg gcuguugccg 1140
gugcugugcc aggaccaugg ccugaccccg gaccaagugg uggcuaucgc cagcaacaau 1200
ggcggcaagc aagcgcucga aacggugcag cggcuguugc cggugcugug ccaggaccau 1260
ggccugaccc cggaccaagu gguggcuauc gccagcaacg guggcggcaa gcaagcgcuc 1320
gaaacggugc agcggcuguu gccggugcug ugccaggacc auggccugac cccggaccaa 1380
gugguggcua ucgccagcaa caauggcggc aagcaagcgc ucgaaacggu gcagcggcug 1440
uugccggugc ugugccagga ccauggccug accccggacc aagugguggc uaucgccagc 1500
aacauuggcg gcaagcaagc gcucgaaagc auuguggccc agcugagccg gccugauccg 1560
gcguuggccg cguugaccaa cgaccaccuc gucgccuugg ccugccucgg cggacguccu 1620
gccauggaug cagugaaaaa gggauugccg cacgcgccgg aauugaucag aagagucaau 1680
cgccguauug gcgaacgcac gucccaucgc guugcgauau cuagaguggg aggaagcucu 1740
cgcagagagu ccaucaaccc auggauucug acugguuucg cugaugccga aggaucauuc 1800
auacuagaca uccgcaaccg aaacaacgaa agcaacagau accgaacuuc gcugagauuc 1860
cagaucaccc ugcacaacaa ggacaaaucg auucuggaga auauccaguc cacuuggaag 1920
gucggcaaga ucacaaacag cagugacaga gccgucaugc ugagggucac ccguuucgaa 1980
gauuugaaag ugauuaucga ccacuucgag aaauauccgc ugauuaccca gaaauugggc 2040
gauuacaagu uguuuaaaca ggcauucagc gucauggaga auaaagaaca ucuuaaggag 2100
aaugggauua aggagcucgu acgaaucaaa gcuaagauga auuggggucu caaugacgaa 2160
uugaaaaaag cauuuccaga gaacauuagc aaagagcgcc cccuuaucaa uaagaacauu 2220
ccgaauuuca aauggcuggc uggauucaca gcuggugaag gcuacuucgg cgugaaucua 2280
aaaaagguaa agggcaccgc aaagguauac gugggacuga gauucucaau cucacagcac 2340
aucagagaca agaaccugau gaauucauug auaacauacc uaggcugugg uuccaucugg 2400
gagaagaaca agucugaguu cagauggcuc gaguucgucg uaaccaaauu cagcgauauc 2460
aacgacaaga ucauuccggu auuccaggaa aauacucuga uuggcgucaa acucgaggac 2520
uuugaagauu ggugcaaggu ugccaaauug aucaaagaga agaaacaccu gaccgaaucc 2580
gguuuggaug agauuaagaa aaucaagcug aacaugaaca aaggucgugg uggaggcggu 2640
agcggaggcg gagggucggc uagcacuccc gacaucauac ugcaacgaac cggaauugac 2700
guucgagcgg ucgagcaagg ggaugaugcc uggcauaagu ugagacuugg ugugauaacu 2760
gcaucagagg uacauaaugu cauugcgaag ccccggagcg ggaaaaagug gcccgacaug 2820
aagaugucau acuuucauac acugcuggcc gaggucugca cggguguugc ucccgagguu 2880
aaugccaaag cacuugcaug ggguaaacag uacgaaaacg acgcucggac ccucuuugag 2940
uucacaucug gugugaaugu gacagagagc ccaaucauuu accgagacga gagcaugaga 3000
acggcuugca guccugaugg ccugugcagc gacgguaacg gacuggagcu aaagugcccg 3060
uuuacuagcc gagacuuuau gaaguuuagg cucggugggu uugaggccau caagagugcu 3120
uacauggcgc aaguacagua uuccaugugg gucacucgca aaaacgcuug guacuuugcu 3180
aacuaugauc cccgaaugaa gagggagggc cugcauuaug uggucauaga acgggacgaa 3240
aaguauaugg cgagcuucga cgaaauugua ccggaguuca uagagaagau ggaugaagca 3300
cucgccgaga uuggguucgu cuuuggggaa caauggaggu ga 3342
<210> 52
<211> 1113
<212> PRT
<213> 人工序列
<220>
<223> 在实验室中制备-低活性TCRα megaTAL-λExo蛋白质构建体
<400> 52
Met Gly Ser Cys Arg Pro Pro Lys Lys Lys Arg Lys Val Val Asp Leu
1 5 10 15
Arg Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys
20 25 30
Val Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val Gly His Gly
35 40 45
Phe Thr His Ala His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu
50 55 60
Gly Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr Ala Leu Pro Glu
65 70 75 80
Ala Thr His Glu Asp Ile Val Gly Val Gly Lys Gln Trp Ser Gly Ala
85 90 95
Arg Ala Leu Glu Ala Leu Leu Thr Asp Ala Gly Glu Leu Arg Gly Pro
100 105 110
Pro Leu Gln Leu Asp Thr Gly Gln Leu Val Lys Ile Ala Lys Arg Gly
115 120 125
Gly Val Thr Ala Met Glu Ala Val His Ala Ser Arg Asn Ala Leu Thr
130 135 140
Gly Ala Pro Leu Asn Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
145 150 155 160
Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
165 170 175
Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
180 185 190
Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
195 200 205
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val
210 215 220
Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
225 230 235 240
Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln
245 250 255
Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr
260 265 270
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro
275 280 285
Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu
290 295 300
Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu
305 310 315 320
Thr Pro Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln
325 330 335
Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His
340 345 350
Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly
355 360 365
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
370 375 380
Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Asn
385 390 395 400
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu
405 410 415
Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
420 425 430
Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
435 440 445
Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
450 455 460
Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
465 470 475 480
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val
485 490 495
Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Ser Ile Val
500 505 510
Ala Gln Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu Thr Asn Asp
515 520 525
His Leu Val Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala Met Asp Ala
530 535 540
Val Lys Lys Gly Leu Pro His Ala Pro Glu Leu Ile Arg Arg Val Asn
545 550 555 560
Arg Arg Ile Gly Glu Arg Thr Ser His Arg Val Ala Ile Ser Arg Val
565 570 575
Gly Gly Ser Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr Gly
580 585 590
Phe Ala Asp Ala Glu Gly Ser Phe Ile Leu Asp Ile Arg Asn Arg Asn
595 600 605
Asn Glu Ser Asn Arg Tyr Arg Thr Ser Leu Arg Phe Gln Ile Thr Leu
610 615 620
His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp Lys
625 630 635 640
Val Gly Lys Ile Thr Asn Ser Ser Asp Arg Ala Val Met Leu Arg Val
645 650 655
Thr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys Tyr
660 665 670
Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln Ala
675 680 685
Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile Lys
690 695 700
Glu Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly Leu Asn Asp Glu
705 710 715 720
Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Pro Leu Ile
725 730 735
Asn Lys Asn Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe Thr Ala Gly
740 745 750
Glu Gly Tyr Phe Gly Val Asn Leu Lys Lys Val Lys Gly Thr Ala Lys
755 760 765
Val Tyr Val Gly Leu Arg Phe Ser Ile Ser Gln His Ile Arg Asp Lys
770 775 780
Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Ser Ile Trp
785 790 795 800
Glu Lys Asn Lys Ser Glu Phe Arg Trp Leu Glu Phe Val Val Thr Lys
805 810 815
Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn Thr
820 825 830
Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val Ala
835 840 845
Lys Leu Ile Lys Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp Glu
850 855 860
Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Gly Gly Gly Gly
865 870 875 880
Ser Gly Gly Gly Gly Ser Ala Ser Thr Pro Asp Ile Ile Leu Gln Arg
885 890 895
Thr Gly Ile Asp Val Arg Ala Val Glu Gln Gly Asp Asp Ala Trp His
900 905 910
Lys Leu Arg Leu Gly Val Ile Thr Ala Ser Glu Val His Asn Val Ile
915 920 925
Ala Lys Pro Arg Ser Gly Lys Lys Trp Pro Asp Met Lys Met Ser Tyr
930 935 940
Phe His Thr Leu Leu Ala Glu Val Cys Thr Gly Val Ala Pro Glu Val
945 950 955 960
Asn Ala Lys Ala Leu Ala Trp Gly Lys Gln Tyr Glu Asn Asp Ala Arg
965 970 975
Thr Leu Phe Glu Phe Thr Ser Gly Val Asn Val Thr Glu Ser Pro Ile
980 985 990
Ile Tyr Arg Asp Glu Ser Met Arg Thr Ala Cys Ser Pro Asp Gly Leu
995 1000 1005
Cys Ser Asp Gly Asn Gly Leu Glu Leu Lys Cys Pro Phe Thr Ser
1010 1015 1020
Arg Asp Phe Met Lys Phe Arg Leu Gly Gly Phe Glu Ala Ile Lys
1025 1030 1035
Ser Ala Tyr Met Ala Gln Val Gln Tyr Ser Met Trp Val Thr Arg
1040 1045 1050
Lys Asn Ala Trp Tyr Phe Ala Asn Tyr Asp Pro Arg Met Lys Arg
1055 1060 1065
Glu Gly Leu His Tyr Val Val Ile Glu Arg Asp Glu Lys Tyr Met
1070 1075 1080
Ala Ser Phe Asp Glu Ile Val Pro Glu Phe Ile Glu Lys Met Asp
1085 1090 1095
Glu Ala Leu Ala Glu Ile Gly Phe Val Phe Gly Glu Gln Trp Arg
1100 1105 1110
<210> 53
<211> 4398
<212> DNA
<213> 人工序列
<220>
<223> 在实验室中制备-低活性TCRα megaTAL-RecJ DNA构建体
<400> 53
atgggatcct gcaggccacc taagaagaaa cgcaaagtcg tggatctacg cacgctcggc 60
tacagtcagc agcagcaaga gaagatcaaa ccgaaggtgc gttcgacagt ggcgcagcac 120
cacgaggcac tggtgggcca tgggtttaca cacgcgcaca tcgttgcgct cagccaacac 180
ccggcagcgt tagggaccgt cgctgtcacg tatcagcaca taatcacggc gttgccagag 240
gcgacacacg aagacatcgt tggcgtcggc aaacagtggt ccggcgcacg cgccctggag 300
gccttgctca cggatgcggg ggagttgaga ggtccgccgt tacagttgga cacaggccaa 360
cttgtgaaga ttgcaaaacg tggcggcgtg accgcaatgg aggcagtgca tgcatcgcgc 420
aatgcactga cgggtgcccc cctgaacctg accccggacc aagtggtggc tatcgccagc 480
aacattggcg gcaagcaagc gctcgaaacg gtgcagcggc tgttgccggt gctgtgccag 540
gaccatggcc tgaccccgga ccaagtggtg gctatcgcca gcaacattgg cggcaagcaa 600
gcgctcgaaa cggtgcagcg gctgttgccg gtgctgtgcc aggaccatgg cctgaccccg 660
gaccaagtgg tggctatcgc cagcaacatt ggcggcaagc aagcgctcga aacggtgcag 720
cggctgttgc cggtgctgtg ccaggaccat ggcctgaccc cggaccaagt ggtggctatc 780
gccagcaacg gtggcggcaa gcaagcgctc gaaacggtgc agcggctgtt gccggtgctg 840
tgccaggacc atggcctgac tccggaccaa gtggtggcta tcgccagcca cgatggcggc 900
aagcaagcgc tcgaaacggt gcagcggctg ttgccggtgc tgtgccagga ccatggcctg 960
actccggacc aagtggtggc tatcgccagc cacgatggcg gcaagcaagc gctcgaaacg 1020
gtgcagcggc tgttgccggt gctgtgccag gaccatggcc tgaccccgga ccaagtggtg 1080
gctatcgcca gcaacattgg cggcaagcaa gcgctcgaaa cggtgcagcg gctgttgccg 1140
gtgctgtgcc aggaccatgg cctgaccccg gaccaagtgg tggctatcgc cagcaacaat 1200
ggcggcaagc aagcgctcga aacggtgcag cggctgttgc cggtgctgtg ccaggaccat 1260
ggcctgaccc cggaccaagt ggtggctatc gccagcaacg gtggcggcaa gcaagcgctc 1320
gaaacggtgc agcggctgtt gccggtgctg tgccaggacc atggcctgac cccggaccaa 1380
gtggtggcta tcgccagcaa caatggcggc aagcaagcgc tcgaaacggt gcagcggctg 1440
ttgccggtgc tgtgccagga ccatggcctg accccggacc aagtggtggc tatcgccagc 1500
aacattggcg gcaagcaagc gctcgaaagc attgtggccc agctgagccg gcctgatccg 1560
gcgttggccg cgttgaccaa cgaccacctc gtcgccttgg cctgcctcgg cggacgtcct 1620
gccatggatg cagtgaaaaa gggattgccg cacgcgccgg aattgatcag aagagtcaat 1680
cgccgtattg gcgaacgcac gtcccatcgc gttgcgatat ctagagtggg aggaagctct 1740
cgcagagagt ccatcaaccc atggattctg actggtttcg ctgatgccga aggatcattc 1800
atactagaca tccgcaaccg aaacaacgaa agcaacagat accgaacttc gctgagattc 1860
cagatcaccc tgcacaacaa ggacaaatcg attctggaga atatccagtc cacttggaag 1920
gtcggcaaga tcacaaacag cagtgacaga gccgtcatgc tgagggtcac ccgtttcgaa 1980
gatttgaaag tgattatcga ccacttcgag aaatatccgc tgattaccca gaaattgggc 2040
gattacaagt tgtttaaaca ggcattcagc gtcatggaga ataaagaaca tcttaaggag 2100
aatgggatta aggagctcgt acgaatcaaa gctaagatga attggggtct caatgacgaa 2160
ttgaaaaaag catttccaga gaacattagc aaagagcgcc cccttatcaa taagaacatt 2220
ccgaatttca aatggctggc tggattcaca gctggtgaag gctacttcgg cgtgaatcta 2280
aaaaaggtaa agggcaccgc aaaggtatac gtgggactga gattctcaat ctcacagcac 2340
atcagagaca agaacctgat gaattcattg ataacatacc taggctgtgg ttccatctgg 2400
gagaagaaca agtctgagtt cagatggctc gagttcgtcg taaccaaatt cagcgatatc 2460
aacgacaaga tcattccggt attccaggaa aatactctga ttggcgtcaa actcgaggac 2520
tttgaagatt ggtgcaaggt tgccaaattg atcaaagaga agaaacacct gaccgaatcc 2580
ggtttggatg agattaagaa aatcaagctg aacatgaaca aaggtcgtgg tggaggcggt 2640
agcggaggcg gagggtcggc tagcaagcaa caaatccatt tgagacgaag ggaggttgac 2700
gagactgctg atctcccggc tgcggagttg cctccgctat tgcgtcgcct gtacgcctct 2760
cggggtgtac gctccgctca agaactggaa cgatcagtaa agggtatgct gccctggcaa 2820
cagctttccg gggtggagaa ggccgtggag atactataca atgcgttccg agaagggacg 2880
agaatcatag tagttgggga ctttgatgcg gatggcgcga cttccactgc cctgtctgtg 2940
ctggcgatgc ggagtcttgg gtgttccaac atagactacc tcgttccaaa tcggtttgaa 3000
gatggatacg gcctttcacc cgaagttgtg gaccaagccc acgcgagggg agcccaactt 3060
atagtgacag tggataacgg aattagctca catgcaggag tagagcacgc aaggtcactg 3120
ggtatccccg tgattgtcac cgaccatcac ctcccaggag acactttgcc tgctgctgag 3180
gccatcatta accctaatct tagggactgc aactttccta gtaagagtct ggctggggtt 3240
ggagtcgcat tctaccttat gctggccctc cgcacttttc tccgagatca aggctggttt 3300
gatgaacgaa acattgctat cccaaatctt gcagaactcc ttgaccttgt ggctttggga 3360
acagtcgccg atgtcgtgcc gctggacgct aacaatcgga tattgacttg gcaggggatg 3420
tcacgcatcc gtgcgggcaa gtgcaggcca ggtatcaagg cattgttgga agtcgccaac 3480
cgggacgcac aaaaacttgc cgcaagcgac ctcggttttg cacttggtcc tcgccttaac 3540
gcagcgggta gattggatga tatgtccgtg ggcgtggcgc tcttgttgtg cgacaacata 3600
ggtgaagcga gagttcttgc aaatgagttg gacgcgctca atcagactag gaaagagata 3660
gaacaaggaa tgcagatcga ggcactgacc ctttgtgaaa agctggagcg atcacgggat 3720
accttgcctg gaggtctcgc aatgtaccat cctgagtggc atcagggcgt cgtaggtatc 3780
ttggcgtcta ggatcaaaga gaggttccat cgcccagtga ttgcatttgc gccagcaggt 3840
gatggcactc tgaaaggatc tggccgctcc atccaaggtc tccacatgag agatgcactt 3900
gagaggttgg acacacttta cccaggcatg atgttgaaat tcggtgggca cgctatggct 3960
gctggtttga gcttggaaga agataagttc aaactgttcc aacagaggtt cggcgaactg 4020
gtaactgagt ggcttgatcc ttcattgctc caaggtgagg tggtatcaga tgggccactc 4080
agcccagccg aaatgactat ggaagtagcc cagctcctca gagatgcagg tccttggggt 4140
caaatgttcc ccgagccctt gttcgacggg catttcaggt tgctccagca gaggttggta 4200
ggcgagcgcc acctcaaagt gatggtcgaa cctgtaggtg gaggtccgct tctggacgga 4260
attgctttca acgtagacac ggcgctgtgg cccgacaatg gcgttaggga ggtacaactt 4320
gcctacaaac tcgacatcaa cgagtttaga ggtaacagaa gcctccagat catcattgac 4380
aatatctggc ccatttga 4398
<210> 54
<211> 4398
<212> RNA
<213> 人工序列
<220>
<223> 在实验室中制备-低活性TCRα megaTAL-RecJ RNA构建体
<400> 54
augggauccu gcaggccacc uaagaagaaa cgcaaagucg uggaucuacg cacgcucggc 60
uacagucagc agcagcaaga gaagaucaaa ccgaaggugc guucgacagu ggcgcagcac 120
cacgaggcac uggugggcca uggguuuaca cacgcgcaca ucguugcgcu cagccaacac 180
ccggcagcgu uagggaccgu cgcugucacg uaucagcaca uaaucacggc guugccagag 240
gcgacacacg aagacaucgu uggcgucggc aaacaguggu ccggcgcacg cgcccuggag 300
gccuugcuca cggaugcggg ggaguugaga gguccgccgu uacaguugga cacaggccaa 360
cuugugaaga uugcaaaacg uggcggcgug accgcaaugg aggcagugca ugcaucgcgc 420
aaugcacuga cgggugcccc ccugaaccug accccggacc aagugguggc uaucgccagc 480
aacauuggcg gcaagcaagc gcucgaaacg gugcagcggc uguugccggu gcugugccag 540
gaccauggcc ugaccccgga ccaaguggug gcuaucgcca gcaacauugg cggcaagcaa 600
gcgcucgaaa cggugcagcg gcuguugccg gugcugugcc aggaccaugg ccugaccccg 660
gaccaagugg uggcuaucgc cagcaacauu ggcggcaagc aagcgcucga aacggugcag 720
cggcuguugc cggugcugug ccaggaccau ggccugaccc cggaccaagu gguggcuauc 780
gccagcaacg guggcggcaa gcaagcgcuc gaaacggugc agcggcuguu gccggugcug 840
ugccaggacc auggccugac uccggaccaa gugguggcua ucgccagcca cgauggcggc 900
aagcaagcgc ucgaaacggu gcagcggcug uugccggugc ugugccagga ccauggccug 960
acuccggacc aagugguggc uaucgccagc cacgauggcg gcaagcaagc gcucgaaacg 1020
gugcagcggc uguugccggu gcugugccag gaccauggcc ugaccccgga ccaaguggug 1080
gcuaucgcca gcaacauugg cggcaagcaa gcgcucgaaa cggugcagcg gcuguugccg 1140
gugcugugcc aggaccaugg ccugaccccg gaccaagugg uggcuaucgc cagcaacaau 1200
ggcggcaagc aagcgcucga aacggugcag cggcuguugc cggugcugug ccaggaccau 1260
ggccugaccc cggaccaagu gguggcuauc gccagcaacg guggcggcaa gcaagcgcuc 1320
gaaacggugc agcggcuguu gccggugcug ugccaggacc auggccugac cccggaccaa 1380
gugguggcua ucgccagcaa caauggcggc aagcaagcgc ucgaaacggu gcagcggcug 1440
uugccggugc ugugccagga ccauggccug accccggacc aagugguggc uaucgccagc 1500
aacauuggcg gcaagcaagc gcucgaaagc auuguggccc agcugagccg gccugauccg 1560
gcguuggccg cguugaccaa cgaccaccuc gucgccuugg ccugccucgg cggacguccu 1620
gccauggaug cagugaaaaa gggauugccg cacgcgccgg aauugaucag aagagucaau 1680
cgccguauug gcgaacgcac gucccaucgc guugcgauau cuagaguggg aggaagcucu 1740
cgcagagagu ccaucaaccc auggauucug acugguuucg cugaugccga aggaucauuc 1800
auacuagaca uccgcaaccg aaacaacgaa agcaacagau accgaacuuc gcugagauuc 1860
cagaucaccc ugcacaacaa ggacaaaucg auucuggaga auauccaguc cacuuggaag 1920
gucggcaaga ucacaaacag cagugacaga gccgucaugc ugagggucac ccguuucgaa 1980
gauuugaaag ugauuaucga ccacuucgag aaauauccgc ugauuaccca gaaauugggc 2040
gauuacaagu uguuuaaaca ggcauucagc gucauggaga auaaagaaca ucuuaaggag 2100
aaugggauua aggagcucgu acgaaucaaa gcuaagauga auuggggucu caaugacgaa 2160
uugaaaaaag cauuuccaga gaacauuagc aaagagcgcc cccuuaucaa uaagaacauu 2220
ccgaauuuca aauggcuggc uggauucaca gcuggugaag gcuacuucgg cgugaaucua 2280
aaaaagguaa agggcaccgc aaagguauac gugggacuga gauucucaau cucacagcac 2340
aucagagaca agaaccugau gaauucauug auaacauacc uaggcugugg uuccaucugg 2400
gagaagaaca agucugaguu cagauggcuc gaguucgucg uaaccaaauu cagcgauauc 2460
aacgacaaga ucauuccggu auuccaggaa aauacucuga uuggcgucaa acucgaggac 2520
uuugaagauu ggugcaaggu ugccaaauug aucaaagaga agaaacaccu gaccgaaucc 2580
gguuuggaug agauuaagaa aaucaagcug aacaugaaca aaggucgugg uggaggcggu 2640
agcggaggcg gagggucggc uagcaagcaa caaauccauu ugagacgaag ggagguugac 2700
gagacugcug aucucccggc ugcggaguug ccuccgcuau ugcgucgccu guacgccucu 2760
cgggguguac gcuccgcuca agaacuggaa cgaucaguaa aggguaugcu gcccuggcaa 2820
cagcuuuccg ggguggagaa ggccguggag auacuauaca augcguuccg agaagggacg 2880
agaaucauag uaguugggga cuuugaugcg gauggcgcga cuuccacugc ccugucugug 2940
cuggcgaugc ggagucuugg guguuccaac auagacuacc ucguuccaaa ucgguuugaa 3000
gauggauacg gccuuucacc cgaaguugug gaccaagccc acgcgagggg agcccaacuu 3060
auagugacag uggauaacgg aauuagcuca caugcaggag uagagcacgc aaggucacug 3120
gguauccccg ugauugucac cgaccaucac cucccaggag acacuuugcc ugcugcugag 3180
gccaucauua acccuaaucu uagggacugc aacuuuccua guaagagucu ggcugggguu 3240
ggagucgcau ucuaccuuau gcuggcccuc cgcacuuuuc uccgagauca aggcugguuu 3300
gaugaacgaa acauugcuau cccaaaucuu gcagaacucc uugaccuugu ggcuuuggga 3360
acagucgccg augucgugcc gcuggacgcu aacaaucgga uauugacuug gcaggggaug 3420
ucacgcaucc gugcgggcaa gugcaggcca gguaucaagg cauuguugga agucgccaac 3480
cgggacgcac aaaaacuugc cgcaagcgac cucgguuuug cacuuggucc ucgccuuaac 3540
gcagcgggua gauuggauga uauguccgug ggcguggcgc ucuuguugug cgacaacaua 3600
ggugaagcga gaguucuugc aaaugaguug gacgcgcuca aucagacuag gaaagagaua 3660
gaacaaggaa ugcagaucga ggcacugacc cuuugugaaa agcuggagcg aucacgggau 3720
accuugccug gaggucucgc aauguaccau ccugaguggc aucagggcgu cguagguauc 3780
uuggcgucua ggaucaaaga gagguuccau cgcccaguga uugcauuugc gccagcaggu 3840
gauggcacuc ugaaaggauc uggccgcucc auccaagguc uccacaugag agaugcacuu 3900
gagagguugg acacacuuua cccaggcaug auguugaaau ucggugggca cgcuauggcu 3960
gcugguuuga gcuuggaaga agauaaguuc aaacuguucc aacagagguu cggcgaacug 4020
guaacugagu ggcuugaucc uucauugcuc caaggugagg ugguaucaga ugggccacuc 4080
agcccagccg aaaugacuau ggaaguagcc cagcuccuca gagaugcagg uccuuggggu 4140
caaauguucc ccgagcccuu guucgacggg cauuucaggu ugcuccagca gagguuggua 4200
ggcgagcgcc accucaaagu gauggucgaa ccuguaggug gagguccgcu ucuggacgga 4260
auugcuuuca acguagacac ggcgcugugg cccgacaaug gcguuaggga gguacaacuu 4320
gccuacaaac ucgacaucaa cgaguuuaga gguaacagaa gccuccagau caucauugac 4380
aauaucuggc ccauuuga 4398
<210> 55
<211> 1465
<212> PRT
<213> 人工序列
<220>
<223> 在实验室中制备-低活性TCRα megaTAL-RecJ蛋白质构建体
<400> 55
Met Gly Ser Cys Arg Pro Pro Lys Lys Lys Arg Lys Val Val Asp Leu
1 5 10 15
Arg Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys
20 25 30
Val Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val Gly His Gly
35 40 45
Phe Thr His Ala His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu
50 55 60
Gly Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr Ala Leu Pro Glu
65 70 75 80
Ala Thr His Glu Asp Ile Val Gly Val Gly Lys Gln Trp Ser Gly Ala
85 90 95
Arg Ala Leu Glu Ala Leu Leu Thr Asp Ala Gly Glu Leu Arg Gly Pro
100 105 110
Pro Leu Gln Leu Asp Thr Gly Gln Leu Val Lys Ile Ala Lys Arg Gly
115 120 125
Gly Val Thr Ala Met Glu Ala Val His Ala Ser Arg Asn Ala Leu Thr
130 135 140
Gly Ala Pro Leu Asn Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
145 150 155 160
Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
165 170 175
Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
180 185 190
Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
195 200 205
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val
210 215 220
Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
225 230 235 240
Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln
245 250 255
Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr
260 265 270
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro
275 280 285
Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu
290 295 300
Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu
305 310 315 320
Thr Pro Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln
325 330 335
Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His
340 345 350
Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly
355 360 365
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
370 375 380
Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Asn
385 390 395 400
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu
405 410 415
Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
420 425 430
Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
435 440 445
Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
450 455 460
Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
465 470 475 480
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val
485 490 495
Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Ser Ile Val
500 505 510
Ala Gln Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu Thr Asn Asp
515 520 525
His Leu Val Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala Met Asp Ala
530 535 540
Val Lys Lys Gly Leu Pro His Ala Pro Glu Leu Ile Arg Arg Val Asn
545 550 555 560
Arg Arg Ile Gly Glu Arg Thr Ser His Arg Val Ala Ile Ser Arg Val
565 570 575
Gly Gly Ser Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr Gly
580 585 590
Phe Ala Asp Ala Glu Gly Ser Phe Ile Leu Asp Ile Arg Asn Arg Asn
595 600 605
Asn Glu Ser Asn Arg Tyr Arg Thr Ser Leu Arg Phe Gln Ile Thr Leu
610 615 620
His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp Lys
625 630 635 640
Val Gly Lys Ile Thr Asn Ser Ser Asp Arg Ala Val Met Leu Arg Val
645 650 655
Thr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys Tyr
660 665 670
Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln Ala
675 680 685
Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile Lys
690 695 700
Glu Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly Leu Asn Asp Glu
705 710 715 720
Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Pro Leu Ile
725 730 735
Asn Lys Asn Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe Thr Ala Gly
740 745 750
Glu Gly Tyr Phe Gly Val Asn Leu Lys Lys Val Lys Gly Thr Ala Lys
755 760 765
Val Tyr Val Gly Leu Arg Phe Ser Ile Ser Gln His Ile Arg Asp Lys
770 775 780
Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Ser Ile Trp
785 790 795 800
Glu Lys Asn Lys Ser Glu Phe Arg Trp Leu Glu Phe Val Val Thr Lys
805 810 815
Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn Thr
820 825 830
Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val Ala
835 840 845
Lys Leu Ile Lys Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp Glu
850 855 860
Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Gly Gly Gly Gly
865 870 875 880
Ser Gly Gly Gly Gly Ser Ala Ser Lys Gln Gln Ile His Leu Arg Arg
885 890 895
Arg Glu Val Asp Glu Thr Ala Asp Leu Pro Ala Ala Glu Leu Pro Pro
900 905 910
Leu Leu Arg Arg Leu Tyr Ala Ser Arg Gly Val Arg Ser Ala Gln Glu
915 920 925
Leu Glu Arg Ser Val Lys Gly Met Leu Pro Trp Gln Gln Leu Ser Gly
930 935 940
Val Glu Lys Ala Val Glu Ile Leu Tyr Asn Ala Phe Arg Glu Gly Thr
945 950 955 960
Arg Ile Ile Val Val Gly Asp Phe Asp Ala Asp Gly Ala Thr Ser Thr
965 970 975
Ala Leu Ser Val Leu Ala Met Arg Ser Leu Gly Cys Ser Asn Ile Asp
980 985 990
Tyr Leu Val Pro Asn Arg Phe Glu Asp Gly Tyr Gly Leu Ser Pro Glu
995 1000 1005
Val Val Asp Gln Ala His Ala Arg Gly Ala Gln Leu Ile Val Thr
1010 1015 1020
Val Asp Asn Gly Ile Ser Ser His Ala Gly Val Glu His Ala Arg
1025 1030 1035
Ser Leu Gly Ile Pro Val Ile Val Thr Asp His His Leu Pro Gly
1040 1045 1050
Asp Thr Leu Pro Ala Ala Glu Ala Ile Ile Asn Pro Asn Leu Arg
1055 1060 1065
Asp Cys Asn Phe Pro Ser Lys Ser Leu Ala Gly Val Gly Val Ala
1070 1075 1080
Phe Tyr Leu Met Leu Ala Leu Arg Thr Phe Leu Arg Asp Gln Gly
1085 1090 1095
Trp Phe Asp Glu Arg Asn Ile Ala Ile Pro Asn Leu Ala Glu Leu
1100 1105 1110
Leu Asp Leu Val Ala Leu Gly Thr Val Ala Asp Val Val Pro Leu
1115 1120 1125
Asp Ala Asn Asn Arg Ile Leu Thr Trp Gln Gly Met Ser Arg Ile
1130 1135 1140
Arg Ala Gly Lys Cys Arg Pro Gly Ile Lys Ala Leu Leu Glu Val
1145 1150 1155
Ala Asn Arg Asp Ala Gln Lys Leu Ala Ala Ser Asp Leu Gly Phe
1160 1165 1170
Ala Leu Gly Pro Arg Leu Asn Ala Ala Gly Arg Leu Asp Asp Met
1175 1180 1185
Ser Val Gly Val Ala Leu Leu Leu Cys Asp Asn Ile Gly Glu Ala
1190 1195 1200
Arg Val Leu Ala Asn Glu Leu Asp Ala Leu Asn Gln Thr Arg Lys
1205 1210 1215
Glu Ile Glu Gln Gly Met Gln Ile Glu Ala Leu Thr Leu Cys Glu
1220 1225 1230
Lys Leu Glu Arg Ser Arg Asp Thr Leu Pro Gly Gly Leu Ala Met
1235 1240 1245
Tyr His Pro Glu Trp His Gln Gly Val Val Gly Ile Leu Ala Ser
1250 1255 1260
Arg Ile Lys Glu Arg Phe His Arg Pro Val Ile Ala Phe Ala Pro
1265 1270 1275
Ala Gly Asp Gly Thr Leu Lys Gly Ser Gly Arg Ser Ile Gln Gly
1280 1285 1290
Leu His Met Arg Asp Ala Leu Glu Arg Leu Asp Thr Leu Tyr Pro
1295 1300 1305
Gly Met Met Leu Lys Phe Gly Gly His Ala Met Ala Ala Gly Leu
1310 1315 1320
Ser Leu Glu Glu Asp Lys Phe Lys Leu Phe Gln Gln Arg Phe Gly
1325 1330 1335
Glu Leu Val Thr Glu Trp Leu Asp Pro Ser Leu Leu Gln Gly Glu
1340 1345 1350
Val Val Ser Asp Gly Pro Leu Ser Pro Ala Glu Met Thr Met Glu
1355 1360 1365
Val Ala Gln Leu Leu Arg Asp Ala Gly Pro Trp Gly Gln Met Phe
1370 1375 1380
Pro Glu Pro Leu Phe Asp Gly His Phe Arg Leu Leu Gln Gln Arg
1385 1390 1395
Leu Val Gly Glu Arg His Leu Lys Val Met Val Glu Pro Val Gly
1400 1405 1410
Gly Gly Pro Leu Leu Asp Gly Ile Ala Phe Asn Val Asp Thr Ala
1415 1420 1425
Leu Trp Pro Asp Asn Gly Val Arg Glu Val Gln Leu Ala Tyr Lys
1430 1435 1440
Leu Asp Ile Asn Glu Phe Arg Gly Asn Arg Ser Leu Gln Ile Ile
1445 1450 1455
Ile Asp Asn Ile Trp Pro Ile
1460 1465
<210> 56
<211> 2733
<212> DNA
<213> 人工序列
<220>
<223> 在实验室中制备-高活性Cb1-b megaTAL DNA构建体
<400> 56
atgggatcct gcaggccacc taagaagaaa cgcaaagtcg tggatctacg cacgctcggc 60
tacagtcagc agcagcaaga gaagatcaaa ccgaaggtgc gttcgacagt ggcgcagcac 120
cacgaggcac tggtgggcca tgggtttaca cacgcgcaca tcgttgcgct cagccaacac 180
ccggcagcgt tagggaccgt cgctgtcacg tatcagcaca taatcacggc gttgccagag 240
gcgacacacg aagacatcgt tggcgtcggc aaacagtggt ccggcgcacg cgccctggag 300
gccttgctca cggatgcggg ggagttgaga ggtccgccgt tacagttgga cacaggccaa 360
cttgtgaaga ttgcaaaacg tggcggcgtg accgcaatgg aggcagtgca tgcatcgcgc 420
aatgcactga cgggtgcccc cctgaactta acacccgatc aagtggtagc gatagcgtca 480
aacggcgggg gtaaacaggc tttggagacg gtacagcggt tattgccggt actctgccag 540
gaccacggat tgacaccgga ccaagtggtg gcgattgcgt ccaacaacgg aggcaagcag 600
gcactagaga ctgtccaacg gcttcttccc gttctttgtc aggatcatgg gctaacccct 660
gatcaggtag tcgctatagc ttcaaatgga gggggcaagc aagcactgga gactgttcaa 720
cgactcctgc cagtgctctg ccaagaccac ggacttacac cagatcaagt ggttgctatt 780
gcctccaatg gtggcgggaa acaagcgttg gaaactgtgc agagactgtt acctgtcttg 840
tgtcaagacc acggcctcac gccagatcag gtggtagcca tagcgtcgaa cattggtggt 900
aagcaagccc ttgaaacggt ccagcgtctt ctgccggtgt tgtgccagga ccacggacta 960
acgccggatc aggtcgtagc cattgcttca aatgggggcg gcaaacaggc gctagagaca 1020
gtccagcgcc tcttgcctgt gttatgccag gatcacggct taaccccaga ccaagttgtg 1080
gctattgcat ctaacaatgg tggcaaacaa gccttggaga cagtgcaacg attactgcct 1140
gtcttatgtc aggatcatgg cctgacgccc gatcaggtag tggcaatcgc atctaacatt 1200
ggaggtaagc aagcactgga gactgtccag agattgttac ccgtactatg tcaagatcat 1260
ggtttgacgc ctgatcaggt tgttgcgata gccagcaata acggagggaa acaggctctt 1320
gaaaccgtac agcgacttct cccagtcttg tgccaagatc acgggcttac tcctgatcaa 1380
gtcgtagcta tcgccagcaa taacggtgga aaacaggccc tggaaaccgt acaacgtctc 1440
ctcccagtac tttgtcaaga ccacgggttg actccggatc aagtcgtcgc gatcgcgagc 1500
aatggtgggg ggaagcaggc gctggaaact gttcagagac tgctgcctgt actttgtcag 1560
gaccatggtc tgacacctga ccaagttgtg gcgatagcca gtaatatcgg gggaaaacag 1620
gcactagaga gcattgtggc ccagctgagc cggcctgatc cggcgttggc cgcgttgacc 1680
aacgaccacc tcgtcgcctt ggcctgcctc ggcggacgtc ctgccatgga tgcagtgaaa 1740
aagggattgc cgcacgcgcc ggaattgatc agaagagtca atcgccgtat tggcgaacgc 1800
acgtcccatc gcgttgcgat atctagagtg ggaggaagct cgcgcagaga gtccatcaac 1860
ccatggattc tgactggttt cgctgatgcc gaaggatgct tccgactaga catccgcaac 1920
gcaaacgatt taagagccgg atacagaact agactggcct tcgaaatcgt actgcacaac 1980
aaggacaaat cgattctgga gaatatccag tcgacttgga aggtcggcac aatctacaac 2040
gcgggcgaca acgcagtcag actgcaagtc acacgtttcg aagatttgaa agtgattatc 2100
gaccacttcg agaaatatcc gctgataaca cagaaattgg gcgattacaa gttgtttaaa 2160
caggcattca gcgtcatgga gaacaaagaa catcttaagg agaatgggat taaggagctc 2220
gtacgaatca aagctaagat gaattggggt ctcaatgacg aattgaaaaa agcatttcca 2280
gagaacatta gcaaagagcg ctcccttatc aataagaaca ttccgaatct caaatggctg 2340
gctggattca catctggtga cggctcgttc gtggtggaac taaagaagag aagaagcccc 2400
gtcaaggtag gagtgcggct gcgattcagc atcacccagc acatcagaga caagaacctg 2460
atgaattcat tgataacata cctaggctgt ggtcgtatcg ttgagaataa caaatctgag 2520
cacagttggc tcgaattcat tgtaacaaaa ttcagcgata tcaacgacaa gatcattccg 2580
gtattccagg aaaatactct gattggcgtc aaactcgagg actttgaaga ttggtgcaag 2640
gttgccaaat tgatcgaaga gaagaaacac ctgaccgaat ccggtttgga tgagattaag 2700
aaaatcaagc tgaacatgaa caaaggtcgt tga 2733
<210> 57
<211> 2733
<212> RNA
<213> 人工序列
<220>
<223> 在实验室中制备-高活性Cb1-b megaTAL RNA构建体
<400> 57
augggauccu gcaggccacc uaagaagaaa cgcaaagucg uggaucuacg cacgcucggc 60
uacagucagc agcagcaaga gaagaucaaa ccgaaggugc guucgacagu ggcgcagcac 120
cacgaggcac uggugggcca uggguuuaca cacgcgcaca ucguugcgcu cagccaacac 180
ccggcagcgu uagggaccgu cgcugucacg uaucagcaca uaaucacggc guugccagag 240
gcgacacacg aagacaucgu uggcgucggc aaacaguggu ccggcgcacg cgcccuggag 300
gccuugcuca cggaugcggg ggaguugaga gguccgccgu uacaguugga cacaggccaa 360
cuugugaaga uugcaaaacg uggcggcgug accgcaaugg aggcagugca ugcaucgcgc 420
aaugcacuga cgggugcccc ccugaacuua acacccgauc aagugguagc gauagcguca 480
aacggcgggg guaaacaggc uuuggagacg guacagcggu uauugccggu acucugccag 540
gaccacggau ugacaccgga ccaaguggug gcgauugcgu ccaacaacgg aggcaagcag 600
gcacuagaga cuguccaacg gcuucuuccc guucuuuguc aggaucaugg gcuaaccccu 660
gaucagguag ucgcuauagc uucaaaugga gggggcaagc aagcacugga gacuguucaa 720
cgacuccugc cagugcucug ccaagaccac ggacuuacac cagaucaagu gguugcuauu 780
gccuccaaug guggcgggaa acaagcguug gaaacugugc agagacuguu accugucuug 840
ugucaagacc acggccucac gccagaucag gugguagcca uagcgucgaa cauugguggu 900
aagcaagccc uugaaacggu ccagcgucuu cugccggugu ugugccagga ccacggacua 960
acgccggauc aggucguagc cauugcuuca aaugggggcg gcaaacaggc gcuagagaca 1020
guccagcgcc ucuugccugu guuaugccag gaucacggcu uaaccccaga ccaaguugug 1080
gcuauugcau cuaacaaugg uggcaaacaa gccuuggaga cagugcaacg auuacugccu 1140
gucuuauguc aggaucaugg ccugacgccc gaucagguag uggcaaucgc aucuaacauu 1200
ggagguaagc aagcacugga gacuguccag agauuguuac ccguacuaug ucaagaucau 1260
gguuugacgc cugaucaggu uguugcgaua gccagcaaua acggagggaa acaggcucuu 1320
gaaaccguac agcgacuucu cccagucuug ugccaagauc acgggcuuac uccugaucaa 1380
gucguagcua ucgccagcaa uaacggugga aaacaggccc uggaaaccgu acaacgucuc 1440
cucccaguac uuugucaaga ccacggguug acuccggauc aagucgucgc gaucgcgagc 1500
aauggugggg ggaagcaggc gcuggaaacu guucagagac ugcugccugu acuuugucag 1560
gaccaugguc ugacaccuga ccaaguugug gcgauagcca guaauaucgg gggaaaacag 1620
gcacuagaga gcauuguggc ccagcugagc cggccugauc cggcguuggc cgcguugacc 1680
aacgaccacc ucgucgccuu ggccugccuc ggcggacguc cugccaugga ugcagugaaa 1740
aagggauugc cgcacgcgcc ggaauugauc agaagaguca aucgccguau uggcgaacgc 1800
acgucccauc gcguugcgau aucuagagug ggaggaagcu cgcgcagaga guccaucaac 1860
ccauggauuc ugacugguuu cgcugaugcc gaaggaugcu uccgacuaga cauccgcaac 1920
gcaaacgauu uaagagccgg auacagaacu agacuggccu ucgaaaucgu acugcacaac 1980
aaggacaaau cgauucugga gaauauccag ucgacuugga aggucggcac aaucuacaac 2040
gcgggcgaca acgcagucag acugcaaguc acacguuucg aagauuugaa agugauuauc 2100
gaccacuucg agaaauaucc gcugauaaca cagaaauugg gcgauuacaa guuguuuaaa 2160
caggcauuca gcgucaugga gaacaaagaa caucuuaagg agaaugggau uaaggagcuc 2220
guacgaauca aagcuaagau gaauuggggu cucaaugacg aauugaaaaa agcauuucca 2280
gagaacauua gcaaagagcg cucccuuauc aauaagaaca uuccgaaucu caaauggcug 2340
gcuggauuca caucugguga cggcucguuc gugguggaac uaaagaagag aagaagcccc 2400
gucaagguag gagugcggcu gcgauucagc aucacccagc acaucagaga caagaaccug 2460
augaauucau ugauaacaua ccuaggcugu ggucguaucg uugagaauaa caaaucugag 2520
cacaguuggc ucgaauucau uguaacaaaa uucagcgaua ucaacgacaa gaucauuccg 2580
guauuccagg aaaauacucu gauuggcguc aaacucgagg acuuugaaga uuggugcaag 2640
guugccaaau ugaucgaaga gaagaaacac cugaccgaau ccgguuugga ugagauuaag 2700
aaaaucaagc ugaacaugaa caaaggucgu uga 2733
<210> 58
<211> 910
<212> PRT
<213> 人工序列
<220>
<223> 在实验室中制备-高活性Cb1-b megaTAL蛋白质构建体
<400> 58
Met Gly Ser Cys Arg Pro Pro Lys Lys Lys Arg Lys Val Val Asp Leu
1 5 10 15
Arg Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys
20 25 30
Val Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val Gly His Gly
35 40 45
Phe Thr His Ala His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu
50 55 60
Gly Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr Ala Leu Pro Glu
65 70 75 80
Ala Thr His Glu Asp Ile Val Gly Val Gly Lys Gln Trp Ser Gly Ala
85 90 95
Arg Ala Leu Glu Ala Leu Leu Thr Asp Ala Gly Glu Leu Arg Gly Pro
100 105 110
Pro Leu Gln Leu Asp Thr Gly Gln Leu Val Lys Ile Ala Lys Arg Gly
115 120 125
Gly Val Thr Ala Met Glu Ala Val His Ala Ser Arg Asn Ala Leu Thr
130 135 140
Gly Ala Pro Leu Asn Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
145 150 155 160
Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
165 170 175
Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
180 185 190
Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
195 200 205
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val
210 215 220
Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
225 230 235 240
Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln
245 250 255
Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr
260 265 270
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro
275 280 285
Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu
290 295 300
Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu
305 310 315 320
Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln
325 330 335
Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His
340 345 350
Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly
355 360 365
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
370 375 380
Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Ile
385 390 395 400
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu
405 410 415
Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
420 425 430
Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
435 440 445
Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
450 455 460
Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
465 470 475 480
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val
485 490 495
Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
500 505 510
Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln
515 520 525
Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Ser
530 535 540
Ile Val Ala Gln Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu Thr
545 550 555 560
Asn Asp His Leu Val Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala Met
565 570 575
Asp Ala Val Lys Lys Gly Leu Pro His Ala Pro Glu Leu Ile Arg Arg
580 585 590
Val Asn Arg Arg Ile Gly Glu Arg Thr Ser His Arg Val Ala Ile Ser
595 600 605
Arg Val Gly Gly Ser Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu
610 615 620
Thr Gly Phe Ala Asp Ala Glu Gly Cys Phe Arg Leu Asp Ile Arg Asn
625 630 635 640
Ala Asn Asp Leu Arg Ala Gly Tyr Arg Thr Arg Leu Ala Phe Glu Ile
645 650 655
Val Leu His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr
660 665 670
Trp Lys Val Gly Thr Ile Tyr Asn Ala Gly Asp Asn Ala Val Arg Leu
675 680 685
Gln Val Thr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu
690 695 700
Lys Tyr Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys
705 710 715 720
Gln Ala Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly
725 730 735
Ile Lys Glu Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly Leu Asn
740 745 750
Asp Glu Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Ser
755 760 765
Leu Ile Asn Lys Asn Ile Pro Asn Leu Lys Trp Leu Ala Gly Phe Thr
770 775 780
Ser Gly Asp Gly Ser Phe Val Val Glu Leu Lys Lys Arg Arg Ser Pro
785 790 795 800
Val Lys Val Gly Val Arg Leu Arg Phe Ser Ile Thr Gln His Ile Arg
805 810 815
Asp Lys Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Arg
820 825 830
Ile Val Glu Asn Asn Lys Ser Glu His Ser Trp Leu Glu Phe Ile Val
835 840 845
Thr Lys Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu
850 855 860
Asn Thr Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys
865 870 875 880
Val Ala Lys Leu Ile Glu Glu Lys Lys His Leu Thr Glu Ser Gly Leu
885 890 895
Asp Glu Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg
900 905 910
<210> 59
<211> 3474
<212> DNA
<213> 人工序列
<220>
<223> 在实验室中制备-高活性Cb1-b megaTAL-Trex2 DNA构建体
<400> 59
atgggatcct gcaggccacc taagaagaaa cgcaaagtcg tggatctacg cacgctcggc 60
tacagtcagc agcagcaaga gaagatcaaa ccgaaggtgc gttcgacagt ggcgcagcac 120
cacgaggcac tggtgggcca tgggtttaca cacgcgcaca tcgttgcgct cagccaacac 180
ccggcagcgt tagggaccgt cgctgtcacg tatcagcaca taatcacggc gttgccagag 240
gcgacacacg aagacatcgt tggcgtcggc aaacagtggt ccggcgcacg cgccctggag 300
gccttgctca cggatgcggg ggagttgaga ggtccgccgt tacagttgga cacaggccaa 360
cttgtgaaga ttgcaaaacg tggcggcgtg accgcaatgg aggcagtgca tgcatcgcgc 420
aatgcactga cgggtgcccc cctgaactta acacccgatc aagtggtagc gatagcgtca 480
aacggcgggg gtaaacaggc tttggagacg gtacagcggt tattgccggt actctgccag 540
gaccacggat tgacaccgga ccaagtggtg gcgattgcgt ccaacaacgg aggcaagcag 600
gcactagaga ctgtccaacg gcttcttccc gttctttgtc aggatcatgg gctaacccct 660
gatcaggtag tcgctatagc ttcaaatgga gggggcaagc aagcactgga gactgttcaa 720
cgactcctgc cagtgctctg ccaagaccac ggacttacac cagatcaagt ggttgctatt 780
gcctccaatg gtggcgggaa acaagcgttg gaaactgtgc agagactgtt acctgtcttg 840
tgtcaagacc acggcctcac gccagatcag gtggtagcca tagcgtcgaa cattggtggt 900
aagcaagccc ttgaaacggt ccagcgtctt ctgccggtgt tgtgccagga ccacggacta 960
acgccggatc aggtcgtagc cattgcttca aatgggggcg gcaaacaggc gctagagaca 1020
gtccagcgcc tcttgcctgt gttatgccag gatcacggct taaccccaga ccaagttgtg 1080
gctattgcat ctaacaatgg tggcaaacaa gccttggaga cagtgcaacg attactgcct 1140
gtcttatgtc aggatcatgg cctgacgccc gatcaggtag tggcaatcgc atctaacatt 1200
ggaggtaagc aagcactgga gactgtccag agattgttac ccgtactatg tcaagatcat 1260
ggtttgacgc ctgatcaggt tgttgcgata gccagcaata acggagggaa acaggctctt 1320
gaaaccgtac agcgacttct cccagtcttg tgccaagatc acgggcttac tcctgatcaa 1380
gtcgtagcta tcgccagcaa taacggtgga aaacaggccc tggaaaccgt acaacgtctc 1440
ctcccagtac tttgtcaaga ccacgggttg actccggatc aagtcgtcgc gatcgcgagc 1500
aatggtgggg ggaagcaggc gctggaaact gttcagagac tgctgcctgt actttgtcag 1560
gaccatggtc tgacacctga ccaagttgtg gcgatagcca gtaatatcgg gggaaaacag 1620
gcactagaga gcattgtggc ccagctgagc cggcctgatc cggcgttggc cgcgttgacc 1680
aacgaccacc tcgtcgcctt ggcctgcctc ggcggacgtc ctgccatgga tgcagtgaaa 1740
aagggattgc cgcacgcgcc ggaattgatc agaagagtca atcgccgtat tggcgaacgc 1800
acgtcccatc gcgttgcgat atctagagtg ggaggaagct cgcgcagaga gtccatcaac 1860
ccatggattc tgactggttt cgctgatgcc gaaggatgct tccgactaga catccgcaac 1920
gcaaacgatt taagagccgg atacagaact agactggcct tcgaaatcgt actgcacaac 1980
aaggacaaat cgattctgga gaatatccag tcgacttgga aggtcggcac aatctacaac 2040
gcgggcgaca acgcagtcag actgcaagtc acacgtttcg aagatttgaa agtgattatc 2100
gaccacttcg agaaatatcc gctgataaca cagaaattgg gcgattacaa gttgtttaaa 2160
caggcattca gcgtcatgga gaacaaagaa catcttaagg agaatgggat taaggagctc 2220
gtacgaatca aagctaagat gaattggggt ctcaatgacg aattgaaaaa agcatttcca 2280
gagaacatta gcaaagagcg ctcccttatc aataagaaca ttccgaatct caaatggctg 2340
gctggattca catctggtga cggctcgttc gtggtggaac taaagaagag aagaagcccc 2400
gtcaaggtag gagtgcggct gcgattcagc atcacccagc acatcagaga caagaacctg 2460
atgaattcat tgataacata cctaggctgt ggtcgtatcg ttgagaataa caaatctgag 2520
cacagttggc tcgaattcat tgtaacaaaa ttcagcgata tcaacgacaa gatcattccg 2580
gtattccagg aaaatactct gattggcgtc aaactcgagg actttgaaga ttggtgcaag 2640
gttgccaaat tgatcgaaga gaagaaacac ctgaccgaat ccggtttgga tgagattaag 2700
aaaatcaagc tgaacatgaa caaaggtcgt ggtggaggcg gtagcggagg cggagggtcg 2760
gctagctccg aagcaccgag agccgaaacc ttcgtgttcc tcgacctgga agccactggc 2820
ctgcccagtg tggagcccga gattgccgag ctgtccctgt tcgccgttca tcggtcctcc 2880
ctggaaaacc ctgagcacga cgagagcgga gcactggtgc tgccaagggt gctggacaag 2940
ctgaccctgt gcatgtgtcc cgagcgtccc ttcactgcca aggcatccga aatcactggt 3000
ctgagttcgg aagggctggc tcgttgtaga aaggctggat tcgatggtgc cgtggttcgg 3060
actttgcagg cattcctgtc caggcaggca ggtccaatct gcttggtcgc ccacaacggt 3120
tttgactacg acttcccact gctctgtgcc gaactgcgtc gcctgggagc acgccttcct 3180
cgggataccg tgtgcctgga caccctaccc gctctgcgtg gactggacag agcccactca 3240
cacggcacta gggcgagagg acgccagggt tacagcctcg gttcactgtt ccatcggtat 3300
ttcagggcag agccttccgc agcccattca gccgaaggcg atgtccacac cttgcttctg 3360
atcttcctgc atcgtgcagc agaactgctc gcctgggcag atgagcaggc tcgtggatgg 3420
gctcacattg agccgatgta cttgccacct gatgacccta gcctggaagc ctga 3474
<210> 60
<211> 3474
<212> RNA
<213> 人工序列
<220>
<223> 在实验室中制备-高活性Cb1-b megaTAL-Trex2 RNA构建体
<400> 60
augggauccu gcaggccacc uaagaagaaa cgcaaagucg uggaucuacg cacgcucggc 60
uacagucagc agcagcaaga gaagaucaaa ccgaaggugc guucgacagu ggcgcagcac 120
cacgaggcac uggugggcca uggguuuaca cacgcgcaca ucguugcgcu cagccaacac 180
ccggcagcgu uagggaccgu cgcugucacg uaucagcaca uaaucacggc guugccagag 240
gcgacacacg aagacaucgu uggcgucggc aaacaguggu ccggcgcacg cgcccuggag 300
gccuugcuca cggaugcggg ggaguugaga gguccgccgu uacaguugga cacaggccaa 360
cuugugaaga uugcaaaacg uggcggcgug accgcaaugg aggcagugca ugcaucgcgc 420
aaugcacuga cgggugcccc ccugaacuua acacccgauc aagugguagc gauagcguca 480
aacggcgggg guaaacaggc uuuggagacg guacagcggu uauugccggu acucugccag 540
gaccacggau ugacaccgga ccaaguggug gcgauugcgu ccaacaacgg aggcaagcag 600
gcacuagaga cuguccaacg gcuucuuccc guucuuuguc aggaucaugg gcuaaccccu 660
gaucagguag ucgcuauagc uucaaaugga gggggcaagc aagcacugga gacuguucaa 720
cgacuccugc cagugcucug ccaagaccac ggacuuacac cagaucaagu gguugcuauu 780
gccuccaaug guggcgggaa acaagcguug gaaacugugc agagacuguu accugucuug 840
ugucaagacc acggccucac gccagaucag gugguagcca uagcgucgaa cauugguggu 900
aagcaagccc uugaaacggu ccagcgucuu cugccggugu ugugccagga ccacggacua 960
acgccggauc aggucguagc cauugcuuca aaugggggcg gcaaacaggc gcuagagaca 1020
guccagcgcc ucuugccugu guuaugccag gaucacggcu uaaccccaga ccaaguugug 1080
gcuauugcau cuaacaaugg uggcaaacaa gccuuggaga cagugcaacg auuacugccu 1140
gucuuauguc aggaucaugg ccugacgccc gaucagguag uggcaaucgc aucuaacauu 1200
ggagguaagc aagcacugga gacuguccag agauuguuac ccguacuaug ucaagaucau 1260
gguuugacgc cugaucaggu uguugcgaua gccagcaaua acggagggaa acaggcucuu 1320
gaaaccguac agcgacuucu cccagucuug ugccaagauc acgggcuuac uccugaucaa 1380
gucguagcua ucgccagcaa uaacggugga aaacaggccc uggaaaccgu acaacgucuc 1440
cucccaguac uuugucaaga ccacggguug acuccggauc aagucgucgc gaucgcgagc 1500
aauggugggg ggaagcaggc gcuggaaacu guucagagac ugcugccugu acuuugucag 1560
gaccaugguc ugacaccuga ccaaguugug gcgauagcca guaauaucgg gggaaaacag 1620
gcacuagaga gcauuguggc ccagcugagc cggccugauc cggcguuggc cgcguugacc 1680
aacgaccacc ucgucgccuu ggccugccuc ggcggacguc cugccaugga ugcagugaaa 1740
aagggauugc cgcacgcgcc ggaauugauc agaagaguca aucgccguau uggcgaacgc 1800
acgucccauc gcguugcgau aucuagagug ggaggaagcu cgcgcagaga guccaucaac 1860
ccauggauuc ugacugguuu cgcugaugcc gaaggaugcu uccgacuaga cauccgcaac 1920
gcaaacgauu uaagagccgg auacagaacu agacuggccu ucgaaaucgu acugcacaac 1980
aaggacaaau cgauucugga gaauauccag ucgacuugga aggucggcac aaucuacaac 2040
gcgggcgaca acgcagucag acugcaaguc acacguuucg aagauuugaa agugauuauc 2100
gaccacuucg agaaauaucc gcugauaaca cagaaauugg gcgauuacaa guuguuuaaa 2160
caggcauuca gcgucaugga gaacaaagaa caucuuaagg agaaugggau uaaggagcuc 2220
guacgaauca aagcuaagau gaauuggggu cucaaugacg aauugaaaaa agcauuucca 2280
gagaacauua gcaaagagcg cucccuuauc aauaagaaca uuccgaaucu caaauggcug 2340
gcuggauuca caucugguga cggcucguuc gugguggaac uaaagaagag aagaagcccc 2400
gucaagguag gagugcggcu gcgauucagc aucacccagc acaucagaga caagaaccug 2460
augaauucau ugauaacaua ccuaggcugu ggucguaucg uugagaauaa caaaucugag 2520
cacaguuggc ucgaauucau uguaacaaaa uucagcgaua ucaacgacaa gaucauuccg 2580
guauuccagg aaaauacucu gauuggcguc aaacucgagg acuuugaaga uuggugcaag 2640
guugccaaau ugaucgaaga gaagaaacac cugaccgaau ccgguuugga ugagauuaag 2700
aaaaucaagc ugaacaugaa caaaggucgu gguggaggcg guagcggagg cggagggucg 2760
gcuagcuccg aagcaccgag agccgaaacc uucguguucc ucgaccugga agccacuggc 2820
cugcccagug uggagcccga gauugccgag cugucccugu ucgccguuca ucgguccucc 2880
cuggaaaacc cugagcacga cgagagcgga gcacuggugc ugccaagggu gcuggacaag 2940
cugacccugu gcaugugucc cgagcguccc uucacugcca aggcauccga aaucacuggu 3000
cugaguucgg aagggcuggc ucguuguaga aaggcuggau ucgauggugc cgugguucgg 3060
acuuugcagg cauuccuguc caggcaggca gguccaaucu gcuuggucgc ccacaacggu 3120
uuugacuacg acuucccacu gcucugugcc gaacugcguc gccugggagc acgccuuccu 3180
cgggauaccg ugugccugga cacccuaccc gcucugcgug gacuggacag agcccacuca 3240
cacggcacua gggcgagagg acgccagggu uacagccucg guucacuguu ccaucgguau 3300
uucagggcag agccuuccgc agcccauuca gccgaaggcg auguccacac cuugcuucug 3360
aucuuccugc aucgugcagc agaacugcuc gccugggcag augagcaggc ucguggaugg 3420
gcucacauug agccgaugua cuugccaccu gaugacccua gccuggaagc cuga 3474
<210> 61
<211> 1157
<212> PRT
<213> 人工序列
<220>
<223> 在实验室中制备-高活性Cb1-b megaTAL-Trex2蛋白质构建体
<400> 61
Met Gly Ser Cys Arg Pro Pro Lys Lys Lys Arg Lys Val Val Asp Leu
1 5 10 15
Arg Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys
20 25 30
Val Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val Gly His Gly
35 40 45
Phe Thr His Ala His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu
50 55 60
Gly Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr Ala Leu Pro Glu
65 70 75 80
Ala Thr His Glu Asp Ile Val Gly Val Gly Lys Gln Trp Ser Gly Ala
85 90 95
Arg Ala Leu Glu Ala Leu Leu Thr Asp Ala Gly Glu Leu Arg Gly Pro
100 105 110
Pro Leu Gln Leu Asp Thr Gly Gln Leu Val Lys Ile Ala Lys Arg Gly
115 120 125
Gly Val Thr Ala Met Glu Ala Val His Ala Ser Arg Asn Ala Leu Thr
130 135 140
Gly Ala Pro Leu Asn Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
145 150 155 160
Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
165 170 175
Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
180 185 190
Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
195 200 205
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val
210 215 220
Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
225 230 235 240
Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln
245 250 255
Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr
260 265 270
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro
275 280 285
Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu
290 295 300
Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu
305 310 315 320
Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln
325 330 335
Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His
340 345 350
Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly
355 360 365
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
370 375 380
Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Ile
385 390 395 400
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu
405 410 415
Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
420 425 430
Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
435 440 445
Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
450 455 460
Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
465 470 475 480
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val
485 490 495
Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
500 505 510
Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln
515 520 525
Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Ser
530 535 540
Ile Val Ala Gln Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu Thr
545 550 555 560
Asn Asp His Leu Val Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala Met
565 570 575
Asp Ala Val Lys Lys Gly Leu Pro His Ala Pro Glu Leu Ile Arg Arg
580 585 590
Val Asn Arg Arg Ile Gly Glu Arg Thr Ser His Arg Val Ala Ile Ser
595 600 605
Arg Val Gly Gly Ser Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu
610 615 620
Thr Gly Phe Ala Asp Ala Glu Gly Cys Phe Arg Leu Asp Ile Arg Asn
625 630 635 640
Ala Asn Asp Leu Arg Ala Gly Tyr Arg Thr Arg Leu Ala Phe Glu Ile
645 650 655
Val Leu His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr
660 665 670
Trp Lys Val Gly Thr Ile Tyr Asn Ala Gly Asp Asn Ala Val Arg Leu
675 680 685
Gln Val Thr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu
690 695 700
Lys Tyr Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys
705 710 715 720
Gln Ala Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly
725 730 735
Ile Lys Glu Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly Leu Asn
740 745 750
Asp Glu Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Ser
755 760 765
Leu Ile Asn Lys Asn Ile Pro Asn Leu Lys Trp Leu Ala Gly Phe Thr
770 775 780
Ser Gly Asp Gly Ser Phe Val Val Glu Leu Lys Lys Arg Arg Ser Pro
785 790 795 800
Val Lys Val Gly Val Arg Leu Arg Phe Ser Ile Thr Gln His Ile Arg
805 810 815
Asp Lys Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Arg
820 825 830
Ile Val Glu Asn Asn Lys Ser Glu His Ser Trp Leu Glu Phe Ile Val
835 840 845
Thr Lys Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu
850 855 860
Asn Thr Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys
865 870 875 880
Val Ala Lys Leu Ile Glu Glu Lys Lys His Leu Thr Glu Ser Gly Leu
885 890 895
Asp Glu Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Gly Gly
900 905 910
Gly Gly Ser Gly Gly Gly Gly Ser Ala Ser Ser Glu Ala Pro Arg Ala
915 920 925
Glu Thr Phe Val Phe Leu Asp Leu Glu Ala Thr Gly Leu Pro Ser Val
930 935 940
Glu Pro Glu Ile Ala Glu Leu Ser Leu Phe Ala Val His Arg Ser Ser
945 950 955 960
Leu Glu Asn Pro Glu His Asp Glu Ser Gly Ala Leu Val Leu Pro Arg
965 970 975
Val Leu Asp Lys Leu Thr Leu Cys Met Cys Pro Glu Arg Pro Phe Thr
980 985 990
Ala Lys Ala Ser Glu Ile Thr Gly Leu Ser Ser Glu Gly Leu Ala Arg
995 1000 1005
Cys Arg Lys Ala Gly Phe Asp Gly Ala Val Val Arg Thr Leu Gln
1010 1015 1020
Ala Phe Leu Ser Arg Gln Ala Gly Pro Ile Cys Leu Val Ala His
1025 1030 1035
Asn Gly Phe Asp Tyr Asp Phe Pro Leu Leu Cys Ala Glu Leu Arg
1040 1045 1050
Arg Leu Gly Ala Arg Leu Pro Arg Asp Thr Val Cys Leu Asp Thr
1055 1060 1065
Leu Pro Ala Leu Arg Gly Leu Asp Arg Ala His Ser His Gly Thr
1070 1075 1080
Arg Ala Arg Gly Arg Gln Gly Tyr Ser Leu Gly Ser Leu Phe His
1085 1090 1095
Arg Tyr Phe Arg Ala Glu Pro Ser Ala Ala His Ser Ala Glu Gly
1100 1105 1110
Asp Val His Thr Leu Leu Leu Ile Phe Leu His Arg Ala Ala Glu
1115 1120 1125
Leu Leu Ala Trp Ala Asp Glu Gln Ala Arg Gly Trp Ala His Ile
1130 1135 1140
Glu Pro Met Tyr Leu Pro Pro Asp Asp Pro Ser Leu Glu Ala
1145 1150 1155
<210> 62
<211> 3426
<212> DNA
<213> 人工序列
<220>
<223> 在实验室中制备-高活性Cb1-b megaTAL-ExoX DNA构建体
<400> 62
atgggatcct gcaggccacc taagaagaaa cgcaaagtcg tggatctacg cacgctcggc 60
tacagtcagc agcagcaaga gaagatcaaa ccgaaggtgc gttcgacagt ggcgcagcac 120
cacgaggcac tggtgggcca tgggtttaca cacgcgcaca tcgttgcgct cagccaacac 180
ccggcagcgt tagggaccgt cgctgtcacg tatcagcaca taatcacggc gttgccagag 240
gcgacacacg aagacatcgt tggcgtcggc aaacagtggt ccggcgcacg cgccctggag 300
gccttgctca cggatgcggg ggagttgaga ggtccgccgt tacagttgga cacaggccaa 360
cttgtgaaga ttgcaaaacg tggcggcgtg accgcaatgg aggcagtgca tgcatcgcgc 420
aatgcactga cgggtgcccc cctgaactta acacccgatc aagtggtagc gatagcgtca 480
aacggcgggg gtaaacaggc tttggagacg gtacagcggt tattgccggt actctgccag 540
gaccacggat tgacaccgga ccaagtggtg gcgattgcgt ccaacaacgg aggcaagcag 600
gcactagaga ctgtccaacg gcttcttccc gttctttgtc aggatcatgg gctaacccct 660
gatcaggtag tcgctatagc ttcaaatgga gggggcaagc aagcactgga gactgttcaa 720
cgactcctgc cagtgctctg ccaagaccac ggacttacac cagatcaagt ggttgctatt 780
gcctccaatg gtggcgggaa acaagcgttg gaaactgtgc agagactgtt acctgtcttg 840
tgtcaagacc acggcctcac gccagatcag gtggtagcca tagcgtcgaa cattggtggt 900
aagcaagccc ttgaaacggt ccagcgtctt ctgccggtgt tgtgccagga ccacggacta 960
acgccggatc aggtcgtagc cattgcttca aatgggggcg gcaaacaggc gctagagaca 1020
gtccagcgcc tcttgcctgt gttatgccag gatcacggct taaccccaga ccaagttgtg 1080
gctattgcat ctaacaatgg tggcaaacaa gccttggaga cagtgcaacg attactgcct 1140
gtcttatgtc aggatcatgg cctgacgccc gatcaggtag tggcaatcgc atctaacatt 1200
ggaggtaagc aagcactgga gactgtccag agattgttac ccgtactatg tcaagatcat 1260
ggtttgacgc ctgatcaggt tgttgcgata gccagcaata acggagggaa acaggctctt 1320
gaaaccgtac agcgacttct cccagtcttg tgccaagatc acgggcttac tcctgatcaa 1380
gtcgtagcta tcgccagcaa taacggtgga aaacaggccc tggaaaccgt acaacgtctc 1440
ctcccagtac tttgtcaaga ccacgggttg actccggatc aagtcgtcgc gatcgcgagc 1500
aatggtgggg ggaagcaggc gctggaaact gttcagagac tgctgcctgt actttgtcag 1560
gaccatggtc tgacacctga ccaagttgtg gcgatagcca gtaatatcgg gggaaaacag 1620
gcactagaga gcattgtggc ccagctgagc cggcctgatc cggcgttggc cgcgttgacc 1680
aacgaccacc tcgtcgcctt ggcctgcctc ggcggacgtc ctgccatgga tgcagtgaaa 1740
aagggattgc cgcacgcgcc ggaattgatc agaagagtca atcgccgtat tggcgaacgc 1800
acgtcccatc gcgttgcgat atctagagtg ggaggaagct cgcgcagaga gtccatcaac 1860
ccatggattc tgactggttt cgctgatgcc gaaggatgct tccgactaga catccgcaac 1920
gcaaacgatt taagagccgg atacagaact agactggcct tcgaaatcgt actgcacaac 1980
aaggacaaat cgattctgga gaatatccag tcgacttgga aggtcggcac aatctacaac 2040
gcgggcgaca acgcagtcag actgcaagtc acacgtttcg aagatttgaa agtgattatc 2100
gaccacttcg agaaatatcc gctgataaca cagaaattgg gcgattacaa gttgtttaaa 2160
caggcattca gcgtcatgga gaacaaagaa catcttaagg agaatgggat taaggagctc 2220
gtacgaatca aagctaagat gaattggggt ctcaatgacg aattgaaaaa agcatttcca 2280
gagaacatta gcaaagagcg ctcccttatc aataagaaca ttccgaatct caaatggctg 2340
gctggattca catctggtga cggctcgttc gtggtggaac taaagaagag aagaagcccc 2400
gtcaaggtag gagtgcggct gcgattcagc atcacccagc acatcagaga caagaacctg 2460
atgaattcat tgataacata cctaggctgt ggtcgtatcg ttgagaataa caaatctgag 2520
cacagttggc tcgaattcat tgtaacaaaa ttcagcgata tcaacgacaa gatcattccg 2580
gtattccagg aaaatactct gattggcgtc aaactcgagg actttgaaga ttggtgcaag 2640
gttgccaaat tgatcgaaga gaagaaacac ctgaccgaat ccggtttgga tgagattaag 2700
aaaatcaagc tgaacatgaa caaaggtcgt ggtggaggcg gtagcggagg cggagggtcg 2760
gctagcctgc ggatcataga cacggaaacc tgtggactcc agggtggtat cgtggagata 2820
gccagtgtcg atgtaattga tgggaaaatc gtaaacccga tgagccacct cgtccgaccg 2880
gataggccga taagtccgca ggctatggct atacatagaa tcacggaggc tatggtagct 2940
gacaagcctt ggatcgagga cgttatccca cactactatg ggagcgaatg gtatgtagcg 3000
cacaacgcat catttgatag gcgcgtgctt cccgaaatgc ctggggagtg gatttgcaca 3060
atgaaactcg ctcgccgact gtggcctggt atcaagtata gtaacatggc actttacaag 3120
acccgcaaac tcaacgtaca gacacctccg ggtctccacc accatagggc actctatgat 3180
tgttacataa ccgctgctct ccttatagac ataatgaata cctccgggtg gaccgccgaa 3240
caaatggcag acataacggg aagacctagt ttgatgacaa cctttacatt tggaaagtat 3300
cggggcaagg cggtgagcga tgtggccgaa agagatcccg gttatcttag atggctcttc 3360
aataacttgg attctatgtc acctgaactt cgccttaccc tgaagcatta ccttgagaat 3420
acctga 3426
<210> 63
<211> 3426
<212> RNA
<213> 人工序列
<220>
<223> 在实验室中制备-高活性Cb1-b megaTAL-ExoX RNA构建体
<400> 63
augggauccu gcaggccacc uaagaagaaa cgcaaagucg uggaucuacg cacgcucggc 60
uacagucagc agcagcaaga gaagaucaaa ccgaaggugc guucgacagu ggcgcagcac 120
cacgaggcac uggugggcca uggguuuaca cacgcgcaca ucguugcgcu cagccaacac 180
ccggcagcgu uagggaccgu cgcugucacg uaucagcaca uaaucacggc guugccagag 240
gcgacacacg aagacaucgu uggcgucggc aaacaguggu ccggcgcacg cgcccuggag 300
gccuugcuca cggaugcggg ggaguugaga gguccgccgu uacaguugga cacaggccaa 360
cuugugaaga uugcaaaacg uggcggcgug accgcaaugg aggcagugca ugcaucgcgc 420
aaugcacuga cgggugcccc ccugaacuua acacccgauc aagugguagc gauagcguca 480
aacggcgggg guaaacaggc uuuggagacg guacagcggu uauugccggu acucugccag 540
gaccacggau ugacaccgga ccaaguggug gcgauugcgu ccaacaacgg aggcaagcag 600
gcacuagaga cuguccaacg gcuucuuccc guucuuuguc aggaucaugg gcuaaccccu 660
gaucagguag ucgcuauagc uucaaaugga gggggcaagc aagcacugga gacuguucaa 720
cgacuccugc cagugcucug ccaagaccac ggacuuacac cagaucaagu gguugcuauu 780
gccuccaaug guggcgggaa acaagcguug gaaacugugc agagacuguu accugucuug 840
ugucaagacc acggccucac gccagaucag gugguagcca uagcgucgaa cauugguggu 900
aagcaagccc uugaaacggu ccagcgucuu cugccggugu ugugccagga ccacggacua 960
acgccggauc aggucguagc cauugcuuca aaugggggcg gcaaacaggc gcuagagaca 1020
guccagcgcc ucuugccugu guuaugccag gaucacggcu uaaccccaga ccaaguugug 1080
gcuauugcau cuaacaaugg uggcaaacaa gccuuggaga cagugcaacg auuacugccu 1140
gucuuauguc aggaucaugg ccugacgccc gaucagguag uggcaaucgc aucuaacauu 1200
ggagguaagc aagcacugga gacuguccag agauuguuac ccguacuaug ucaagaucau 1260
gguuugacgc cugaucaggu uguugcgaua gccagcaaua acggagggaa acaggcucuu 1320
gaaaccguac agcgacuucu cccagucuug ugccaagauc acgggcuuac uccugaucaa 1380
gucguagcua ucgccagcaa uaacggugga aaacaggccc uggaaaccgu acaacgucuc 1440
cucccaguac uuugucaaga ccacggguug acuccggauc aagucgucgc gaucgcgagc 1500
aauggugggg ggaagcaggc gcuggaaacu guucagagac ugcugccugu acuuugucag 1560
gaccaugguc ugacaccuga ccaaguugug gcgauagcca guaauaucgg gggaaaacag 1620
gcacuagaga gcauuguggc ccagcugagc cggccugauc cggcguuggc cgcguugacc 1680
aacgaccacc ucgucgccuu ggccugccuc ggcggacguc cugccaugga ugcagugaaa 1740
aagggauugc cgcacgcgcc ggaauugauc agaagaguca aucgccguau uggcgaacgc 1800
acgucccauc gcguugcgau aucuagagug ggaggaagcu cgcgcagaga guccaucaac 1860
ccauggauuc ugacugguuu cgcugaugcc gaaggaugcu uccgacuaga cauccgcaac 1920
gcaaacgauu uaagagccgg auacagaacu agacuggccu ucgaaaucgu acugcacaac 1980
aaggacaaau cgauucugga gaauauccag ucgacuugga aggucggcac aaucuacaac 2040
gcgggcgaca acgcagucag acugcaaguc acacguuucg aagauuugaa agugauuauc 2100
gaccacuucg agaaauaucc gcugauaaca cagaaauugg gcgauuacaa guuguuuaaa 2160
caggcauuca gcgucaugga gaacaaagaa caucuuaagg agaaugggau uaaggagcuc 2220
guacgaauca aagcuaagau gaauuggggu cucaaugacg aauugaaaaa agcauuucca 2280
gagaacauua gcaaagagcg cucccuuauc aauaagaaca uuccgaaucu caaauggcug 2340
gcuggauuca caucugguga cggcucguuc gugguggaac uaaagaagag aagaagcccc 2400
gucaagguag gagugcggcu gcgauucagc aucacccagc acaucagaga caagaaccug 2460
augaauucau ugauaacaua ccuaggcugu ggucguaucg uugagaauaa caaaucugag 2520
cacaguuggc ucgaauucau uguaacaaaa uucagcgaua ucaacgacaa gaucauuccg 2580
guauuccagg aaaauacucu gauuggcguc aaacucgagg acuuugaaga uuggugcaag 2640
guugccaaau ugaucgaaga gaagaaacac cugaccgaau ccgguuugga ugagauuaag 2700
aaaaucaagc ugaacaugaa caaaggucgu gguggaggcg guagcggagg cggagggucg 2760
gcuagccugc ggaucauaga cacggaaacc uguggacucc agggugguau cguggagaua 2820
gccagugucg auguaauuga ugggaaaauc guaaacccga ugagccaccu cguccgaccg 2880
gauaggccga uaaguccgca ggcuauggcu auacauagaa ucacggaggc uaugguagcu 2940
gacaagccuu ggaucgagga cguuauccca cacuacuaug ggagcgaaug guauguagcg 3000
cacaacgcau cauuugauag gcgcgugcuu cccgaaaugc cuggggagug gauuugcaca 3060
augaaacucg cucgccgacu guggccuggu aucaaguaua guaacauggc acuuuacaag 3120
acccgcaaac ucaacguaca gacaccuccg ggucuccacc accauagggc acucuaugau 3180
uguuacauaa ccgcugcucu ccuuauagac auaaugaaua ccuccgggug gaccgccgaa 3240
caaauggcag acauaacggg aagaccuagu uugaugacaa ccuuuacauu uggaaaguau 3300
cggggcaagg cggugagcga uguggccgaa agagaucccg guuaucuuag auggcucuuc 3360
aauaacuugg auucuauguc accugaacuu cgccuuaccc ugaagcauua ccuugagaau 3420
accuga 3426
<210> 64
<211> 1141
<212> PRT
<213> 人工序列
<220>
<223> 在实验室中制备-高活性Cb1-b megaTAL-ExoX蛋白质构建体
<400> 64
Met Gly Ser Cys Arg Pro Pro Lys Lys Lys Arg Lys Val Val Asp Leu
1 5 10 15
Arg Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys
20 25 30
Val Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val Gly His Gly
35 40 45
Phe Thr His Ala His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu
50 55 60
Gly Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr Ala Leu Pro Glu
65 70 75 80
Ala Thr His Glu Asp Ile Val Gly Val Gly Lys Gln Trp Ser Gly Ala
85 90 95
Arg Ala Leu Glu Ala Leu Leu Thr Asp Ala Gly Glu Leu Arg Gly Pro
100 105 110
Pro Leu Gln Leu Asp Thr Gly Gln Leu Val Lys Ile Ala Lys Arg Gly
115 120 125
Gly Val Thr Ala Met Glu Ala Val His Ala Ser Arg Asn Ala Leu Thr
130 135 140
Gly Ala Pro Leu Asn Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
145 150 155 160
Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
165 170 175
Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
180 185 190
Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
195 200 205
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val
210 215 220
Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
225 230 235 240
Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln
245 250 255
Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr
260 265 270
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro
275 280 285
Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu
290 295 300
Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu
305 310 315 320
Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln
325 330 335
Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His
340 345 350
Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly
355 360 365
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
370 375 380
Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Ile
385 390 395 400
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu
405 410 415
Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
420 425 430
Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
435 440 445
Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
450 455 460
Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
465 470 475 480
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val
485 490 495
Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
500 505 510
Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln
515 520 525
Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Ser
530 535 540
Ile Val Ala Gln Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu Thr
545 550 555 560
Asn Asp His Leu Val Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala Met
565 570 575
Asp Ala Val Lys Lys Gly Leu Pro His Ala Pro Glu Leu Ile Arg Arg
580 585 590
Val Asn Arg Arg Ile Gly Glu Arg Thr Ser His Arg Val Ala Ile Ser
595 600 605
Arg Val Gly Gly Ser Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu
610 615 620
Thr Gly Phe Ala Asp Ala Glu Gly Cys Phe Arg Leu Asp Ile Arg Asn
625 630 635 640
Ala Asn Asp Leu Arg Ala Gly Tyr Arg Thr Arg Leu Ala Phe Glu Ile
645 650 655
Val Leu His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr
660 665 670
Trp Lys Val Gly Thr Ile Tyr Asn Ala Gly Asp Asn Ala Val Arg Leu
675 680 685
Gln Val Thr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu
690 695 700
Lys Tyr Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys
705 710 715 720
Gln Ala Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly
725 730 735
Ile Lys Glu Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly Leu Asn
740 745 750
Asp Glu Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Ser
755 760 765
Leu Ile Asn Lys Asn Ile Pro Asn Leu Lys Trp Leu Ala Gly Phe Thr
770 775 780
Ser Gly Asp Gly Ser Phe Val Val Glu Leu Lys Lys Arg Arg Ser Pro
785 790 795 800
Val Lys Val Gly Val Arg Leu Arg Phe Ser Ile Thr Gln His Ile Arg
805 810 815
Asp Lys Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Arg
820 825 830
Ile Val Glu Asn Asn Lys Ser Glu His Ser Trp Leu Glu Phe Ile Val
835 840 845
Thr Lys Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu
850 855 860
Asn Thr Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys
865 870 875 880
Val Ala Lys Leu Ile Glu Glu Lys Lys His Leu Thr Glu Ser Gly Leu
885 890 895
Asp Glu Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Gly Gly
900 905 910
Gly Gly Ser Gly Gly Gly Gly Ser Ala Ser Leu Arg Ile Ile Asp Thr
915 920 925
Glu Thr Cys Gly Leu Gln Gly Gly Ile Val Glu Ile Ala Ser Val Asp
930 935 940
Val Ile Asp Gly Lys Ile Val Asn Pro Met Ser His Leu Val Arg Pro
945 950 955 960
Asp Arg Pro Ile Ser Pro Gln Ala Met Ala Ile His Arg Ile Thr Glu
965 970 975
Ala Met Val Ala Asp Lys Pro Trp Ile Glu Asp Val Ile Pro His Tyr
980 985 990
Tyr Gly Ser Glu Trp Tyr Val Ala His Asn Ala Ser Phe Asp Arg Arg
995 1000 1005
Val Leu Pro Glu Met Pro Gly Glu Trp Ile Cys Thr Met Lys Leu
1010 1015 1020
Ala Arg Arg Leu Trp Pro Gly Ile Lys Tyr Ser Asn Met Ala Leu
1025 1030 1035
Tyr Lys Thr Arg Lys Leu Asn Val Gln Thr Pro Pro Gly Leu His
1040 1045 1050
His His Arg Ala Leu Tyr Asp Cys Tyr Ile Thr Ala Ala Leu Leu
1055 1060 1065
Ile Asp Ile Met Asn Thr Ser Gly Trp Thr Ala Glu Gln Met Ala
1070 1075 1080
Asp Ile Thr Gly Arg Pro Ser Leu Met Thr Thr Phe Thr Phe Gly
1085 1090 1095
Lys Tyr Arg Gly Lys Ala Val Ser Asp Val Ala Glu Arg Asp Pro
1100 1105 1110
Gly Tyr Leu Arg Trp Leu Phe Asn Asn Leu Asp Ser Met Ser Pro
1115 1120 1125
Glu Leu Arg Leu Thr Leu Lys His Tyr Leu Glu Asn Thr
1130 1135 1140
<210> 65
<211> 2748
<212> DNA
<213> 人工序列
<220>
<223> 在实验室中制备-低活性Cb1-b megaTAL DNA构建体
<400> 65
atgggatcct gcaggccacc taagaagaaa cgcaaagtcg tggatctacg cacgctcggc 60
tacagtcagc agcagcaaga gaagatcaaa ccgaaggtgc gttcgacagt ggcgcagcac 120
cacgaggcac tggtgggcca tgggtttaca cacgcgcaca tcgttgcgct cagccaacac 180
ccggcagcgt tagggaccgt cgctgtcacg tatcagcaca taatcacggc gttgccagag 240
gcgacacacg aagacatcgt tggcgtcggc aaacagtggt ccggcgcacg cgccctggag 300
gccttgctca cggatgcggg ggagttgaga ggtccgccgt tacagttgga cacaggccaa 360
cttgtgaaga ttgcaaaacg tggcggcgtg accgcaatgg aggcagtgca tgcatcgcgc 420
aatgcactga cgggtgcccc cctgaactta acacccgatc aagtggtagc gatagcgtca 480
aacggcgggg gtaaacaggc tttggagacg gtacagcggt tattgccggt actctgccag 540
gaccacggat tgacaccgga ccaagtggtg gcgattgcgt ccaacaacgg aggcaagcag 600
gcactagaga ctgtccaacg gcttcttccc gttctttgtc aggatcatgg gctaacccct 660
gatcaggtag tcgctatagc ttcaaatgga gggggcaagc aagcactgga gactgttcaa 720
cgactcctgc cagtgctctg ccaagaccac ggacttacac cagatcaagt ggttgctatt 780
gcctccaatg gtggcgggaa acaagcgttg gaaactgtgc agagactgtt acctgtcttg 840
tgtcaagacc acggcctcac gccagatcag gtggtagcca tagcgtcgaa cattggtggt 900
aagcaagccc ttgaaacggt ccagcgtctt ctgccggtgt tgtgccagga ccacggacta 960
acgccggatc aggtcgtagc cattgcttca aatgggggcg gcaaacaggc gctagagaca 1020
gtccagcgcc tcttgcctgt gttatgccag gatcacggct taaccccaga ccaagttgtg 1080
gctattgcat ctaacaatgg tggcaaacaa gccttggaga cagtgcaacg attactgcct 1140
gtcttatgtc aggatcatgg cctgacgccc gatcaggtag tggcaatcgc atctaacatt 1200
ggaggtaagc aagcactgga gactgtccag agattgttac ccgtactatg tcaagatcat 1260
ggtttgacgc ctgatcaggt tgttgcgata gccagcaata acggagggaa acaggctctt 1320
gaaaccgtac agcgacttct cccagtcttg tgccaagatc acgggcttac tcctgatcaa 1380
gtcgtagcta tcgccagcaa taacggtgga aaacaggccc tggaaaccgt acaacgtctc 1440
ctcccagtac tttgtcaaga ccacgggttg actccggatc aagtcgtcgc gatcgcgagc 1500
aatggtgggg ggaagcaggc gctggaaact gttcagagac tgctgcctgt actttgtcag 1560
gaccatggtc tgacacctga ccaagttgtg gcgatagcca gtaatatcgg gggaaaacag 1620
gcactagaga gcattgtggc ccagctgagc cggcctgatc cggcgttggc cgcgttgacc 1680
aacgaccacc tcgtcgcctt ggcctgcctc ggcggacgtc ctgccatgga tgcagtgaaa 1740
aagggattgc cgcacgcgcc ggaattgatc agaagagtca atcgccgtat tggcgaacgc 1800
acgtcccatc gcgttgcgat atctagagtg ggaggaagct cgcgcagaga gtccatcaac 1860
ccatggattc tgactggttt cgctgatgcc gaaggatgct tccgactaga catccgcaac 1920
gcaaactcca tgcgtgtaaa atacagaact agactgtcct tcgaaatcgt actgcacaac 1980
aaggacaaat cgattctgga gaatatccag tcgacttgga aggtcggcaa aatcacaaac 2040
tacggcgaca gaggagtccg cctgagagtc ggccgtttcg aagatttgaa agtgattatc 2100
gaccacttcg agaaatatcc gctgataaca cagaaattgg gcgattacaa gttgtttaaa 2160
caggcattca gcgtcatgga gaacaaagaa catcttaagg agaatgggat taaggagctc 2220
gtacgaatca aagctaagat gaattggggt ctcaatgacg aattgaaaaa agcatttcca 2280
gagaacatta gcaaagagcg cccccttatc aataagaaca tccccaattt caaatggctg 2340
gctggattca catctggtga cggctcgttc atggtggaac taatgaagaa taagaataac 2400
gttattgtac gtgtgcgtct gagattctca atctcccagc acatcagaga caagaacctg 2460
atgaattcat tgataacata cctaggctgt ggtcgtatcg ttgagaataa caaatctgag 2520
cacagttggc tcgaattcat tgtaacaaaa ttcagcgata tcaacgacaa gatcattccg 2580
gtattccagg aaaatactct gattggcgtc aaactcgagg actttgaaga ttggtgcaag 2640
gttgccaaat tgatcgaaga gaagaaacac ctgaccgaat ccggtttgga tgagattaag 2700
aaaatcaagc tgaacatgaa caaaggtcgt gtcttcagcg gccgctga 2748
<210> 66
<211> 2748
<212> RNA
<213> 人工序列
<220>
<223> 在实验室中制备-低活性Cb1-b megaTAL RNA构建体
<400> 66
augggauccu gcaggccacc uaagaagaaa cgcaaagucg uggaucuacg cacgcucggc 60
uacagucagc agcagcaaga gaagaucaaa ccgaaggugc guucgacagu ggcgcagcac 120
cacgaggcac uggugggcca uggguuuaca cacgcgcaca ucguugcgcu cagccaacac 180
ccggcagcgu uagggaccgu cgcugucacg uaucagcaca uaaucacggc guugccagag 240
gcgacacacg aagacaucgu uggcgucggc aaacaguggu ccggcgcacg cgcccuggag 300
gccuugcuca cggaugcggg ggaguugaga gguccgccgu uacaguugga cacaggccaa 360
cuugugaaga uugcaaaacg uggcggcgug accgcaaugg aggcagugca ugcaucgcgc 420
aaugcacuga cgggugcccc ccugaacuua acacccgauc aagugguagc gauagcguca 480
aacggcgggg guaaacaggc uuuggagacg guacagcggu uauugccggu acucugccag 540
gaccacggau ugacaccgga ccaaguggug gcgauugcgu ccaacaacgg aggcaagcag 600
gcacuagaga cuguccaacg gcuucuuccc guucuuuguc aggaucaugg gcuaaccccu 660
gaucagguag ucgcuauagc uucaaaugga gggggcaagc aagcacugga gacuguucaa 720
cgacuccugc cagugcucug ccaagaccac ggacuuacac cagaucaagu gguugcuauu 780
gccuccaaug guggcgggaa acaagcguug gaaacugugc agagacuguu accugucuug 840
ugucaagacc acggccucac gccagaucag gugguagcca uagcgucgaa cauugguggu 900
aagcaagccc uugaaacggu ccagcgucuu cugccggugu ugugccagga ccacggacua 960
acgccggauc aggucguagc cauugcuuca aaugggggcg gcaaacaggc gcuagagaca 1020
guccagcgcc ucuugccugu guuaugccag gaucacggcu uaaccccaga ccaaguugug 1080
gcuauugcau cuaacaaugg uggcaaacaa gccuuggaga cagugcaacg auuacugccu 1140
gucuuauguc aggaucaugg ccugacgccc gaucagguag uggcaaucgc aucuaacauu 1200
ggagguaagc aagcacugga gacuguccag agauuguuac ccguacuaug ucaagaucau 1260
gguuugacgc cugaucaggu uguugcgaua gccagcaaua acggagggaa acaggcucuu 1320
gaaaccguac agcgacuucu cccagucuug ugccaagauc acgggcuuac uccugaucaa 1380
gucguagcua ucgccagcaa uaacggugga aaacaggccc uggaaaccgu acaacgucuc 1440
cucccaguac uuugucaaga ccacggguug acuccggauc aagucgucgc gaucgcgagc 1500
aauggugggg ggaagcaggc gcuggaaacu guucagagac ugcugccugu acuuugucag 1560
gaccaugguc ugacaccuga ccaaguugug gcgauagcca guaauaucgg gggaaaacag 1620
gcacuagaga gcauuguggc ccagcugagc cggccugauc cggcguuggc cgcguugacc 1680
aacgaccacc ucgucgccuu ggccugccuc ggcggacguc cugccaugga ugcagugaaa 1740
aagggauugc cgcacgcgcc ggaauugauc agaagaguca aucgccguau uggcgaacgc 1800
acgucccauc gcguugcgau aucuagagug ggaggaagcu cgcgcagaga guccaucaac 1860
ccauggauuc ugacugguuu cgcugaugcc gaaggaugcu uccgacuaga cauccgcaac 1920
gcaaacucca ugcguguaaa auacagaacu agacuguccu ucgaaaucgu acugcacaac 1980
aaggacaaau cgauucugga gaauauccag ucgacuugga aggucggcaa aaucacaaac 2040
uacggcgaca gaggaguccg ccugagaguc ggccguuucg aagauuugaa agugauuauc 2100
gaccacuucg agaaauaucc gcugauaaca cagaaauugg gcgauuacaa guuguuuaaa 2160
caggcauuca gcgucaugga gaacaaagaa caucuuaagg agaaugggau uaaggagcuc 2220
guacgaauca aagcuaagau gaauuggggu cucaaugacg aauugaaaaa agcauuucca 2280
gagaacauua gcaaagagcg cccccuuauc aauaagaaca uccccaauuu caaauggcug 2340
gcuggauuca caucugguga cggcucguuc augguggaac uaaugaagaa uaagaauaac 2400
guuauuguac gugugcgucu gagauucuca aucucccagc acaucagaga caagaaccug 2460
augaauucau ugauaacaua ccuaggcugu ggucguaucg uugagaauaa caaaucugag 2520
cacaguuggc ucgaauucau uguaacaaaa uucagcgaua ucaacgacaa gaucauuccg 2580
guauuccagg aaaauacucu gauuggcguc aaacucgagg acuuugaaga uuggugcaag 2640
guugccaaau ugaucgaaga gaagaaacac cugaccgaau ccgguuugga ugagauuaag 2700
aaaaucaagc ugaacaugaa caaaggucgu gucuucagcg gccgcuga 2748
<210> 67
<211> 915
<212> PRT
<213> 人工序列
<220>
<223> 在实验室中制备-低活性Cb1-b megaTAL蛋白质构建体
<400> 67
Met Gly Ser Cys Arg Pro Pro Lys Lys Lys Arg Lys Val Val Asp Leu
1 5 10 15
Arg Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys
20 25 30
Val Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val Gly His Gly
35 40 45
Phe Thr His Ala His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu
50 55 60
Gly Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr Ala Leu Pro Glu
65 70 75 80
Ala Thr His Glu Asp Ile Val Gly Val Gly Lys Gln Trp Ser Gly Ala
85 90 95
Arg Ala Leu Glu Ala Leu Leu Thr Asp Ala Gly Glu Leu Arg Gly Pro
100 105 110
Pro Leu Gln Leu Asp Thr Gly Gln Leu Val Lys Ile Ala Lys Arg Gly
115 120 125
Gly Val Thr Ala Met Glu Ala Val His Ala Ser Arg Asn Ala Leu Thr
130 135 140
Gly Ala Pro Leu Asn Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
145 150 155 160
Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
165 170 175
Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
180 185 190
Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
195 200 205
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val
210 215 220
Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
225 230 235 240
Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln
245 250 255
Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr
260 265 270
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro
275 280 285
Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu
290 295 300
Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu
305 310 315 320
Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln
325 330 335
Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His
340 345 350
Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly
355 360 365
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
370 375 380
Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Ile
385 390 395 400
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu
405 410 415
Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
420 425 430
Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
435 440 445
Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
450 455 460
Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
465 470 475 480
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val
485 490 495
Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
500 505 510
Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln
515 520 525
Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Ser
530 535 540
Ile Val Ala Gln Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu Thr
545 550 555 560
Asn Asp His Leu Val Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala Met
565 570 575
Asp Ala Val Lys Lys Gly Leu Pro His Ala Pro Glu Leu Ile Arg Arg
580 585 590
Val Asn Arg Arg Ile Gly Glu Arg Thr Ser His Arg Val Ala Ile Ser
595 600 605
Arg Val Gly Gly Ser Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu
610 615 620
Thr Gly Phe Ala Asp Ala Glu Gly Cys Phe Arg Leu Asp Ile Arg Asn
625 630 635 640
Ala Asn Ser Met Arg Val Lys Tyr Arg Thr Arg Leu Ser Phe Glu Ile
645 650 655
Val Leu His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr
660 665 670
Trp Lys Val Gly Lys Ile Thr Asn Tyr Gly Asp Arg Gly Val Arg Leu
675 680 685
Arg Val Gly Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu
690 695 700
Lys Tyr Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys
705 710 715 720
Gln Ala Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly
725 730 735
Ile Lys Glu Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly Leu Asn
740 745 750
Asp Glu Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Pro
755 760 765
Leu Ile Asn Lys Asn Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe Thr
770 775 780
Ser Gly Asp Gly Ser Phe Met Val Glu Leu Met Lys Asn Lys Asn Asn
785 790 795 800
Val Ile Val Arg Val Arg Leu Arg Phe Ser Ile Ser Gln His Ile Arg
805 810 815
Asp Lys Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Arg
820 825 830
Ile Val Glu Asn Asn Lys Ser Glu His Ser Trp Leu Glu Phe Ile Val
835 840 845
Thr Lys Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu
850 855 860
Asn Thr Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys
865 870 875 880
Val Ala Lys Leu Ile Glu Glu Lys Lys His Leu Thr Glu Ser Gly Leu
885 890 895
Asp Glu Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Val Phe
900 905 910
Ser Gly Arg
915
<210> 68
<211> 3474
<212> DNA
<213> 人工序列
<220>
<223> 在实验室中制备-低活性Cb1-b megaTAL-Trex2 DNA构建体
<400> 68
atgggatcct gcaggccacc taagaagaaa cgcaaagtcg tggatctacg cacgctcggc 60
tacagtcagc agcagcaaga gaagatcaaa ccgaaggtgc gttcgacagt ggcgcagcac 120
cacgaggcac tggtgggcca tgggtttaca cacgcgcaca tcgttgcgct cagccaacac 180
ccggcagcgt tagggaccgt cgctgtcacg tatcagcaca taatcacggc gttgccagag 240
gcgacacacg aagacatcgt tggcgtcggc aaacagtggt ccggcgcacg cgccctggag 300
gccttgctca cggatgcggg ggagttgaga ggtccgccgt tacagttgga cacaggccaa 360
cttgtgaaga ttgcaaaacg tggcggcgtg accgcaatgg aggcagtgca tgcatcgcgc 420
aatgcactga cgggtgcccc cctgaactta acacccgatc aagtggtagc gatagcgtca 480
aacggcgggg gtaaacaggc tttggagacg gtacagcggt tattgccggt actctgccag 540
gaccacggat tgacaccgga ccaagtggtg gcgattgcgt ccaacaacgg aggcaagcag 600
gcactagaga ctgtccaacg gcttcttccc gttctttgtc aggatcatgg gctaacccct 660
gatcaggtag tcgctatagc ttcaaatgga gggggcaagc aagcactgga gactgttcaa 720
cgactcctgc cagtgctctg ccaagaccac ggacttacac cagatcaagt ggttgctatt 780
gcctccaatg gtggcgggaa acaagcgttg gaaactgtgc agagactgtt acctgtcttg 840
tgtcaagacc acggcctcac gccagatcag gtggtagcca tagcgtcgaa cattggtggt 900
aagcaagccc ttgaaacggt ccagcgtctt ctgccggtgt tgtgccagga ccacggacta 960
acgccggatc aggtcgtagc cattgcttca aatgggggcg gcaaacaggc gctagagaca 1020
gtccagcgcc tcttgcctgt gttatgccag gatcacggct taaccccaga ccaagttgtg 1080
gctattgcat ctaacaatgg tggcaaacaa gccttggaga cagtgcaacg attactgcct 1140
gtcttatgtc aggatcatgg cctgacgccc gatcaggtag tggcaatcgc atctaacatt 1200
ggaggtaagc aagcactgga gactgtccag agattgttac ccgtactatg tcaagatcat 1260
ggtttgacgc ctgatcaggt tgttgcgata gccagcaata acggagggaa acaggctctt 1320
gaaaccgtac agcgacttct cccagtcttg tgccaagatc acgggcttac tcctgatcaa 1380
gtcgtagcta tcgccagcaa taacggtgga aaacaggccc tggaaaccgt acaacgtctc 1440
ctcccagtac tttgtcaaga ccacgggttg actccggatc aagtcgtcgc gatcgcgagc 1500
aatggtgggg ggaagcaggc gctggaaact gttcagagac tgctgcctgt actttgtcag 1560
gaccatggtc tgacacctga ccaagttgtg gcgatagcca gtaatatcgg gggaaaacag 1620
gcactagaga gcattgtggc ccagctgagc cggcctgatc cggcgttggc cgcgttgacc 1680
aacgaccacc tcgtcgcctt ggcctgcctc ggcggacgtc ctgccatgga tgcagtgaaa 1740
aagggattgc cgcacgcgcc ggaattgatc agaagagtca atcgccgtat tggcgaacgc 1800
acgtcccatc gcgttgcgat atctagagtg ggaggaagct cgcgcagaga gtccatcaac 1860
ccatggattc tgactggttt cgctgatgcc gaaggatgct tccgactaga catccgcaac 1920
gcaaactcca tgcgtgtaaa atacagaact agactgtcct tcgaaatcgt actgcacaac 1980
aaggacaaat cgattctgga gaatatccag tcgacttgga aggtcggcaa aatcacaaac 2040
tacggcgaca gaggagtccg cctgagagtc ggccgtttcg aagatttgaa agtgattatc 2100
gaccacttcg agaaatatcc gctgataaca cagaaattgg gcgattacaa gttgtttaaa 2160
caggcattca gcgtcatgga gaacaaagaa catcttaagg agaatgggat taaggagctc 2220
gtacgaatca aagctaagat gaattggggt ctcaatgacg aattgaaaaa agcatttcca 2280
gagaacatta gcaaagagcg cccccttatc aataagaaca tccccaattt caaatggctg 2340
gctggattca catctggtga cggctcgttc atggtggaac taatgaagaa taagaataac 2400
gttattgtac gtgtgcgtct gagattctca atctcccagc acatcagaga caagaacctg 2460
atgaattcat tgataacata cctaggctgt ggtcgtatcg ttgagaataa caaatctgag 2520
cacagttggc tcgaattcat tgtaacaaaa ttcagcgata tcaacgacaa gatcattccg 2580
gtattccagg aaaatactct gattggcgtc aaactcgagg actttgaaga ttggtgcaag 2640
gttgccaaat tgatcgaaga gaagaaacac ctgaccgaat ccggtttgga tgagattaag 2700
aaaatcaagc tgaacatgaa caaaggtcgt ggtggaggcg gtagcggagg cggagggtcg 2760
gctagctccg aagcaccgag agccgaaacc ttcgtgttcc tcgacctgga agccactggc 2820
ctgcccagtg tggagcccga gattgccgag ctgtccctgt tcgccgttca tcggtcctcc 2880
ctggaaaacc ctgagcacga cgagagcgga gcactggtgc tgccaagggt gctggacaag 2940
ctgaccctgt gcatgtgtcc cgagcgtccc ttcactgcca aggcatccga aatcactggt 3000
ctgagttcgg aagggctggc tcgttgtaga aaggctggat tcgatggtgc cgtggttcgg 3060
actttgcagg cattcctgtc caggcaggca ggtccaatct gcttggtcgc ccacaacggt 3120
tttgactacg acttcccact gctctgtgcc gaactgcgtc gcctgggagc acgccttcct 3180
cgggataccg tgtgcctgga caccctaccc gctctgcgtg gactggacag agcccactca 3240
cacggcacta gggcgagagg acgccagggt tacagcctcg gttcactgtt ccatcggtat 3300
ttcagggcag agccttccgc agcccattca gccgaaggcg atgtccacac cttgcttctg 3360
atcttcctgc atcgtgcagc agaactgctc gcctgggcag atgagcaggc tcgtggatgg 3420
gctcacattg agccgatgta cttgccacct gatgacccta gcctggaagc ctga 3474
<210> 69
<211> 3474
<212> RNA
<213> 人工序列
<220>
<223> 在实验室中制备-低活性Cb1-b megaTAL-Trex2 RNA构建体
<400> 69
augggauccu gcaggccacc uaagaagaaa cgcaaagucg uggaucuacg cacgcucggc 60
uacagucagc agcagcaaga gaagaucaaa ccgaaggugc guucgacagu ggcgcagcac 120
cacgaggcac uggugggcca uggguuuaca cacgcgcaca ucguugcgcu cagccaacac 180
ccggcagcgu uagggaccgu cgcugucacg uaucagcaca uaaucacggc guugccagag 240
gcgacacacg aagacaucgu uggcgucggc aaacaguggu ccggcgcacg cgcccuggag 300
gccuugcuca cggaugcggg ggaguugaga gguccgccgu uacaguugga cacaggccaa 360
cuugugaaga uugcaaaacg uggcggcgug accgcaaugg aggcagugca ugcaucgcgc 420
aaugcacuga cgggugcccc ccugaacuua acacccgauc aagugguagc gauagcguca 480
aacggcgggg guaaacaggc uuuggagacg guacagcggu uauugccggu acucugccag 540
gaccacggau ugacaccgga ccaaguggug gcgauugcgu ccaacaacgg aggcaagcag 600
gcacuagaga cuguccaacg gcuucuuccc guucuuuguc aggaucaugg gcuaaccccu 660
gaucagguag ucgcuauagc uucaaaugga gggggcaagc aagcacugga gacuguucaa 720
cgacuccugc cagugcucug ccaagaccac ggacuuacac cagaucaagu gguugcuauu 780
gccuccaaug guggcgggaa acaagcguug gaaacugugc agagacuguu accugucuug 840
ugucaagacc acggccucac gccagaucag gugguagcca uagcgucgaa cauugguggu 900
aagcaagccc uugaaacggu ccagcgucuu cugccggugu ugugccagga ccacggacua 960
acgccggauc aggucguagc cauugcuuca aaugggggcg gcaaacaggc gcuagagaca 1020
guccagcgcc ucuugccugu guuaugccag gaucacggcu uaaccccaga ccaaguugug 1080
gcuauugcau cuaacaaugg uggcaaacaa gccuuggaga cagugcaacg auuacugccu 1140
gucuuauguc aggaucaugg ccugacgccc gaucagguag uggcaaucgc aucuaacauu 1200
ggagguaagc aagcacugga gacuguccag agauuguuac ccguacuaug ucaagaucau 1260
gguuugacgc cugaucaggu uguugcgaua gccagcaaua acggagggaa acaggcucuu 1320
gaaaccguac agcgacuucu cccagucuug ugccaagauc acgggcuuac uccugaucaa 1380
gucguagcua ucgccagcaa uaacggugga aaacaggccc uggaaaccgu acaacgucuc 1440
cucccaguac uuugucaaga ccacggguug acuccggauc aagucgucgc gaucgcgagc 1500
aauggugggg ggaagcaggc gcuggaaacu guucagagac ugcugccugu acuuugucag 1560
gaccaugguc ugacaccuga ccaaguugug gcgauagcca guaauaucgg gggaaaacag 1620
gcacuagaga gcauuguggc ccagcugagc cggccugauc cggcguuggc cgcguugacc 1680
aacgaccacc ucgucgccuu ggccugccuc ggcggacguc cugccaugga ugcagugaaa 1740
aagggauugc cgcacgcgcc ggaauugauc agaagaguca aucgccguau uggcgaacgc 1800
acgucccauc gcguugcgau aucuagagug ggaggaagcu cgcgcagaga guccaucaac 1860
ccauggauuc ugacugguuu cgcugaugcc gaaggaugcu uccgacuaga cauccgcaac 1920
gcaaacucca ugcguguaaa auacagaacu agacuguccu ucgaaaucgu acugcacaac 1980
aaggacaaau cgauucugga gaauauccag ucgacuugga aggucggcaa aaucacaaac 2040
uacggcgaca gaggaguccg ccugagaguc ggccguuucg aagauuugaa agugauuauc 2100
gaccacuucg agaaauaucc gcugauaaca cagaaauugg gcgauuacaa guuguuuaaa 2160
caggcauuca gcgucaugga gaacaaagaa caucuuaagg agaaugggau uaaggagcuc 2220
guacgaauca aagcuaagau gaauuggggu cucaaugacg aauugaaaaa agcauuucca 2280
gagaacauua gcaaagagcg cccccuuauc aauaagaaca uccccaauuu caaauggcug 2340
gcuggauuca caucugguga cggcucguuc augguggaac uaaugaagaa uaagaauaac 2400
guuauuguac gugugcgucu gagauucuca aucucccagc acaucagaga caagaaccug 2460
augaauucau ugauaacaua ccuaggcugu ggucguaucg uugagaauaa caaaucugag 2520
cacaguuggc ucgaauucau uguaacaaaa uucagcgaua ucaacgacaa gaucauuccg 2580
guauuccagg aaaauacucu gauuggcguc aaacucgagg acuuugaaga uuggugcaag 2640
guugccaaau ugaucgaaga gaagaaacac cugaccgaau ccgguuugga ugagauuaag 2700
aaaaucaagc ugaacaugaa caaaggucgu gguggaggcg guagcggagg cggagggucg 2760
gcuagcuccg aagcaccgag agccgaaacc uucguguucc ucgaccugga agccacuggc 2820
cugcccagug uggagcccga gauugccgag cugucccugu ucgccguuca ucgguccucc 2880
cuggaaaacc cugagcacga cgagagcgga gcacuggugc ugccaagggu gcuggacaag 2940
cugacccugu gcaugugucc cgagcguccc uucacugcca aggcauccga aaucacuggu 3000
cugaguucgg aagggcuggc ucguuguaga aaggcuggau ucgauggugc cgugguucgg 3060
acuuugcagg cauuccuguc caggcaggca gguccaaucu gcuuggucgc ccacaacggu 3120
uuugacuacg acuucccacu gcucugugcc gaacugcguc gccugggagc acgccuuccu 3180
cgggauaccg ugugccugga cacccuaccc gcucugcgug gacuggacag agcccacuca 3240
cacggcacua gggcgagagg acgccagggu uacagccucg guucacuguu ccaucgguau 3300
uucagggcag agccuuccgc agcccauuca gccgaaggcg auguccacac cuugcuucug 3360
aucuuccugc aucgugcagc agaacugcuc gccugggcag augagcaggc ucguggaugg 3420
gcucacauug agccgaugua cuugccaccu gaugacccua gccuggaagc cuga 3474
<210> 70
<211> 1157
<212> PRT
<213> 人工序列
<220>
<223> 在实验室中制备-低活性Cb1-b megaTAL-Trex2蛋白质构建体
<400> 70
Met Gly Ser Cys Arg Pro Pro Lys Lys Lys Arg Lys Val Val Asp Leu
1 5 10 15
Arg Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys
20 25 30
Val Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val Gly His Gly
35 40 45
Phe Thr His Ala His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu
50 55 60
Gly Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr Ala Leu Pro Glu
65 70 75 80
Ala Thr His Glu Asp Ile Val Gly Val Gly Lys Gln Trp Ser Gly Ala
85 90 95
Arg Ala Leu Glu Ala Leu Leu Thr Asp Ala Gly Glu Leu Arg Gly Pro
100 105 110
Pro Leu Gln Leu Asp Thr Gly Gln Leu Val Lys Ile Ala Lys Arg Gly
115 120 125
Gly Val Thr Ala Met Glu Ala Val His Ala Ser Arg Asn Ala Leu Thr
130 135 140
Gly Ala Pro Leu Asn Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
145 150 155 160
Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
165 170 175
Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
180 185 190
Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
195 200 205
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val
210 215 220
Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
225 230 235 240
Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln
245 250 255
Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr
260 265 270
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro
275 280 285
Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu
290 295 300
Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu
305 310 315 320
Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln
325 330 335
Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His
340 345 350
Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly
355 360 365
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
370 375 380
Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Ile
385 390 395 400
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu
405 410 415
Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
420 425 430
Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
435 440 445
Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
450 455 460
Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
465 470 475 480
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val
485 490 495
Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
500 505 510
Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln
515 520 525
Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Ser
530 535 540
Ile Val Ala Gln Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu Thr
545 550 555 560
Asn Asp His Leu Val Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala Met
565 570 575
Asp Ala Val Lys Lys Gly Leu Pro His Ala Pro Glu Leu Ile Arg Arg
580 585 590
Val Asn Arg Arg Ile Gly Glu Arg Thr Ser His Arg Val Ala Ile Ser
595 600 605
Arg Val Gly Gly Ser Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu
610 615 620
Thr Gly Phe Ala Asp Ala Glu Gly Cys Phe Arg Leu Asp Ile Arg Asn
625 630 635 640
Ala Asn Ser Met Arg Val Lys Tyr Arg Thr Arg Leu Ser Phe Glu Ile
645 650 655
Val Leu His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr
660 665 670
Trp Lys Val Gly Lys Ile Thr Asn Tyr Gly Asp Arg Gly Val Arg Leu
675 680 685
Arg Val Gly Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu
690 695 700
Lys Tyr Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys
705 710 715 720
Gln Ala Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly
725 730 735
Ile Lys Glu Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly Leu Asn
740 745 750
Asp Glu Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Pro
755 760 765
Leu Ile Asn Lys Asn Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe Thr
770 775 780
Ser Gly Asp Gly Ser Phe Met Val Glu Leu Met Lys Asn Lys Asn Asn
785 790 795 800
Val Ile Val Arg Val Arg Leu Arg Phe Ser Ile Ser Gln His Ile Arg
805 810 815
Asp Lys Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Arg
820 825 830
Ile Val Glu Asn Asn Lys Ser Glu His Ser Trp Leu Glu Phe Ile Val
835 840 845
Thr Lys Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu
850 855 860
Asn Thr Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys
865 870 875 880
Val Ala Lys Leu Ile Glu Glu Lys Lys His Leu Thr Glu Ser Gly Leu
885 890 895
Asp Glu Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Gly Gly
900 905 910
Gly Gly Ser Gly Gly Gly Gly Ser Ala Ser Ser Glu Ala Pro Arg Ala
915 920 925
Glu Thr Phe Val Phe Leu Asp Leu Glu Ala Thr Gly Leu Pro Ser Val
930 935 940
Glu Pro Glu Ile Ala Glu Leu Ser Leu Phe Ala Val His Arg Ser Ser
945 950 955 960
Leu Glu Asn Pro Glu His Asp Glu Ser Gly Ala Leu Val Leu Pro Arg
965 970 975
Val Leu Asp Lys Leu Thr Leu Cys Met Cys Pro Glu Arg Pro Phe Thr
980 985 990
Ala Lys Ala Ser Glu Ile Thr Gly Leu Ser Ser Glu Gly Leu Ala Arg
995 1000 1005
Cys Arg Lys Ala Gly Phe Asp Gly Ala Val Val Arg Thr Leu Gln
1010 1015 1020
Ala Phe Leu Ser Arg Gln Ala Gly Pro Ile Cys Leu Val Ala His
1025 1030 1035
Asn Gly Phe Asp Tyr Asp Phe Pro Leu Leu Cys Ala Glu Leu Arg
1040 1045 1050
Arg Leu Gly Ala Arg Leu Pro Arg Asp Thr Val Cys Leu Asp Thr
1055 1060 1065
Leu Pro Ala Leu Arg Gly Leu Asp Arg Ala His Ser His Gly Thr
1070 1075 1080
Arg Ala Arg Gly Arg Gln Gly Tyr Ser Leu Gly Ser Leu Phe His
1085 1090 1095
Arg Tyr Phe Arg Ala Glu Pro Ser Ala Ala His Ser Ala Glu Gly
1100 1105 1110
Asp Val His Thr Leu Leu Leu Ile Phe Leu His Arg Ala Ala Glu
1115 1120 1125
Leu Leu Ala Trp Ala Asp Glu Gln Ala Arg Gly Trp Ala His Ile
1130 1135 1140
Glu Pro Met Tyr Leu Pro Pro Asp Asp Pro Ser Leu Glu Ala
1145 1150 1155
<210> 71
<211> 3426
<212> DNA
<213> 人工序列
<220>
<223> 在实验室中制备-低活性Cb1-b megaTAL-ExoX DNA构建体
<400> 71
atgggatcct gcaggccacc taagaagaaa cgcaaagtcg tggatctacg cacgctcggc 60
tacagtcagc agcagcaaga gaagatcaaa ccgaaggtgc gttcgacagt ggcgcagcac 120
cacgaggcac tggtgggcca tgggtttaca cacgcgcaca tcgttgcgct cagccaacac 180
ccggcagcgt tagggaccgt cgctgtcacg tatcagcaca taatcacggc gttgccagag 240
gcgacacacg aagacatcgt tggcgtcggc aaacagtggt ccggcgcacg cgccctggag 300
gccttgctca cggatgcggg ggagttgaga ggtccgccgt tacagttgga cacaggccaa 360
cttgtgaaga ttgcaaaacg tggcggcgtg accgcaatgg aggcagtgca tgcatcgcgc 420
aatgcactga cgggtgcccc cctgaactta acacccgatc aagtggtagc gatagcgtca 480
aacggcgggg gtaaacaggc tttggagacg gtacagcggt tattgccggt actctgccag 540
gaccacggat tgacaccgga ccaagtggtg gcgattgcgt ccaacaacgg aggcaagcag 600
gcactagaga ctgtccaacg gcttcttccc gttctttgtc aggatcatgg gctaacccct 660
gatcaggtag tcgctatagc ttcaaatgga gggggcaagc aagcactgga gactgttcaa 720
cgactcctgc cagtgctctg ccaagaccac ggacttacac cagatcaagt ggttgctatt 780
gcctccaatg gtggcgggaa acaagcgttg gaaactgtgc agagactgtt acctgtcttg 840
tgtcaagacc acggcctcac gccagatcag gtggtagcca tagcgtcgaa cattggtggt 900
aagcaagccc ttgaaacggt ccagcgtctt ctgccggtgt tgtgccagga ccacggacta 960
acgccggatc aggtcgtagc cattgcttca aatgggggcg gcaaacaggc gctagagaca 1020
gtccagcgcc tcttgcctgt gttatgccag gatcacggct taaccccaga ccaagttgtg 1080
gctattgcat ctaacaatgg tggcaaacaa gccttggaga cagtgcaacg attactgcct 1140
gtcttatgtc aggatcatgg cctgacgccc gatcaggtag tggcaatcgc atctaacatt 1200
ggaggtaagc aagcactgga gactgtccag agattgttac ccgtactatg tcaagatcat 1260
ggtttgacgc ctgatcaggt tgttgcgata gccagcaata acggagggaa acaggctctt 1320
gaaaccgtac agcgacttct cccagtcttg tgccaagatc acgggcttac tcctgatcaa 1380
gtcgtagcta tcgccagcaa taacggtgga aaacaggccc tggaaaccgt acaacgtctc 1440
ctcccagtac tttgtcaaga ccacgggttg actccggatc aagtcgtcgc gatcgcgagc 1500
aatggtgggg ggaagcaggc gctggaaact gttcagagac tgctgcctgt actttgtcag 1560
gaccatggtc tgacacctga ccaagttgtg gcgatagcca gtaatatcgg gggaaaacag 1620
gcactagaga gcattgtggc ccagctgagc cggcctgatc cggcgttggc cgcgttgacc 1680
aacgaccacc tcgtcgcctt ggcctgcctc ggcggacgtc ctgccatgga tgcagtgaaa 1740
aagggattgc cgcacgcgcc ggaattgatc agaagagtca atcgccgtat tggcgaacgc 1800
acgtcccatc gcgttgcgat atctagagtg ggaggaagct cgcgcagaga gtccatcaac 1860
ccatggattc tgactggttt cgctgatgcc gaaggatgct tccgactaga catccgcaac 1920
gcaaactcca tgcgtgtaaa atacagaact agactgtcct tcgaaatcgt actgcacaac 1980
aaggacaaat cgattctgga gaatatccag tcgacttgga aggtcggcaa aatcacaaac 2040
tacggcgaca gaggagtccg cctgagagtc ggccgtttcg aagatttgaa agtgattatc 2100
gaccacttcg agaaatatcc gctgataaca cagaaattgg gcgattacaa gttgtttaaa 2160
caggcattca gcgtcatgga gaacaaagaa catcttaagg agaatgggat taaggagctc 2220
gtacgaatca aagctaagat gaattggggt ctcaatgacg aattgaaaaa agcatttcca 2280
gagaacatta gcaaagagcg cccccttatc aataagaaca tccccaattt caaatggctg 2340
gctggattca catctggtga cggctcgttc atggtggaac taatgaagaa taagaataac 2400
gttattgtac gtgtgcgtct gagattctca atctcccagc acatcagaga caagaacctg 2460
atgaattcat tgataacata cctaggctgt ggtcgtatcg ttgagaataa caaatctgag 2520
cacagttggc tcgaattcat tgtaacaaaa ttcagcgata tcaacgacaa gatcattccg 2580
gtattccagg aaaatactct gattggcgtc aaactcgagg actttgaaga ttggtgcaag 2640
gttgccaaat tgatcgaaga gaagaaacac ctgaccgaat ccggtttgga tgagattaag 2700
aaaatcaagc tgaacatgaa caaaggtcgt ggtggaggcg gtagcggagg cggagggtcg 2760
gctagcctgc ggatcataga cacggaaacc tgtggactcc agggtggtat cgtggagata 2820
gccagtgtcg atgtaattga tgggaaaatc gtaaacccga tgagccacct cgtccgaccg 2880
gataggccga taagtccgca ggctatggct atacatagaa tcacggaggc tatggtagct 2940
gacaagcctt ggatcgagga cgttatccca cactactatg ggagcgaatg gtatgtagcg 3000
cacaacgcat catttgatag gcgcgtgctt cccgaaatgc ctggggagtg gatttgcaca 3060
atgaaactcg ctcgccgact gtggcctggt atcaagtata gtaacatggc actttacaag 3120
acccgcaaac tcaacgtaca gacacctccg ggtctccacc accatagggc actctatgat 3180
tgttacataa ccgctgctct ccttatagac ataatgaata cctccgggtg gaccgccgaa 3240
caaatggcag acataacggg aagacctagt ttgatgacaa cctttacatt tggaaagtat 3300
cggggcaagg cggtgagcga tgtggccgaa agagatcccg gttatcttag atggctcttc 3360
aataacttgg attctatgtc acctgaactt cgccttaccc tgaagcatta ccttgagaat 3420
acctga 3426
<210> 72
<211> 3426
<212> RNA
<213> 人工序列
<220>
<223> 在实验室中制备-低活性Cb1-b megaTAL-ExoX RNA构建体
<400> 72
augggauccu gcaggccacc uaagaagaaa cgcaaagucg uggaucuacg cacgcucggc 60
uacagucagc agcagcaaga gaagaucaaa ccgaaggugc guucgacagu ggcgcagcac 120
cacgaggcac uggugggcca uggguuuaca cacgcgcaca ucguugcgcu cagccaacac 180
ccggcagcgu uagggaccgu cgcugucacg uaucagcaca uaaucacggc guugccagag 240
gcgacacacg aagacaucgu uggcgucggc aaacaguggu ccggcgcacg cgcccuggag 300
gccuugcuca cggaugcggg ggaguugaga gguccgccgu uacaguugga cacaggccaa 360
cuugugaaga uugcaaaacg uggcggcgug accgcaaugg aggcagugca ugcaucgcgc 420
aaugcacuga cgggugcccc ccugaacuua acacccgauc aagugguagc gauagcguca 480
aacggcgggg guaaacaggc uuuggagacg guacagcggu uauugccggu acucugccag 540
gaccacggau ugacaccgga ccaaguggug gcgauugcgu ccaacaacgg aggcaagcag 600
gcacuagaga cuguccaacg gcuucuuccc guucuuuguc aggaucaugg gcuaaccccu 660
gaucagguag ucgcuauagc uucaaaugga gggggcaagc aagcacugga gacuguucaa 720
cgacuccugc cagugcucug ccaagaccac ggacuuacac cagaucaagu gguugcuauu 780
gccuccaaug guggcgggaa acaagcguug gaaacugugc agagacuguu accugucuug 840
ugucaagacc acggccucac gccagaucag gugguagcca uagcgucgaa cauugguggu 900
aagcaagccc uugaaacggu ccagcgucuu cugccggugu ugugccagga ccacggacua 960
acgccggauc aggucguagc cauugcuuca aaugggggcg gcaaacaggc gcuagagaca 1020
guccagcgcc ucuugccugu guuaugccag gaucacggcu uaaccccaga ccaaguugug 1080
gcuauugcau cuaacaaugg uggcaaacaa gccuuggaga cagugcaacg auuacugccu 1140
gucuuauguc aggaucaugg ccugacgccc gaucagguag uggcaaucgc aucuaacauu 1200
ggagguaagc aagcacugga gacuguccag agauuguuac ccguacuaug ucaagaucau 1260
gguuugacgc cugaucaggu uguugcgaua gccagcaaua acggagggaa acaggcucuu 1320
gaaaccguac agcgacuucu cccagucuug ugccaagauc acgggcuuac uccugaucaa 1380
gucguagcua ucgccagcaa uaacggugga aaacaggccc uggaaaccgu acaacgucuc 1440
cucccaguac uuugucaaga ccacggguug acuccggauc aagucgucgc gaucgcgagc 1500
aauggugggg ggaagcaggc gcuggaaacu guucagagac ugcugccugu acuuugucag 1560
gaccaugguc ugacaccuga ccaaguugug gcgauagcca guaauaucgg gggaaaacag 1620
gcacuagaga gcauuguggc ccagcugagc cggccugauc cggcguuggc cgcguugacc 1680
aacgaccacc ucgucgccuu ggccugccuc ggcggacguc cugccaugga ugcagugaaa 1740
aagggauugc cgcacgcgcc ggaauugauc agaagaguca aucgccguau uggcgaacgc 1800
acgucccauc gcguugcgau aucuagagug ggaggaagcu cgcgcagaga guccaucaac 1860
ccauggauuc ugacugguuu cgcugaugcc gaaggaugcu uccgacuaga cauccgcaac 1920
gcaaacucca ugcguguaaa auacagaacu agacuguccu ucgaaaucgu acugcacaac 1980
aaggacaaau cgauucugga gaauauccag ucgacuugga aggucggcaa aaucacaaac 2040
uacggcgaca gaggaguccg ccugagaguc ggccguuucg aagauuugaa agugauuauc 2100
gaccacuucg agaaauaucc gcugauaaca cagaaauugg gcgauuacaa guuguuuaaa 2160
caggcauuca gcgucaugga gaacaaagaa caucuuaagg agaaugggau uaaggagcuc 2220
guacgaauca aagcuaagau gaauuggggu cucaaugacg aauugaaaaa agcauuucca 2280
gagaacauua gcaaagagcg cccccuuauc aauaagaaca uccccaauuu caaauggcug 2340
gcuggauuca caucugguga cggcucguuc augguggaac uaaugaagaa uaagaauaac 2400
guuauuguac gugugcgucu gagauucuca aucucccagc acaucagaga caagaaccug 2460
augaauucau ugauaacaua ccuaggcugu ggucguaucg uugagaauaa caaaucugag 2520
cacaguuggc ucgaauucau uguaacaaaa uucagcgaua ucaacgacaa gaucauuccg 2580
guauuccagg aaaauacucu gauuggcguc aaacucgagg acuuugaaga uuggugcaag 2640
guugccaaau ugaucgaaga gaagaaacac cugaccgaau ccgguuugga ugagauuaag 2700
aaaaucaagc ugaacaugaa caaaggucgu gguggaggcg guagcggagg cggagggucg 2760
gcuagccugc ggaucauaga cacggaaacc uguggacucc agggugguau cguggagaua 2820
gccagugucg auguaauuga ugggaaaauc guaaacccga ugagccaccu cguccgaccg 2880
gauaggccga uaaguccgca ggcuauggcu auacauagaa ucacggaggc uaugguagcu 2940
gacaagccuu ggaucgagga cguuauccca cacuacuaug ggagcgaaug guauguagcg 3000
cacaacgcau cauuugauag gcgcgugcuu cccgaaaugc cuggggagug gauuugcaca 3060
augaaacucg cucgccgacu guggccuggu aucaaguaua guaacauggc acuuuacaag 3120
acccgcaaac ucaacguaca gacaccuccg ggucuccacc accauagggc acucuaugau 3180
uguuacauaa ccgcugcucu ccuuauagac auaaugaaua ccuccgggug gaccgccgaa 3240
caaauggcag acauaacggg aagaccuagu uugaugacaa ccuuuacauu uggaaaguau 3300
cggggcaagg cggugagcga uguggccgaa agagaucccg guuaucuuag auggcucuuc 3360
aauaacuugg auucuauguc accugaacuu cgccuuaccc ugaagcauua ccuugagaau 3420
accuga 3426
<210> 73
<211> 1141
<212> PRT
<213> 人工序列
<220>
<223> 在实验室中制备-低活性Cb1-b megaTAL-ExoX蛋白质构建体
<400> 73
Met Gly Ser Cys Arg Pro Pro Lys Lys Lys Arg Lys Val Val Asp Leu
1 5 10 15
Arg Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys
20 25 30
Val Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val Gly His Gly
35 40 45
Phe Thr His Ala His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu
50 55 60
Gly Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr Ala Leu Pro Glu
65 70 75 80
Ala Thr His Glu Asp Ile Val Gly Val Gly Lys Gln Trp Ser Gly Ala
85 90 95
Arg Ala Leu Glu Ala Leu Leu Thr Asp Ala Gly Glu Leu Arg Gly Pro
100 105 110
Pro Leu Gln Leu Asp Thr Gly Gln Leu Val Lys Ile Ala Lys Arg Gly
115 120 125
Gly Val Thr Ala Met Glu Ala Val His Ala Ser Arg Asn Ala Leu Thr
130 135 140
Gly Ala Pro Leu Asn Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
145 150 155 160
Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
165 170 175
Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
180 185 190
Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
195 200 205
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val
210 215 220
Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
225 230 235 240
Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln
245 250 255
Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr
260 265 270
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro
275 280 285
Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu
290 295 300
Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu
305 310 315 320
Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln
325 330 335
Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His
340 345 350
Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly
355 360 365
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
370 375 380
Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Ile
385 390 395 400
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu
405 410 415
Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
420 425 430
Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
435 440 445
Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
450 455 460
Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
465 470 475 480
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val
485 490 495
Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
500 505 510
Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln
515 520 525
Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Ser
530 535 540
Ile Val Ala Gln Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu Thr
545 550 555 560
Asn Asp His Leu Val Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala Met
565 570 575
Asp Ala Val Lys Lys Gly Leu Pro His Ala Pro Glu Leu Ile Arg Arg
580 585 590
Val Asn Arg Arg Ile Gly Glu Arg Thr Ser His Arg Val Ala Ile Ser
595 600 605
Arg Val Gly Gly Ser Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu
610 615 620
Thr Gly Phe Ala Asp Ala Glu Gly Cys Phe Arg Leu Asp Ile Arg Asn
625 630 635 640
Ala Asn Ser Met Arg Val Lys Tyr Arg Thr Arg Leu Ser Phe Glu Ile
645 650 655
Val Leu His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr
660 665 670
Trp Lys Val Gly Lys Ile Thr Asn Tyr Gly Asp Arg Gly Val Arg Leu
675 680 685
Arg Val Gly Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu
690 695 700
Lys Tyr Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys
705 710 715 720
Gln Ala Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly
725 730 735
Ile Lys Glu Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly Leu Asn
740 745 750
Asp Glu Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Pro
755 760 765
Leu Ile Asn Lys Asn Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe Thr
770 775 780
Ser Gly Asp Gly Ser Phe Met Val Glu Leu Met Lys Asn Lys Asn Asn
785 790 795 800
Val Ile Val Arg Val Arg Leu Arg Phe Ser Ile Ser Gln His Ile Arg
805 810 815
Asp Lys Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Arg
820 825 830
Ile Val Glu Asn Asn Lys Ser Glu His Ser Trp Leu Glu Phe Ile Val
835 840 845
Thr Lys Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu
850 855 860
Asn Thr Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys
865 870 875 880
Val Ala Lys Leu Ile Glu Glu Lys Lys His Leu Thr Glu Ser Gly Leu
885 890 895
Asp Glu Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Gly Gly
900 905 910
Gly Gly Ser Gly Gly Gly Gly Ser Ala Ser Leu Arg Ile Ile Asp Thr
915 920 925
Glu Thr Cys Gly Leu Gln Gly Gly Ile Val Glu Ile Ala Ser Val Asp
930 935 940
Val Ile Asp Gly Lys Ile Val Asn Pro Met Ser His Leu Val Arg Pro
945 950 955 960
Asp Arg Pro Ile Ser Pro Gln Ala Met Ala Ile His Arg Ile Thr Glu
965 970 975
Ala Met Val Ala Asp Lys Pro Trp Ile Glu Asp Val Ile Pro His Tyr
980 985 990
Tyr Gly Ser Glu Trp Tyr Val Ala His Asn Ala Ser Phe Asp Arg Arg
995 1000 1005
Val Leu Pro Glu Met Pro Gly Glu Trp Ile Cys Thr Met Lys Leu
1010 1015 1020
Ala Arg Arg Leu Trp Pro Gly Ile Lys Tyr Ser Asn Met Ala Leu
1025 1030 1035
Tyr Lys Thr Arg Lys Leu Asn Val Gln Thr Pro Pro Gly Leu His
1040 1045 1050
His His Arg Ala Leu Tyr Asp Cys Tyr Ile Thr Ala Ala Leu Leu
1055 1060 1065
Ile Asp Ile Met Asn Thr Ser Gly Trp Thr Ala Glu Gln Met Ala
1070 1075 1080
Asp Ile Thr Gly Arg Pro Ser Leu Met Thr Thr Phe Thr Phe Gly
1085 1090 1095
Lys Tyr Arg Gly Lys Ala Val Ser Asp Val Ala Glu Arg Asp Pro
1100 1105 1110
Gly Tyr Leu Arg Trp Leu Phe Asn Asn Leu Asp Ser Met Ser Pro
1115 1120 1125
Glu Leu Arg Leu Thr Leu Lys His Tyr Leu Glu Asn Thr
1130 1135 1140
<210> 74
<211> 2835
<212> DNA
<213> 人工序列
<220>
<223> 在实验室中制备-PD-1 megaTAL DNA构建体
<400> 74
atgggatcct gcaggccacc taagaagaaa cgcaaagtcg tggatctacg cacgctcggc 60
tacagtcagc agcagcaaga gaagatcaaa ccgaaggtgc gttcgacagt ggcgcagcac 120
cacgaggcac tggtgggcca tgggtttaca cacgcgcaca tcgttgcgct cagccaacac 180
ccggcagcgt tagggaccgt cgctgtcacg tatcagcaca taatcacggc gttgccagag 240
gcgacacacg aagacatcgt tggcgtcggc aaacagtggt ccggcgcacg cgccctggag 300
gccttgctca cggatgcggg ggagttgaga ggtccgccgt tacagttgga cacaggccaa 360
cttgtgaaga ttgcaaaacg tggcggcgtg accgcaatgg aggcagtgca tgcatcgcgc 420
aatgcactga cgggtgcccc cctgaaccta acccctgatc aggtagtcgc tatagcttca 480
aacaacgggg gcaagcaagc actggagacc gttcaacgac tcctgccagt gctctgccaa 540
gaccacggac ttacgccaga tcaggtggtt gctattgcct ccaacaatgg cgggaaacaa 600
gcgttggaaa ctgtgcagag actgttacct gtcttgtgtc aagaccacgg cctcacgcca 660
gatcaggtgg tagccatagc gtcgaatgga ggtggtaagc aagcccttga aacggtccag 720
cgtcttctgc cggtgttgtg ccaggaccac ggactaacgc cggatcaggt cgtagccatt 780
gcttcaaata acggcggcaa acaggcgcta gagacagtcc agcgcctctt gcctgtgtta 840
tgccaggatc acggcttaac cccagaccaa gttgtggcta ttgcatctaa caatggtggc 900
aaacaagcct tggagacagt gcaacgatta ctgcctgtct tatgtcagga tcatggcctg 960
acgcccgatc aggtagtggc aatcgcatct aataatggag gtaagcaagc actggagact 1020
gtccagagat tgttacccgt actatgtcaa gatcatggtt tgacgcctga tcaggttgtt 1080
gcgatagcca gcaacaacgg agggaaacag gctcttgaaa ccgtacagcg acttctccca 1140
gtcttgtgcc aagatcacgg gcttactcct gatcaagtcg tagctatcgc cagccacgac 1200
ggtgggaaac aggccctgga aaccgtacaa cgtctcctcc cagtactttg tcaagaccac 1260
gggttgactc cggatcaagt cgtcgcgatc gcgagcaatg gaggggggaa gcaggcgctg 1320
gaaactgttc agagactgct gcctgtactt tgtcaggacc atggtctgac acctgaccaa 1380
gttgtggcga tagccagtaa caatggggga aaacaggcac tagagacggt tcaaaggttg 1440
ttgcccgttc tgtgccagga ccacggcttg acaccggatc aggtggtagc tatcgcttca 1500
cacgatggcg gaaaacaggc tttagaaaca gtccaaagac ttctcccagt cctttgtcag 1560
gaccacggat tgactccaga tcaagtcgtt gctattgcaa gtaatggtgg tggtaagcaa 1620
gctttagaaa ccgtacagag gcttttgcca gtgctgtgcc aggaccatgg actgacccct 1680
gatcaagtgg tagcaattgc atctcatgat ggaggaaaac aagctctgga aagcattgtg 1740
gcccagctga gccggcctga tccggcgttg gccgcgttga ccaacgacca cctcgtcgcc 1800
ttggcctgcc tcggcggacg tcctgccatg gatgcagtga aaaagggatt gccgcacgcg 1860
ccggaattga tcagaagagt caatcgccgt attggcgaac gcacgtccca tcgcgttgcg 1920
atatctagag tgggaggaag ctctcgcaga gagtccatca acccatggac tctgactggt 1980
ttcgctgatg ccgaaggatc attcgggcta agcatcctca acagaaacag aggtactgct 2040
agataccaca ctcgactgtc attcacaatc atgctgcaca acaaggacaa atcgattctg 2100
gagaatatcc agtcgacttg gaaggtcggc agcatcctca acaatggcga ccactacgtc 2160
tcgctggtgg tctaccgttt cgaagatttg aaagtgatta tcgaccactt cgagaaatat 2220
ccgctgataa cacagaaatt gggcgattac aagttgttta aacaggcatt cagcgtcatg 2280
gagaacaaag aacatcttaa ggagaatggg attaaggagc tcgtacgaat caaagctaag 2340
atgaattggg gtctcaatga cgaattgaaa aaagcatttc cagaggtgat tagcagggag 2400
cgccccctta tcaataagaa cattccgaat gggaaatggc tggctggatt cacatctggt 2460
gatggctcct tcttcgtgcg cctaagaaag tctaatgtta atgctagagt acgtgtgcaa 2520
ctggtattcg agatctcaca gcacatcaga gacaagaacc tgatgaattc attgataaca 2580
tacctaggct gtggtcacat ctacgaggga aacaaatctg agcgcagttg gctccaattc 2640
agagtagaaa aattcagcga tatcaacgac aagatcattc cggtattcca ggaaaatact 2700
ctgattggca tgaaactcga ggactttgaa gattggtgca aggttgccaa attgatcgaa 2760
gagaagaaac acctgaccga atccggtttg gatgagatta agaaaatcaa gctgaacatg 2820
aacaaaagac gttga 2835
<210> 75
<211> 2835
<212> RNA
<213> 人工序列
<220>
<223> 在实验室中制备-PD-1 megaTAL RNA构建体
<400> 75
augggauccu gcaggccacc uaagaagaaa cgcaaagucg uggaucuacg cacgcucggc 60
uacagucagc agcagcaaga gaagaucaaa ccgaaggugc guucgacagu ggcgcagcac 120
cacgaggcac uggugggcca uggguuuaca cacgcgcaca ucguugcgcu cagccaacac 180
ccggcagcgu uagggaccgu cgcugucacg uaucagcaca uaaucacggc guugccagag 240
gcgacacacg aagacaucgu uggcgucggc aaacaguggu ccggcgcacg cgcccuggag 300
gccuugcuca cggaugcggg ggaguugaga gguccgccgu uacaguugga cacaggccaa 360
cuugugaaga uugcaaaacg uggcggcgug accgcaaugg aggcagugca ugcaucgcgc 420
aaugcacuga cgggugcccc ccugaaccua accccugauc agguagucgc uauagcuuca 480
aacaacgggg gcaagcaagc acuggagacc guucaacgac uccugccagu gcucugccaa 540
gaccacggac uuacgccaga ucaggugguu gcuauugccu ccaacaaugg cgggaaacaa 600
gcguuggaaa cugugcagag acuguuaccu gucuuguguc aagaccacgg ccucacgcca 660
gaucaggugg uagccauagc gucgaaugga ggugguaagc aagcccuuga aacgguccag 720
cgucuucugc cgguguugug ccaggaccac ggacuaacgc cggaucaggu cguagccauu 780
gcuucaaaua acggcggcaa acaggcgcua gagacagucc agcgccucuu gccuguguua 840
ugccaggauc acggcuuaac cccagaccaa guuguggcua uugcaucuaa caaugguggc 900
aaacaagccu uggagacagu gcaacgauua cugccugucu uaugucagga ucauggccug 960
acgcccgauc agguaguggc aaucgcaucu aauaauggag guaagcaagc acuggagacu 1020
guccagagau uguuacccgu acuaugucaa gaucaugguu ugacgccuga ucagguuguu 1080
gcgauagcca gcaacaacgg agggaaacag gcucuugaaa ccguacagcg acuucuccca 1140
gucuugugcc aagaucacgg gcuuacuccu gaucaagucg uagcuaucgc cagccacgac 1200
ggugggaaac aggcccugga aaccguacaa cgucuccucc caguacuuug ucaagaccac 1260
ggguugacuc cggaucaagu cgucgcgauc gcgagcaaug gaggggggaa gcaggcgcug 1320
gaaacuguuc agagacugcu gccuguacuu ugucaggacc auggucugac accugaccaa 1380
guuguggcga uagccaguaa caauggggga aaacaggcac uagagacggu ucaaagguug 1440
uugcccguuc ugugccagga ccacggcuug acaccggauc aggugguagc uaucgcuuca 1500
cacgauggcg gaaaacaggc uuuagaaaca guccaaagac uucucccagu ccuuugucag 1560
gaccacggau ugacuccaga ucaagucguu gcuauugcaa guaauggugg ugguaagcaa 1620
gcuuuagaaa ccguacagag gcuuuugcca gugcugugcc aggaccaugg acugaccccu 1680
gaucaagugg uagcaauugc aucucaugau ggaggaaaac aagcucugga aagcauugug 1740
gcccagcuga gccggccuga uccggcguug gccgcguuga ccaacgacca ccucgucgcc 1800
uuggccugcc ucggcggacg uccugccaug gaugcaguga aaaagggauu gccgcacgcg 1860
ccggaauuga ucagaagagu caaucgccgu auuggcgaac gcacguccca ucgcguugcg 1920
auaucuagag ugggaggaag cucucgcaga gaguccauca acccauggac ucugacuggu 1980
uucgcugaug ccgaaggauc auucgggcua agcauccuca acagaaacag agguacugcu 2040
agauaccaca cucgacuguc auucacaauc augcugcaca acaaggacaa aucgauucug 2100
gagaauaucc agucgacuug gaaggucggc agcauccuca acaauggcga ccacuacguc 2160
ucgcuggugg ucuaccguuu cgaagauuug aaagugauua ucgaccacuu cgagaaauau 2220
ccgcugauaa cacagaaauu gggcgauuac aaguuguuua aacaggcauu cagcgucaug 2280
gagaacaaag aacaucuuaa ggagaauggg auuaaggagc ucguacgaau caaagcuaag 2340
augaauuggg gucucaauga cgaauugaaa aaagcauuuc cagaggugau uagcagggag 2400
cgcccccuua ucaauaagaa cauuccgaau gggaaauggc uggcuggauu cacaucuggu 2460
gauggcuccu ucuucgugcg ccuaagaaag ucuaauguua augcuagagu acgugugcaa 2520
cugguauucg agaucucaca gcacaucaga gacaagaacc ugaugaauuc auugauaaca 2580
uaccuaggcu guggucacau cuacgaggga aacaaaucug agcgcaguug gcuccaauuc 2640
agaguagaaa aauucagcga uaucaacgac aagaucauuc cgguauucca ggaaaauacu 2700
cugauuggca ugaaacucga ggacuuugaa gauuggugca agguugccaa auugaucgaa 2760
gagaagaaac accugaccga auccgguuug gaugagauua agaaaaucaa gcugaacaug 2820
aacaaaagac guuga 2835
<210> 76
<211> 944
<212> PRT
<213> 人工序列
<220>
<223> 在实验室中制备-PD-1 megaTAL蛋白质构建体
<400> 76
Met Gly Ser Cys Arg Pro Pro Lys Lys Lys Arg Lys Val Val Asp Leu
1 5 10 15
Arg Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys
20 25 30
Val Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val Gly His Gly
35 40 45
Phe Thr His Ala His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu
50 55 60
Gly Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr Ala Leu Pro Glu
65 70 75 80
Ala Thr His Glu Asp Ile Val Gly Val Gly Lys Gln Trp Ser Gly Ala
85 90 95
Arg Ala Leu Glu Ala Leu Leu Thr Asp Ala Gly Glu Leu Arg Gly Pro
100 105 110
Pro Leu Gln Leu Asp Thr Gly Gln Leu Val Lys Ile Ala Lys Arg Gly
115 120 125
Gly Val Thr Ala Met Glu Ala Val His Ala Ser Arg Asn Ala Leu Thr
130 135 140
Gly Ala Pro Leu Asn Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
145 150 155 160
Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
165 170 175
Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
180 185 190
Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
195 200 205
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val
210 215 220
Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
225 230 235 240
Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln
245 250 255
Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr
260 265 270
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro
275 280 285
Asp Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu
290 295 300
Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu
305 310 315 320
Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln
325 330 335
Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His
340 345 350
Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly
355 360 365
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
370 375 380
Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser His Asp
385 390 395 400
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu
405 410 415
Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
420 425 430
Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
435 440 445
Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
450 455 460
Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
465 470 475 480
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val
485 490 495
Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
500 505 510
Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln
515 520 525
Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr
530 535 540
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro
545 550 555 560
Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu
565 570 575
Glu Ser Ile Val Ala Gln Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala
580 585 590
Leu Thr Asn Asp His Leu Val Ala Leu Ala Cys Leu Gly Gly Arg Pro
595 600 605
Ala Met Asp Ala Val Lys Lys Gly Leu Pro His Ala Pro Glu Leu Ile
610 615 620
Arg Arg Val Asn Arg Arg Ile Gly Glu Arg Thr Ser His Arg Val Ala
625 630 635 640
Ile Ser Arg Val Gly Gly Ser Ser Arg Arg Glu Ser Ile Asn Pro Trp
645 650 655
Thr Leu Thr Gly Phe Ala Asp Ala Glu Gly Ser Phe Gly Leu Ser Ile
660 665 670
Leu Asn Arg Asn Arg Gly Thr Ala Arg Tyr His Thr Arg Leu Ser Phe
675 680 685
Thr Ile Met Leu His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln
690 695 700
Ser Thr Trp Lys Val Gly Ser Ile Leu Asn Asn Gly Asp His Tyr Val
705 710 715 720
Ser Leu Val Val Tyr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His
725 730 735
Phe Glu Lys Tyr Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu
740 745 750
Phe Lys Gln Ala Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu
755 760 765
Asn Gly Ile Lys Glu Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly
770 775 780
Leu Asn Asp Glu Leu Lys Lys Ala Phe Pro Glu Val Ile Ser Arg Glu
785 790 795 800
Arg Pro Leu Ile Asn Lys Asn Ile Pro Asn Gly Lys Trp Leu Ala Gly
805 810 815
Phe Thr Ser Gly Asp Gly Ser Phe Phe Val Arg Leu Arg Lys Ser Asn
820 825 830
Val Asn Ala Arg Val Arg Val Gln Leu Val Phe Glu Ile Ser Gln His
835 840 845
Ile Arg Asp Lys Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys
850 855 860
Gly His Ile Tyr Glu Gly Asn Lys Ser Glu Arg Ser Trp Leu Gln Phe
865 870 875 880
Arg Val Glu Lys Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe
885 890 895
Gln Glu Asn Thr Leu Ile Gly Met Lys Leu Glu Asp Phe Glu Asp Trp
900 905 910
Cys Lys Val Ala Lys Leu Ile Glu Glu Lys Lys His Leu Thr Glu Ser
915 920 925
Gly Leu Asp Glu Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Arg Arg
930 935 940
<210> 77
<211> 3576
<212> DNA
<213> 人工序列
<220>
<223> 在实验室中制备-PD-1 megaTAL-Trex2 DNA构建体
<400> 77
atgggatcct gcaggccacc taagaagaaa cgcaaagtcg tggatctacg cacgctcggc 60
tacagtcagc agcagcaaga gaagatcaaa ccgaaggtgc gttcgacagt ggcgcagcac 120
cacgaggcac tggtgggcca tgggtttaca cacgcgcaca tcgttgcgct cagccaacac 180
ccggcagcgt tagggaccgt cgctgtcacg tatcagcaca taatcacggc gttgccagag 240
gcgacacacg aagacatcgt tggcgtcggc aaacagtggt ccggcgcacg cgccctggag 300
gccttgctca cggatgcggg ggagttgaga ggtccgccgt tacagttgga cacaggccaa 360
cttgtgaaga ttgcaaaacg tggcggcgtg accgcaatgg aggcagtgca tgcatcgcgc 420
aatgcactga cgggtgcccc cctgaaccta acccctgatc aggtagtcgc tatagcttca 480
aacaacgggg gcaagcaagc actggagacc gttcaacgac tcctgccagt gctctgccaa 540
gaccacggac ttacgccaga tcaggtggtt gctattgcct ccaacaatgg cgggaaacaa 600
gcgttggaaa ctgtgcagag actgttacct gtcttgtgtc aagaccacgg cctcacgcca 660
gatcaggtgg tagccatagc gtcgaatgga ggtggtaagc aagcccttga aacggtccag 720
cgtcttctgc cggtgttgtg ccaggaccac ggactaacgc cggatcaggt cgtagccatt 780
gcttcaaata acggcggcaa acaggcgcta gagacagtcc agcgcctctt gcctgtgtta 840
tgccaggatc acggcttaac cccagaccaa gttgtggcta ttgcatctaa caatggtggc 900
aaacaagcct tggagacagt gcaacgatta ctgcctgtct tatgtcagga tcatggcctg 960
acgcccgatc aggtagtggc aatcgcatct aataatggag gtaagcaagc actggagact 1020
gtccagagat tgttacccgt actatgtcaa gatcatggtt tgacgcctga tcaggttgtt 1080
gcgatagcca gcaacaacgg agggaaacag gctcttgaaa ccgtacagcg acttctccca 1140
gtcttgtgcc aagatcacgg gcttactcct gatcaagtcg tagctatcgc cagccacgac 1200
ggtgggaaac aggccctgga aaccgtacaa cgtctcctcc cagtactttg tcaagaccac 1260
gggttgactc cggatcaagt cgtcgcgatc gcgagcaatg gaggggggaa gcaggcgctg 1320
gaaactgttc agagactgct gcctgtactt tgtcaggacc atggtctgac acctgaccaa 1380
gttgtggcga tagccagtaa caatggggga aaacaggcac tagagacggt tcaaaggttg 1440
ttgcccgttc tgtgccagga ccacggcttg acaccggatc aggtggtagc tatcgcttca 1500
cacgatggcg gaaaacaggc tttagaaaca gtccaaagac ttctcccagt cctttgtcag 1560
gaccacggat tgactccaga tcaagtcgtt gctattgcaa gtaatggtgg tggtaagcaa 1620
gctttagaaa ccgtacagag gcttttgcca gtgctgtgcc aggaccatgg actgacccct 1680
gatcaagtgg tagcaattgc atctcatgat ggaggaaaac aagctctgga aagcattgtg 1740
gcccagctga gccggcctga tccggcgttg gccgcgttga ccaacgacca cctcgtcgcc 1800
ttggcctgcc tcggcggacg tcctgccatg gatgcagtga aaaagggatt gccgcacgcg 1860
ccggaattga tcagaagagt caatcgccgt attggcgaac gcacgtccca tcgcgttgcg 1920
atatctagag tgggaggaag ctctcgcaga gagtccatca acccatggac tctgactggt 1980
ttcgctgatg ccgaaggatc attcgggcta agcatcctca acagaaacag aggtactgct 2040
agataccaca ctcgactgtc attcacaatc atgctgcaca acaaggacaa atcgattctg 2100
gagaatatcc agtcgacttg gaaggtcggc agcatcctca acaatggcga ccactacgtc 2160
tcgctggtgg tctaccgttt cgaagatttg aaagtgatta tcgaccactt cgagaaatat 2220
ccgctgataa cacagaaatt gggcgattac aagttgttta aacaggcatt cagcgtcatg 2280
gagaacaaag aacatcttaa ggagaatggg attaaggagc tcgtacgaat caaagctaag 2340
atgaattggg gtctcaatga cgaattgaaa aaagcatttc cagaggtgat tagcagggag 2400
cgccccctta tcaataagaa cattccgaat gggaaatggc tggctggatt cacatctggt 2460
gatggctcct tcttcgtgcg cctaagaaag tctaatgtta atgctagagt acgtgtgcaa 2520
ctggtattcg agatctcaca gcacatcaga gacaagaacc tgatgaattc attgataaca 2580
tacctaggct gtggtcacat ctacgaggga aacaaatctg agcgcagttg gctccaattc 2640
agagtagaaa aattcagcga tatcaacgac aagatcattc cggtattcca ggaaaatact 2700
ctgattggca tgaaactcga ggactttgaa gattggtgca aggttgccaa attgatcgaa 2760
gagaagaaac acctgaccga atccggtttg gatgagatta agaaaatcaa gctgaacatg 2820
aacaaaagac gtggtggagg cggtagcgga ggcggagggt cggctagctc cgaagcaccg 2880
agagccgaaa ccttcgtgtt cctcgacctg gaagccactg gcctgcccag tgtggagccc 2940
gagattgccg agctgtccct gttcgccgtt catcggtcct ccctggaaaa ccctgagcac 3000
gacgagagcg gagcactggt gctgccaagg gtgctggaca agctgaccct gtgcatgtgt 3060
cccgagcgtc ccttcactgc caaggcatcc gaaatcactg gtctgagttc ggaagggctg 3120
gctcgttgta gaaaggctgg attcgatggt gccgtggttc ggactttgca ggcattcctg 3180
tccaggcagg caggtccaat ctgcttggtc gcccacaacg gttttgacta cgacttccca 3240
ctgctctgtg ccgaactgcg tcgcctggga gcacgccttc ctcgggatac cgtgtgcctg 3300
gacaccctac ccgctctgcg tggactggac agagcccact cacacggcac tagggcgaga 3360
ggacgccagg gttacagcct cggttcactg ttccatcggt atttcagggc agagccttcc 3420
gcagcccatt cagccgaagg cgatgtccac accttgcttc tgatcttcct gcatcgtgca 3480
gcagaactgc tcgcctgggc agatgagcag gctcgtggat gggctcacat tgagccgatg 3540
tacttgccac ctgatgaccc tagcctggaa gcctga 3576
<210> 78
<211> 3576
<212> RNA
<213> 人工序列
<220>
<223> 在实验室中制备-PD-1 megaTAL-Trex2 RNA构建体
<400> 78
augggauccu gcaggccacc uaagaagaaa cgcaaagucg uggaucuacg cacgcucggc 60
uacagucagc agcagcaaga gaagaucaaa ccgaaggugc guucgacagu ggcgcagcac 120
cacgaggcac uggugggcca uggguuuaca cacgcgcaca ucguugcgcu cagccaacac 180
ccggcagcgu uagggaccgu cgcugucacg uaucagcaca uaaucacggc guugccagag 240
gcgacacacg aagacaucgu uggcgucggc aaacaguggu ccggcgcacg cgcccuggag 300
gccuugcuca cggaugcggg ggaguugaga gguccgccgu uacaguugga cacaggccaa 360
cuugugaaga uugcaaaacg uggcggcgug accgcaaugg aggcagugca ugcaucgcgc 420
aaugcacuga cgggugcccc ccugaaccua accccugauc agguagucgc uauagcuuca 480
aacaacgggg gcaagcaagc acuggagacc guucaacgac uccugccagu gcucugccaa 540
gaccacggac uuacgccaga ucaggugguu gcuauugccu ccaacaaugg cgggaaacaa 600
gcguuggaaa cugugcagag acuguuaccu gucuuguguc aagaccacgg ccucacgcca 660
gaucaggugg uagccauagc gucgaaugga ggugguaagc aagcccuuga aacgguccag 720
cgucuucugc cgguguugug ccaggaccac ggacuaacgc cggaucaggu cguagccauu 780
gcuucaaaua acggcggcaa acaggcgcua gagacagucc agcgccucuu gccuguguua 840
ugccaggauc acggcuuaac cccagaccaa guuguggcua uugcaucuaa caaugguggc 900
aaacaagccu uggagacagu gcaacgauua cugccugucu uaugucagga ucauggccug 960
acgcccgauc agguaguggc aaucgcaucu aauaauggag guaagcaagc acuggagacu 1020
guccagagau uguuacccgu acuaugucaa gaucaugguu ugacgccuga ucagguuguu 1080
gcgauagcca gcaacaacgg agggaaacag gcucuugaaa ccguacagcg acuucuccca 1140
gucuugugcc aagaucacgg gcuuacuccu gaucaagucg uagcuaucgc cagccacgac 1200
ggugggaaac aggcccugga aaccguacaa cgucuccucc caguacuuug ucaagaccac 1260
ggguugacuc cggaucaagu cgucgcgauc gcgagcaaug gaggggggaa gcaggcgcug 1320
gaaacuguuc agagacugcu gccuguacuu ugucaggacc auggucugac accugaccaa 1380
guuguggcga uagccaguaa caauggggga aaacaggcac uagagacggu ucaaagguug 1440
uugcccguuc ugugccagga ccacggcuug acaccggauc aggugguagc uaucgcuuca 1500
cacgauggcg gaaaacaggc uuuagaaaca guccaaagac uucucccagu ccuuugucag 1560
gaccacggau ugacuccaga ucaagucguu gcuauugcaa guaauggugg ugguaagcaa 1620
gcuuuagaaa ccguacagag gcuuuugcca gugcugugcc aggaccaugg acugaccccu 1680
gaucaagugg uagcaauugc aucucaugau ggaggaaaac aagcucugga aagcauugug 1740
gcccagcuga gccggccuga uccggcguug gccgcguuga ccaacgacca ccucgucgcc 1800
uuggccugcc ucggcggacg uccugccaug gaugcaguga aaaagggauu gccgcacgcg 1860
ccggaauuga ucagaagagu caaucgccgu auuggcgaac gcacguccca ucgcguugcg 1920
auaucuagag ugggaggaag cucucgcaga gaguccauca acccauggac ucugacuggu 1980
uucgcugaug ccgaaggauc auucgggcua agcauccuca acagaaacag agguacugcu 2040
agauaccaca cucgacuguc auucacaauc augcugcaca acaaggacaa aucgauucug 2100
gagaauaucc agucgacuug gaaggucggc agcauccuca acaauggcga ccacuacguc 2160
ucgcuggugg ucuaccguuu cgaagauuug aaagugauua ucgaccacuu cgagaaauau 2220
ccgcugauaa cacagaaauu gggcgauuac aaguuguuua aacaggcauu cagcgucaug 2280
gagaacaaag aacaucuuaa ggagaauggg auuaaggagc ucguacgaau caaagcuaag 2340
augaauuggg gucucaauga cgaauugaaa aaagcauuuc cagaggugau uagcagggag 2400
cgcccccuua ucaauaagaa cauuccgaau gggaaauggc uggcuggauu cacaucuggu 2460
gauggcuccu ucuucgugcg ccuaagaaag ucuaauguua augcuagagu acgugugcaa 2520
cugguauucg agaucucaca gcacaucaga gacaagaacc ugaugaauuc auugauaaca 2580
uaccuaggcu guggucacau cuacgaggga aacaaaucug agcgcaguug gcuccaauuc 2640
agaguagaaa aauucagcga uaucaacgac aagaucauuc cgguauucca ggaaaauacu 2700
cugauuggca ugaaacucga ggacuuugaa gauuggugca agguugccaa auugaucgaa 2760
gagaagaaac accugaccga auccgguuug gaugagauua agaaaaucaa gcugaacaug 2820
aacaaaagac gugguggagg cgguagcgga ggcggagggu cggcuagcuc cgaagcaccg 2880
agagccgaaa ccuucguguu ccucgaccug gaagccacug gccugcccag uguggagccc 2940
gagauugccg agcugucccu guucgccguu caucgguccu cccuggaaaa cccugagcac 3000
gacgagagcg gagcacuggu gcugccaagg gugcuggaca agcugacccu gugcaugugu 3060
cccgagcguc ccuucacugc caaggcaucc gaaaucacug gucugaguuc ggaagggcug 3120
gcucguugua gaaaggcugg auucgauggu gccgugguuc ggacuuugca ggcauuccug 3180
uccaggcagg cagguccaau cugcuugguc gcccacaacg guuuugacua cgacuuccca 3240
cugcucugug ccgaacugcg ucgccuggga gcacgccuuc cucgggauac cgugugccug 3300
gacacccuac ccgcucugcg uggacuggac agagcccacu cacacggcac uagggcgaga 3360
ggacgccagg guuacagccu cgguucacug uuccaucggu auuucagggc agagccuucc 3420
gcagcccauu cagccgaagg cgauguccac accuugcuuc ugaucuuccu gcaucgugca 3480
gcagaacugc ucgccugggc agaugagcag gcucguggau gggcucacau ugagccgaug 3540
uacuugccac cugaugaccc uagccuggaa gccuga 3576
<210> 79
<211> 1191
<212> PRT
<213> 人工序列
<220>
<223> 在实验室中制备-PD-1 megaTAL-Trex2蛋白质构建体
<400> 79
Met Gly Ser Cys Arg Pro Pro Lys Lys Lys Arg Lys Val Val Asp Leu
1 5 10 15
Arg Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys
20 25 30
Val Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val Gly His Gly
35 40 45
Phe Thr His Ala His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu
50 55 60
Gly Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr Ala Leu Pro Glu
65 70 75 80
Ala Thr His Glu Asp Ile Val Gly Val Gly Lys Gln Trp Ser Gly Ala
85 90 95
Arg Ala Leu Glu Ala Leu Leu Thr Asp Ala Gly Glu Leu Arg Gly Pro
100 105 110
Pro Leu Gln Leu Asp Thr Gly Gln Leu Val Lys Ile Ala Lys Arg Gly
115 120 125
Gly Val Thr Ala Met Glu Ala Val His Ala Ser Arg Asn Ala Leu Thr
130 135 140
Gly Ala Pro Leu Asn Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
145 150 155 160
Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
165 170 175
Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
180 185 190
Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
195 200 205
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val
210 215 220
Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
225 230 235 240
Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln
245 250 255
Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr
260 265 270
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro
275 280 285
Asp Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu
290 295 300
Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu
305 310 315 320
Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln
325 330 335
Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His
340 345 350
Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly
355 360 365
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
370 375 380
Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser His Asp
385 390 395 400
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu
405 410 415
Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
420 425 430
Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
435 440 445
Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
450 455 460
Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
465 470 475 480
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val
485 490 495
Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
500 505 510
Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln
515 520 525
Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr
530 535 540
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro
545 550 555 560
Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu
565 570 575
Glu Ser Ile Val Ala Gln Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala
580 585 590
Leu Thr Asn Asp His Leu Val Ala Leu Ala Cys Leu Gly Gly Arg Pro
595 600 605
Ala Met Asp Ala Val Lys Lys Gly Leu Pro His Ala Pro Glu Leu Ile
610 615 620
Arg Arg Val Asn Arg Arg Ile Gly Glu Arg Thr Ser His Arg Val Ala
625 630 635 640
Ile Ser Arg Val Gly Gly Ser Ser Arg Arg Glu Ser Ile Asn Pro Trp
645 650 655
Thr Leu Thr Gly Phe Ala Asp Ala Glu Gly Ser Phe Gly Leu Ser Ile
660 665 670
Leu Asn Arg Asn Arg Gly Thr Ala Arg Tyr His Thr Arg Leu Ser Phe
675 680 685
Thr Ile Met Leu His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln
690 695 700
Ser Thr Trp Lys Val Gly Ser Ile Leu Asn Asn Gly Asp His Tyr Val
705 710 715 720
Ser Leu Val Val Tyr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His
725 730 735
Phe Glu Lys Tyr Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu
740 745 750
Phe Lys Gln Ala Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu
755 760 765
Asn Gly Ile Lys Glu Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly
770 775 780
Leu Asn Asp Glu Leu Lys Lys Ala Phe Pro Glu Val Ile Ser Arg Glu
785 790 795 800
Arg Pro Leu Ile Asn Lys Asn Ile Pro Asn Gly Lys Trp Leu Ala Gly
805 810 815
Phe Thr Ser Gly Asp Gly Ser Phe Phe Val Arg Leu Arg Lys Ser Asn
820 825 830
Val Asn Ala Arg Val Arg Val Gln Leu Val Phe Glu Ile Ser Gln His
835 840 845
Ile Arg Asp Lys Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys
850 855 860
Gly His Ile Tyr Glu Gly Asn Lys Ser Glu Arg Ser Trp Leu Gln Phe
865 870 875 880
Arg Val Glu Lys Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe
885 890 895
Gln Glu Asn Thr Leu Ile Gly Met Lys Leu Glu Asp Phe Glu Asp Trp
900 905 910
Cys Lys Val Ala Lys Leu Ile Glu Glu Lys Lys His Leu Thr Glu Ser
915 920 925
Gly Leu Asp Glu Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Arg Arg
930 935 940
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ser Ser Glu Ala Pro
945 950 955 960
Arg Ala Glu Thr Phe Val Phe Leu Asp Leu Glu Ala Thr Gly Leu Pro
965 970 975
Ser Val Glu Pro Glu Ile Ala Glu Leu Ser Leu Phe Ala Val His Arg
980 985 990
Ser Ser Leu Glu Asn Pro Glu His Asp Glu Ser Gly Ala Leu Val Leu
995 1000 1005
Pro Arg Val Leu Asp Lys Leu Thr Leu Cys Met Cys Pro Glu Arg
1010 1015 1020
Pro Phe Thr Ala Lys Ala Ser Glu Ile Thr Gly Leu Ser Ser Glu
1025 1030 1035
Gly Leu Ala Arg Cys Arg Lys Ala Gly Phe Asp Gly Ala Val Val
1040 1045 1050
Arg Thr Leu Gln Ala Phe Leu Ser Arg Gln Ala Gly Pro Ile Cys
1055 1060 1065
Leu Val Ala His Asn Gly Phe Asp Tyr Asp Phe Pro Leu Leu Cys
1070 1075 1080
Ala Glu Leu Arg Arg Leu Gly Ala Arg Leu Pro Arg Asp Thr Val
1085 1090 1095
Cys Leu Asp Thr Leu Pro Ala Leu Arg Gly Leu Asp Arg Ala His
1100 1105 1110
Ser His Gly Thr Arg Ala Arg Gly Arg Gln Gly Tyr Ser Leu Gly
1115 1120 1125
Ser Leu Phe His Arg Tyr Phe Arg Ala Glu Pro Ser Ala Ala His
1130 1135 1140
Ser Ala Glu Gly Asp Val His Thr Leu Leu Leu Ile Phe Leu His
1145 1150 1155
Arg Ala Ala Glu Leu Leu Ala Trp Ala Asp Glu Gln Ala Arg Gly
1160 1165 1170
Trp Ala His Ile Glu Pro Met Tyr Leu Pro Pro Asp Asp Pro Ser
1175 1180 1185
Leu Glu Ala
1190
<210> 80
<211> 3528
<212> DNA
<213> 人工序列
<220>
<223> 在实验室中制备-PD-1 megaTAL-ExoX DNA构建体
<400> 80
atgggatcct gcaggccacc taagaagaaa cgcaaagtcg tggatctacg cacgctcggc 60
tacagtcagc agcagcaaga gaagatcaaa ccgaaggtgc gttcgacagt ggcgcagcac 120
cacgaggcac tggtgggcca tgggtttaca cacgcgcaca tcgttgcgct cagccaacac 180
ccggcagcgt tagggaccgt cgctgtcacg tatcagcaca taatcacggc gttgccagag 240
gcgacacacg aagacatcgt tggcgtcggc aaacagtggt ccggcgcacg cgccctggag 300
gccttgctca cggatgcggg ggagttgaga ggtccgccgt tacagttgga cacaggccaa 360
cttgtgaaga ttgcaaaacg tggcggcgtg accgcaatgg aggcagtgca tgcatcgcgc 420
aatgcactga cgggtgcccc cctgaaccta acccctgatc aggtagtcgc tatagcttca 480
aacaacgggg gcaagcaagc actggagacc gttcaacgac tcctgccagt gctctgccaa 540
gaccacggac ttacgccaga tcaggtggtt gctattgcct ccaacaatgg cgggaaacaa 600
gcgttggaaa ctgtgcagag actgttacct gtcttgtgtc aagaccacgg cctcacgcca 660
gatcaggtgg tagccatagc gtcgaatgga ggtggtaagc aagcccttga aacggtccag 720
cgtcttctgc cggtgttgtg ccaggaccac ggactaacgc cggatcaggt cgtagccatt 780
gcttcaaata acggcggcaa acaggcgcta gagacagtcc agcgcctctt gcctgtgtta 840
tgccaggatc acggcttaac cccagaccaa gttgtggcta ttgcatctaa caatggtggc 900
aaacaagcct tggagacagt gcaacgatta ctgcctgtct tatgtcagga tcatggcctg 960
acgcccgatc aggtagtggc aatcgcatct aataatggag gtaagcaagc actggagact 1020
gtccagagat tgttacccgt actatgtcaa gatcatggtt tgacgcctga tcaggttgtt 1080
gcgatagcca gcaacaacgg agggaaacag gctcttgaaa ccgtacagcg acttctccca 1140
gtcttgtgcc aagatcacgg gcttactcct gatcaagtcg tagctatcgc cagccacgac 1200
ggtgggaaac aggccctgga aaccgtacaa cgtctcctcc cagtactttg tcaagaccac 1260
gggttgactc cggatcaagt cgtcgcgatc gcgagcaatg gaggggggaa gcaggcgctg 1320
gaaactgttc agagactgct gcctgtactt tgtcaggacc atggtctgac acctgaccaa 1380
gttgtggcga tagccagtaa caatggggga aaacaggcac tagagacggt tcaaaggttg 1440
ttgcccgttc tgtgccagga ccacggcttg acaccggatc aggtggtagc tatcgcttca 1500
cacgatggcg gaaaacaggc tttagaaaca gtccaaagac ttctcccagt cctttgtcag 1560
gaccacggat tgactccaga tcaagtcgtt gctattgcaa gtaatggtgg tggtaagcaa 1620
gctttagaaa ccgtacagag gcttttgcca gtgctgtgcc aggaccatgg actgacccct 1680
gatcaagtgg tagcaattgc atctcatgat ggaggaaaac aagctctgga aagcattgtg 1740
gcccagctga gccggcctga tccggcgttg gccgcgttga ccaacgacca cctcgtcgcc 1800
ttggcctgcc tcggcggacg tcctgccatg gatgcagtga aaaagggatt gccgcacgcg 1860
ccggaattga tcagaagagt caatcgccgt attggcgaac gcacgtccca tcgcgttgcg 1920
atatctagag tgggaggaag ctctcgcaga gagtccatca acccatggac tctgactggt 1980
ttcgctgatg ccgaaggatc attcgggcta agcatcctca acagaaacag aggtactgct 2040
agataccaca ctcgactgtc attcacaatc atgctgcaca acaaggacaa atcgattctg 2100
gagaatatcc agtcgacttg gaaggtcggc agcatcctca acaatggcga ccactacgtc 2160
tcgctggtgg tctaccgttt cgaagatttg aaagtgatta tcgaccactt cgagaaatat 2220
ccgctgataa cacagaaatt gggcgattac aagttgttta aacaggcatt cagcgtcatg 2280
gagaacaaag aacatcttaa ggagaatggg attaaggagc tcgtacgaat caaagctaag 2340
atgaattggg gtctcaatga cgaattgaaa aaagcatttc cagaggtgat tagcagggag 2400
cgccccctta tcaataagaa cattccgaat gggaaatggc tggctggatt cacatctggt 2460
gatggctcct tcttcgtgcg cctaagaaag tctaatgtta atgctagagt acgtgtgcaa 2520
ctggtattcg agatctcaca gcacatcaga gacaagaacc tgatgaattc attgataaca 2580
tacctaggct gtggtcacat ctacgaggga aacaaatctg agcgcagttg gctccaattc 2640
agagtagaaa aattcagcga tatcaacgac aagatcattc cggtattcca ggaaaatact 2700
ctgattggca tgaaactcga ggactttgaa gattggtgca aggttgccaa attgatcgaa 2760
gagaagaaac acctgaccga atccggtttg gatgagatta agaaaatcaa gctgaacatg 2820
aacaaaagac gtggtggagg cggtagcgga ggcggagggt cggctagcct gcggatcata 2880
gacacggaaa cctgtggact ccagggtggt atcgtggaga tagccagtgt cgatgtaatt 2940
gatgggaaaa tcgtaaaccc gatgagccac ctcgtccgac cggataggcc gataagtccg 3000
caggctatgg ctatacatag aatcacggag gctatggtag ctgacaagcc ttggatcgag 3060
gacgttatcc cacactacta tgggagcgaa tggtatgtag cgcacaacgc atcatttgat 3120
aggcgcgtgc ttcccgaaat gcctggggag tggatttgca caatgaaact cgctcgccga 3180
ctgtggcctg gtatcaagta tagtaacatg gcactttaca agacccgcaa actcaacgta 3240
cagacacctc cgggtctcca ccaccatagg gcactctatg attgttacat aaccgctgct 3300
ctccttatag acataatgaa tacctccggg tggaccgccg aacaaatggc agacataacg 3360
ggaagaccta gtttgatgac aacctttaca tttggaaagt atcggggcaa ggcggtgagc 3420
gatgtggccg aaagagatcc cggttatctt agatggctct tcaataactt ggattctatg 3480
tcacctgaac ttcgccttac cctgaagcat taccttgaga atacctga 3528
<210> 81
<211> 3528
<212> RNA
<213> 人工序列
<220>
<223> 在实验室中制备-PD-1 megaTAL-ExoX RNA构建体
<400> 81
augggauccu gcaggccacc uaagaagaaa cgcaaagucg uggaucuacg cacgcucggc 60
uacagucagc agcagcaaga gaagaucaaa ccgaaggugc guucgacagu ggcgcagcac 120
cacgaggcac uggugggcca uggguuuaca cacgcgcaca ucguugcgcu cagccaacac 180
ccggcagcgu uagggaccgu cgcugucacg uaucagcaca uaaucacggc guugccagag 240
gcgacacacg aagacaucgu uggcgucggc aaacaguggu ccggcgcacg cgcccuggag 300
gccuugcuca cggaugcggg ggaguugaga gguccgccgu uacaguugga cacaggccaa 360
cuugugaaga uugcaaaacg uggcggcgug accgcaaugg aggcagugca ugcaucgcgc 420
aaugcacuga cgggugcccc ccugaaccua accccugauc agguagucgc uauagcuuca 480
aacaacgggg gcaagcaagc acuggagacc guucaacgac uccugccagu gcucugccaa 540
gaccacggac uuacgccaga ucaggugguu gcuauugccu ccaacaaugg cgggaaacaa 600
gcguuggaaa cugugcagag acuguuaccu gucuuguguc aagaccacgg ccucacgcca 660
gaucaggugg uagccauagc gucgaaugga ggugguaagc aagcccuuga aacgguccag 720
cgucuucugc cgguguugug ccaggaccac ggacuaacgc cggaucaggu cguagccauu 780
gcuucaaaua acggcggcaa acaggcgcua gagacagucc agcgccucuu gccuguguua 840
ugccaggauc acggcuuaac cccagaccaa guuguggcua uugcaucuaa caaugguggc 900
aaacaagccu uggagacagu gcaacgauua cugccugucu uaugucagga ucauggccug 960
acgcccgauc agguaguggc aaucgcaucu aauaauggag guaagcaagc acuggagacu 1020
guccagagau uguuacccgu acuaugucaa gaucaugguu ugacgccuga ucagguuguu 1080
gcgauagcca gcaacaacgg agggaaacag gcucuugaaa ccguacagcg acuucuccca 1140
gucuugugcc aagaucacgg gcuuacuccu gaucaagucg uagcuaucgc cagccacgac 1200
ggugggaaac aggcccugga aaccguacaa cgucuccucc caguacuuug ucaagaccac 1260
ggguugacuc cggaucaagu cgucgcgauc gcgagcaaug gaggggggaa gcaggcgcug 1320
gaaacuguuc agagacugcu gccuguacuu ugucaggacc auggucugac accugaccaa 1380
guuguggcga uagccaguaa caauggggga aaacaggcac uagagacggu ucaaagguug 1440
uugcccguuc ugugccagga ccacggcuug acaccggauc aggugguagc uaucgcuuca 1500
cacgauggcg gaaaacaggc uuuagaaaca guccaaagac uucucccagu ccuuugucag 1560
gaccacggau ugacuccaga ucaagucguu gcuauugcaa guaauggugg ugguaagcaa 1620
gcuuuagaaa ccguacagag gcuuuugcca gugcugugcc aggaccaugg acugaccccu 1680
gaucaagugg uagcaauugc aucucaugau ggaggaaaac aagcucugga aagcauugug 1740
gcccagcuga gccggccuga uccggcguug gccgcguuga ccaacgacca ccucgucgcc 1800
uuggccugcc ucggcggacg uccugccaug gaugcaguga aaaagggauu gccgcacgcg 1860
ccggaauuga ucagaagagu caaucgccgu auuggcgaac gcacguccca ucgcguugcg 1920
auaucuagag ugggaggaag cucucgcaga gaguccauca acccauggac ucugacuggu 1980
uucgcugaug ccgaaggauc auucgggcua agcauccuca acagaaacag agguacugcu 2040
agauaccaca cucgacuguc auucacaauc augcugcaca acaaggacaa aucgauucug 2100
gagaauaucc agucgacuug gaaggucggc agcauccuca acaauggcga ccacuacguc 2160
ucgcuggugg ucuaccguuu cgaagauuug aaagugauua ucgaccacuu cgagaaauau 2220
ccgcugauaa cacagaaauu gggcgauuac aaguuguuua aacaggcauu cagcgucaug 2280
gagaacaaag aacaucuuaa ggagaauggg auuaaggagc ucguacgaau caaagcuaag 2340
augaauuggg gucucaauga cgaauugaaa aaagcauuuc cagaggugau uagcagggag 2400
cgcccccuua ucaauaagaa cauuccgaau gggaaauggc uggcuggauu cacaucuggu 2460
gauggcuccu ucuucgugcg ccuaagaaag ucuaauguua augcuagagu acgugugcaa 2520
cugguauucg agaucucaca gcacaucaga gacaagaacc ugaugaauuc auugauaaca 2580
uaccuaggcu guggucacau cuacgaggga aacaaaucug agcgcaguug gcuccaauuc 2640
agaguagaaa aauucagcga uaucaacgac aagaucauuc cgguauucca ggaaaauacu 2700
cugauuggca ugaaacucga ggacuuugaa gauuggugca agguugccaa auugaucgaa 2760
gagaagaaac accugaccga auccgguuug gaugagauua agaaaaucaa gcugaacaug 2820
aacaaaagac gugguggagg cgguagcgga ggcggagggu cggcuagccu gcggaucaua 2880
gacacggaaa ccuguggacu ccaggguggu aucguggaga uagccagugu cgauguaauu 2940
gaugggaaaa ucguaaaccc gaugagccac cucguccgac cggauaggcc gauaaguccg 3000
caggcuaugg cuauacauag aaucacggag gcuaugguag cugacaagcc uuggaucgag 3060
gacguuaucc cacacuacua ugggagcgaa ugguauguag cgcacaacgc aucauuugau 3120
aggcgcgugc uucccgaaau gccuggggag uggauuugca caaugaaacu cgcucgccga 3180
cuguggccug guaucaagua uaguaacaug gcacuuuaca agacccgcaa acucaacgua 3240
cagacaccuc cgggucucca ccaccauagg gcacucuaug auuguuacau aaccgcugcu 3300
cuccuuauag acauaaugaa uaccuccggg uggaccgccg aacaaauggc agacauaacg 3360
ggaagaccua guuugaugac aaccuuuaca uuuggaaagu aucggggcaa ggcggugagc 3420
gauguggccg aaagagaucc cgguuaucuu agauggcucu ucaauaacuu ggauucuaug 3480
ucaccugaac uucgccuuac ccugaagcau uaccuugaga auaccuga 3528
<210> 82
<211> 1175
<212> PRT
<213> 人工序列
<220>
<223> 在实验室中制备-PD-1 megaTAL-ExoX蛋白质构建体
<400> 82
Met Gly Ser Cys Arg Pro Pro Lys Lys Lys Arg Lys Val Val Asp Leu
1 5 10 15
Arg Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys
20 25 30
Val Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val Gly His Gly
35 40 45
Phe Thr His Ala His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu
50 55 60
Gly Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr Ala Leu Pro Glu
65 70 75 80
Ala Thr His Glu Asp Ile Val Gly Val Gly Lys Gln Trp Ser Gly Ala
85 90 95
Arg Ala Leu Glu Ala Leu Leu Thr Asp Ala Gly Glu Leu Arg Gly Pro
100 105 110
Pro Leu Gln Leu Asp Thr Gly Gln Leu Val Lys Ile Ala Lys Arg Gly
115 120 125
Gly Val Thr Ala Met Glu Ala Val His Ala Ser Arg Asn Ala Leu Thr
130 135 140
Gly Ala Pro Leu Asn Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
145 150 155 160
Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
165 170 175
Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
180 185 190
Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
195 200 205
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val
210 215 220
Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
225 230 235 240
Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln
245 250 255
Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr
260 265 270
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro
275 280 285
Asp Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu
290 295 300
Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu
305 310 315 320
Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln
325 330 335
Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His
340 345 350
Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly
355 360 365
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
370 375 380
Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser His Asp
385 390 395 400
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu
405 410 415
Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser
420 425 430
Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
435 440 445
Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile
450 455 460
Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu
465 470 475 480
Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val
485 490 495
Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln
500 505 510
Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln
515 520 525
Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr
530 535 540
Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro
545 550 555 560
Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu
565 570 575
Glu Ser Ile Val Ala Gln Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala
580 585 590
Leu Thr Asn Asp His Leu Val Ala Leu Ala Cys Leu Gly Gly Arg Pro
595 600 605
Ala Met Asp Ala Val Lys Lys Gly Leu Pro His Ala Pro Glu Leu Ile
610 615 620
Arg Arg Val Asn Arg Arg Ile Gly Glu Arg Thr Ser His Arg Val Ala
625 630 635 640
Ile Ser Arg Val Gly Gly Ser Ser Arg Arg Glu Ser Ile Asn Pro Trp
645 650 655
Thr Leu Thr Gly Phe Ala Asp Ala Glu Gly Ser Phe Gly Leu Ser Ile
660 665 670
Leu Asn Arg Asn Arg Gly Thr Ala Arg Tyr His Thr Arg Leu Ser Phe
675 680 685
Thr Ile Met Leu His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln
690 695 700
Ser Thr Trp Lys Val Gly Ser Ile Leu Asn Asn Gly Asp His Tyr Val
705 710 715 720
Ser Leu Val Val Tyr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His
725 730 735
Phe Glu Lys Tyr Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu
740 745 750
Phe Lys Gln Ala Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu
755 760 765
Asn Gly Ile Lys Glu Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly
770 775 780
Leu Asn Asp Glu Leu Lys Lys Ala Phe Pro Glu Val Ile Ser Arg Glu
785 790 795 800
Arg Pro Leu Ile Asn Lys Asn Ile Pro Asn Gly Lys Trp Leu Ala Gly
805 810 815
Phe Thr Ser Gly Asp Gly Ser Phe Phe Val Arg Leu Arg Lys Ser Asn
820 825 830
Val Asn Ala Arg Val Arg Val Gln Leu Val Phe Glu Ile Ser Gln His
835 840 845
Ile Arg Asp Lys Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys
850 855 860
Gly His Ile Tyr Glu Gly Asn Lys Ser Glu Arg Ser Trp Leu Gln Phe
865 870 875 880
Arg Val Glu Lys Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe
885 890 895
Gln Glu Asn Thr Leu Ile Gly Met Lys Leu Glu Asp Phe Glu Asp Trp
900 905 910
Cys Lys Val Ala Lys Leu Ile Glu Glu Lys Lys His Leu Thr Glu Ser
915 920 925
Gly Leu Asp Glu Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Arg Arg
930 935 940
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ser Leu Arg Ile Ile
945 950 955 960
Asp Thr Glu Thr Cys Gly Leu Gln Gly Gly Ile Val Glu Ile Ala Ser
965 970 975
Val Asp Val Ile Asp Gly Lys Ile Val Asn Pro Met Ser His Leu Val
980 985 990
Arg Pro Asp Arg Pro Ile Ser Pro Gln Ala Met Ala Ile His Arg Ile
995 1000 1005
Thr Glu Ala Met Val Ala Asp Lys Pro Trp Ile Glu Asp Val Ile
1010 1015 1020
Pro His Tyr Tyr Gly Ser Glu Trp Tyr Val Ala His Asn Ala Ser
1025 1030 1035
Phe Asp Arg Arg Val Leu Pro Glu Met Pro Gly Glu Trp Ile Cys
1040 1045 1050
Thr Met Lys Leu Ala Arg Arg Leu Trp Pro Gly Ile Lys Tyr Ser
1055 1060 1065
Asn Met Ala Leu Tyr Lys Thr Arg Lys Leu Asn Val Gln Thr Pro
1070 1075 1080
Pro Gly Leu His His His Arg Ala Leu Tyr Asp Cys Tyr Ile Thr
1085 1090 1095
Ala Ala Leu Leu Ile Asp Ile Met Asn Thr Ser Gly Trp Thr Ala
1100 1105 1110
Glu Gln Met Ala Asp Ile Thr Gly Arg Pro Ser Leu Met Thr Thr
1115 1120 1125
Phe Thr Phe Gly Lys Tyr Arg Gly Lys Ala Val Ser Asp Val Ala
1130 1135 1140
Glu Arg Asp Pro Gly Tyr Leu Arg Trp Leu Phe Asn Asn Leu Asp
1145 1150 1155
Ser Met Ser Pro Glu Leu Arg Leu Thr Leu Lys His Tyr Leu Glu
1160 1165 1170
Asn Thr
1175
<210> 83
<211> 870
<212> RNA
<213> 智人(Homo sapiens)
<400> 83
augguccaga ucccacaggc gcccuggcca gucgucuggg cggugcuaca acugggcugg 60
cggccaggau gguucuuaga cuccccagac aggcccugga acccccccac cuucucccca 120
gcccugcucg uggugaccga aggggacaac gccaccuuca ccugcagcuu cuccaacaca 180
ucggagagcu ucgugcuaaa cugguaccgc augagcccca gcaaccagac ggacaagcug 240
gccgccuucc ccgaggaccg cagccagccc ggccaggacu gccgcuuccg ugucacacaa 300
cugcccaacg ggcgugacuu ccacaugagc guggucaggg cccggcgcaa ugacagcggc 360
accuaccucu guggggccau cucccuggcc cccaaggcgc agaucaaaga gagccugcgg 420
gcagagcuca gggugacaga gagaagggca gaagugccca cagcccaccc cagccccuca 480
cccaggccag ccggccaguu ccaaacccug gugguuggug ucgugggcgg ccugcugggc 540
agccuggugc ugcuagucug gguccuggcc gucaucugcu cccgggccgc acgagggaca 600
auaggagcca ggcgcaccgg ccagccccug aaggaggacc ccucagccgu gccuguguuc 660
ucuguggacu auggggagcu ggauuuccag uggcgagaga agaccccgga gccccccgug 720
cccugugucc cugagcagac ggaguaugcc accauugucu uuccuagcgg aaugggcacc 780
ucaucccccg cccgcagggg cucagcugac ggcccucgga gugcccagcc acugaggccu 840
gaggauggac acugcucuug gccccucuga 870
<210> 84
<211> 869
<212> RNA
<213> 人工序列
<220>
<223> 具有缺失的模拟物编辑的PD-1开放阅读框
<400> 84
augguccaga cccacaggcg cccuggccag ucgucugggc ggugcuacaa cugggcuggc 60
ggccaggaug guucuuagac uccccagaca ggcccuggaa cccccccacc uucuccccag 120
cccugcucgu ggugaccgaa ggggacaacg ccaccuucac cugcagcuuc uccaacacau 180
cggagagcuu cgugcuaaac ugguaccgca ugagccccag caaccagacg gacaagcugg 240
ccgccuuccc cgaggaccgc agccagcccg gccaggacug ccgcuuccgu gucacacaac 300
ugcccaacgg gcgugacuuc cacaugagcg uggucagggc ccggcgcaau gacagcggca 360
ccuaccucug uggggccauc ucccuggccc ccaaggcgca gaucaaagag agccugcggg 420
cagagcucag ggugacagag agaagggcag aagugcccac agcccacccc agccccucac 480
ccaggccagc cggccaguuc caaacccugg ugguuggugu cgugggcggc cugcugggca 540
gccuggugcu gcuagucugg guccuggccg ucaucugcuc ccgggccgca cgagggacaa 600
uaggagccag gcgcaccggc cagccccuga aggaggaccc cucagccgug ccuguguucu 660
cuguggacua uggggagcug gauuuccagu ggcgagagaa gaccccggag ccccccgugc 720
ccuguguccc ugagcagacg gaguaugcca ccauugucuu uccuagcgga augggcaccu 780
caucccccgc ccgcaggggc ucagcugacg gcccucggag ugcccagcca cugaggccug 840
aggauggaca cugcucuugg ccccucuga 869
<210> 85
<211> 868
<212> RNA
<213> 人工序列
<220>
<223> 具有缺失的模拟物编辑的PD-1开放阅读框
<400> 85
augguccaga ccacaggcgc ccuggccagu cgucugggcg gugcuacaac ugggcuggcg 60
gccaggaugg uucuuagacu ccccagacag gcccuggaac ccccccaccu ucuccccagc 120
ccugcucgug gugaccgaag gggacaacgc caccuucacc ugcagcuucu ccaacacauc 180
ggagagcuuc gugcuaaacu gguaccgcau gagccccagc aaccagacgg acaagcuggc 240
cgccuucccc gaggaccgca gccagcccgg ccaggacugc cgcuuccgug ucacacaacu 300
gcccaacggg cgugacuucc acaugagcgu ggucagggcc cggcgcaaug acagcggcac 360
cuaccucugu ggggccaucu cccuggcccc caaggcgcag aucaaagaga gccugcgggc 420
agagcucagg gugacagaga gaagggcaga agugcccaca gcccacccca gccccucacc 480
caggccagcc ggccaguucc aaacccuggu gguugguguc gugggcggcc ugcugggcag 540
ccuggugcug cuagucuggg uccuggccgu caucugcucc cgggccgcac gagggacaau 600
aggagccagg cgcaccggcc agccccugaa ggaggacccc ucagccgugc cuguguucuc 660
uguggacuau ggggagcugg auuuccagug gcgagagaag accccggagc cccccgugcc 720
cugugucccu gagcagacgg aguaugccac cauugucuuu ccuagcggaa ugggcaccuc 780
aucccccgcc cgcaggggcu cagcugacgg cccucggagu gcccagccac ugaggccuga 840
ggauggacac ugcucuuggc cccucuga 868
<210> 86
<211> 867
<212> RNA
<213> 人工序列
<220>
<223> 具有缺失的模拟物编辑的PD-1开放阅读框
<400> 86
augguccagc cacaggcgcc cuggccaguc gucugggcgg ugcuacaacu gggcuggcgg 60
ccaggauggu ucuuagacuc cccagacagg cccuggaacc cccccaccuu cuccccagcc 120
cugcucgugg ugaccgaagg ggacaacgcc accuucaccu gcagcuucuc caacacaucg 180
gagagcuucg ugcuaaacug guaccgcaug agccccagca accagacgga caagcuggcc 240
gccuuccccg aggaccgcag ccagcccggc caggacugcc gcuuccgugu cacacaacug 300
cccaacgggc gugacuucca caugagcgug gucagggccc ggcgcaauga cagcggcacc 360
uaccucugug gggccaucuc ccuggccccc aaggcgcaga ucaaagagag ccugcgggca 420
gagcucaggg ugacagagag aagggcagaa gugcccacag cccaccccag ccccucaccc 480
aggccagccg gccaguucca aacccuggug guuggugucg ugggcggccu gcugggcagc 540
cuggugcugc uagucugggu ccuggccguc aucugcuccc gggccgcacg agggacaaua 600
ggagccaggc gcaccggcca gccccugaag gaggaccccu cagccgugcc uguguucucu 660
guggacuaug gggagcugga uuuccagugg cgagagaaga ccccggagcc ccccgugccc 720
ugugucccug agcagacgga guaugccacc auugucuuuc cuagcggaau gggcaccuca 780
ucccccgccc gcaggggcuc agcugacggc ccucggagug cccagccacu gaggccugag 840
gauggacacu gcucuuggcc ccucuga 867
<210> 87
<211> 891
<212> DNA
<213> 人工序列
<220>
<223> I-OnuI LHE的工程化变体
<400> 87
tctcgcagag agtccatcaa cccatggatt ctgactggtt tcgctgatgc cgaaggatca 60
ttcatactag acatccgcaa ccgaaacaac gaaagcaaca gataccgaac ttcgctgaga 120
ttccagatca ccctgcacaa caaggacaaa tcgattctgg agaatatcca gtccacttgg 180
aaggtcggca agatcacaaa cagcagtgac agagccgtca tgctgagggt cacccgtttc 240
gaagatttga aagtgattat cgaccacttc gagaaatatc cgctgattac ccagaaattg 300
ggcgattaca agttgtttaa acaggcattc agcgtcatgg agaataaaga acatcttaag 360
gagaatggga ttaaggagct cgtacgaatc aaagctaaga tgaattgggg tctcaatgac 420
gaattgaaaa aagcatttcc agagaacatt agcaaagagc gcccccttat caataagaac 480
attccgaatt tcaaatggct ggctggattc acagctggtg aaggctactt cggcgtgaat 540
ctaaaaaagg taaagggcac cgcaaaggta tacgtgggac tgagattctc aatctcacag 600
cacatcagag acaagaacct gatgaattca ttgataacat acctaggctg tggttccatc 660
tgggagaaga acaagtctga gttcagatgg ctcgagttcg tcgtaaccaa attcagcgat 720
atcaacgaca agatcattcc ggtattccag gaaaatactc tgattggcgt caaactcgag 780
gactttgaag attggtgcaa ggttgccaaa ttgatcaaag agaagaaaca cctgaccgaa 840
tccggtttgg atgagattaa gaaaatcaag ctgaacatga acaaaggtcg t 891
<210> 88
<211> 891
<212> RNA
<213> 人工序列
<220>
<223> I-OnuI LHE的工程化变体
<400> 88
ucucgcagag aguccaucaa cccauggauu cugacugguu ucgcugaugc cgaaggauca 60
uucauacuag acauccgcaa ccgaaacaac gaaagcaaca gauaccgaac uucgcugaga 120
uuccagauca cccugcacaa caaggacaaa ucgauucugg agaauaucca guccacuugg 180
aaggucggca agaucacaaa cagcagugac agagccguca ugcugagggu cacccguuuc 240
gaagauuuga aagugauuau cgaccacuuc gagaaauauc cgcugauuac ccagaaauug 300
ggcgauuaca aguuguuuaa acaggcauuc agcgucaugg agaauaaaga acaucuuaag 360
gagaauggga uuaaggagcu cguacgaauc aaagcuaaga ugaauugggg ucucaaugac 420
gaauugaaaa aagcauuucc agagaacauu agcaaagagc gcccccuuau caauaagaac 480
auuccgaauu ucaaauggcu ggcuggauuc acagcuggug aaggcuacuu cggcgugaau 540
cuaaaaaagg uaaagggcac cgcaaaggua uacgugggac ugagauucuc aaucucacag 600
cacaucagag acaagaaccu gaugaauuca uugauaacau accuaggcug ugguuccauc 660
ugggagaaga acaagucuga guucagaugg cucgaguucg ucguaaccaa auucagcgau 720
aucaacgaca agaucauucc gguauuccag gaaaauacuc ugauuggcgu caaacucgag 780
gacuuugaag auuggugcaa gguugccaaa uugaucaaag agaagaaaca ccugaccgaa 840
uccgguuugg augagauuaa gaaaaucaag cugaacauga acaaaggucg u 891
<210> 89
<211> 303
<212> PRT
<213> 人工序列
<220>
<223> I-OnuI LHE的工程化变体
<220>
<221> MOD_RES
<222> (1)..(1)
<223> Xaa是任何氨基酸或不存在
<220>
<221> MOD_RES
<222> (2)..(2)
<223> Xaa是任何氨基酸或不存在
<220>
<221> MOD_RES
<222> (3)..(3)
<223> Xaa是任何氨基酸或不存在
<220>
<221> MOD_RES
<222> (4)..(4)
<223> Xaa是任何氨基酸或不存在
<220>
<221> MOD_RES
<222> (302)..(302)
<223> Xaa是任何氨基酸或不存在
<220>
<221> MOD_RES
<222> (303)..(303)
<223> Xaa是任何氨基酸或不存在
<400> 89
Xaa Xaa Xaa Xaa Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr
1 5 10 15
Gly Phe Ala Asp Ala Glu Gly Ser Phe Ile Leu Asp Ile Arg Asn Arg
20 25 30
Asn Asn Glu Ser Asn Arg Tyr Arg Thr Ser Leu Arg Phe Gln Ile Thr
35 40 45
Leu His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp
50 55 60
Lys Val Gly Lys Ile Thr Asn Ser Ser Asp Arg Ala Val Met Leu Arg
65 70 75 80
Val Thr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys
85 90 95
Tyr Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln
100 105 110
Ala Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile
115 120 125
Lys Glu Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly Leu Asn Asp
130 135 140
Glu Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Pro Leu
145 150 155 160
Ile Asn Lys Asn Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe Thr Ala
165 170 175
Gly Glu Gly Tyr Phe Gly Val Asn Leu Lys Lys Val Lys Gly Thr Ala
180 185 190
Lys Val Tyr Val Gly Leu Arg Phe Ser Ile Ser Gln His Ile Arg Asp
195 200 205
Lys Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Ser Ile
210 215 220
Trp Glu Lys Asn Lys Ser Glu Phe Arg Trp Leu Glu Phe Val Val Thr
225 230 235 240
Lys Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn
245 250 255
Thr Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val
260 265 270
Ala Lys Leu Ile Lys Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp
275 280 285
Glu Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Xaa Xaa
290 295 300
<210> 90
<211> 882
<212> DNA
<213> 人工序列
<220>
<223> I-OnuI LHE的工程化变体
<400> 90
tccatcaacc catggattct gactggtttc gctgatgccg aaggatcatt catactagac 60
atccgcaacc gaaacaacga aagcaacaga taccgaactt cgctgagatt ccagatcacc 120
ctgcacaaca aggacaaatc gattctggag aatatccagt ccacttggaa ggtcggcaag 180
atcacaaaca gcagtgacag agccgtcatg ctgagggtca cccgtttcga agatttgaaa 240
gtgattatcg accacttcga gaaatatccg ctgattaccc agaaattggg cgattacaag 300
ttgtttaaac aggcattcag cgtcatggag aataaagaac atcttaagga gaatgggatt 360
aaggagctcg tacgaatcaa agctaagatg aattggggtc tcaatgacga attgaaaaaa 420
gcatttccag agaacattag caaagagcgc ccccttatca ataagaacat tccgaatttc 480
aaatggctgg ctggattcac agctggtgat ggccatttcg gcgtgaatct aaaaaaggta 540
aagggcaccg caaaggtata cgtgggactg agattcgcta tctcacagca catcagagac 600
aagaacctga tgaattcatt gataacatac ctaggctgtg gttccatcag agagaagaac 660
aagtctgagt tcagatggct cgagttcgaa gtaaccaaat tcagcgatat caacgacaag 720
atcattccgg tattccagga aaatactctg attggcgtca aactcgagga ctttgaagat 780
tggtgcaagg ttgccaaatt gatcgaagag aagaaacacc tgaccgaatc cggtttggat 840
gagattaaga aaatcaagct gaacatgaac aaaggtcgtt ga 882
<210> 91
<211> 882
<212> RNA
<213> 人工序列
<220>
<223> I-OnuI LHE的工程化变体
<400> 91
uccaucaacc cauggauucu gacugguuuc gcugaugccg aaggaucauu cauacuagac 60
auccgcaacc gaaacaacga aagcaacaga uaccgaacuu cgcugagauu ccagaucacc 120
cugcacaaca aggacaaauc gauucuggag aauauccagu ccacuuggaa ggucggcaag 180
aucacaaaca gcagugacag agccgucaug cugaggguca cccguuucga agauuugaaa 240
gugauuaucg accacuucga gaaauauccg cugauuaccc agaaauuggg cgauuacaag 300
uuguuuaaac aggcauucag cgucauggag aauaaagaac aucuuaagga gaaugggauu 360
aaggagcucg uacgaaucaa agcuaagaug aauugggguc ucaaugacga auugaaaaaa 420
gcauuuccag agaacauuag caaagagcgc ccccuuauca auaagaacau uccgaauuuc 480
aaauggcugg cuggauucac agcuggugau ggccauuucg gcgugaaucu aaaaaaggua 540
aagggcaccg caaagguaua cgugggacug agauucgcua ucucacagca caucagagac 600
aagaaccuga ugaauucauu gauaacauac cuaggcugug guuccaucag agagaagaac 660
aagucugagu ucagauggcu cgaguucgaa guaaccaaau ucagcgauau caacgacaag 720
aucauuccgg uauuccagga aaauacucug auuggcguca aacucgagga cuuugaagau 780
uggugcaagg uugccaaauu gaucgaagag aagaaacacc ugaccgaauc cgguuuggau 840
gagauuaaga aaaucaagcu gaacaugaac aaaggucguu ga 882
<210> 92
<211> 303
<212> PRT
<213> 人工序列
<220>
<223> I-OnuI LHE的工程化变体
<220>
<221> MOD_RES
<222> (1)..(1)
<223> Xaa是任何氨基酸或不存在
<220>
<221> MOD_RES
<222> (2)..(2)
<223> Xaa是任何氨基酸或不存在
<220>
<221> MOD_RES
<222> (3)..(3)
<223> Xaa是任何氨基酸或不存在
<220>
<221> MOD_RES
<222> (4)..(4)
<223> Xaa是任何氨基酸或不存在
<220>
<221> MOD_RES
<222> (5)..(5)
<223> Xaa是任何氨基酸或不存在
<220>
<221> MOD_RES
<222> (6)..(6)
<223> Xaa是任何氨基酸或不存在
<220>
<221> MOD_RES
<222> (7)..(7)
<223> Xaa是任何氨基酸或不存在
<220>
<221> MOD_RES
<222> (8)..(8)
<223> Xaa是任何氨基酸或不存在
<220>
<221> MOD_RES
<222> (302)..(302)
<223> Xaa是任何氨基酸或不存在
<220>
<221> MOD_RES
<222> (303)..(303)
<223> Xaa是任何氨基酸或不存在
<400> 92
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Ile Asn Pro Trp Ile Leu Thr
1 5 10 15
Gly Phe Ala Asp Ala Glu Gly Ser Phe Ile Leu Asp Ile Arg Asn Arg
20 25 30
Asn Asn Glu Ser Asn Arg Tyr Arg Thr Ser Leu Arg Phe Gln Ile Thr
35 40 45
Leu His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp
50 55 60
Lys Val Gly Lys Ile Thr Asn Ser Ser Asp Arg Ala Val Met Leu Arg
65 70 75 80
Val Thr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys
85 90 95
Tyr Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln
100 105 110
Ala Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile
115 120 125
Lys Glu Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly Leu Asn Asp
130 135 140
Glu Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Pro Leu
145 150 155 160
Ile Asn Lys Asn Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe Thr Ala
165 170 175
Gly Asp Gly His Phe Gly Val Asn Leu Lys Lys Val Lys Gly Thr Ala
180 185 190
Lys Val Tyr Val Gly Leu Arg Phe Ala Ile Ser Gln His Ile Arg Asp
195 200 205
Lys Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Ser Ile
210 215 220
Arg Glu Lys Asn Lys Ser Glu Phe Arg Trp Leu Glu Phe Glu Val Thr
225 230 235 240
Lys Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn
245 250 255
Thr Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val
260 265 270
Ala Lys Leu Ile Glu Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp
275 280 285
Glu Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Xaa Xaa
290 295 300
<210> 93
<211> 891
<212> DNA
<213> 人工序列
<220>
<223> I-OnuI LHE的工程化变体
<400> 93
tcgcgcagag agtccatcaa cccatggatt ctgactggtt tcgctgatgc cgaaggatgc 60
ttccgactag acatccgcaa cgcaaactcc atgcgtgtaa aatacagaac tagactgtcc 120
ttcgaaatcg tactgcacaa caaggacaaa tcgattctgg agaatatcca gtcgacttgg 180
aaggtcggca aaatcacaaa ctacggcgac agaggagtcc gcctgagagt cggccgtttc 240
gaagatttga aagtgattat cgaccacttc gagaaatatc cgctgataac acagaaattg 300
ggcgattaca agttgtttaa acaggcattc agcgtcatgg agaacaaaga acatcttaag 360
gagaatggga ttaaggagct cgtacgaatc aaagctaaga tgaattgggg tctcaatgac 420
gaattgaaaa aagcatttcc agagaacatt agcaaagagc gcccccttat caataagaac 480
atccccaatt tcaaatggct ggctggattc acatctggtg acggctcgtt catggtggaa 540
ctaatgaaga ataagaataa cgttattgta cgtgtgcgtc tgagattctc aatctcccag 600
cacatcagag acaagaacct gatgaattca ttgataacat acctaggctg tggtcgtatc 660
gttgagaata acaaatctga gcacagttgg ctcgaattca ttgtaacaaa attcagcgat 720
atcaacgaca agatcattcc ggtattccag gaaaatactc tgattggcgt caaactcgag 780
gactttgaag attggtgcaa ggttgccaaa ttgatcgaag agaagaaaca cctgaccgaa 840
tccggtttgg atgagattaa gaaaatcaag ctgaacatga acaaaggtcg t 891
<210> 94
<211> 891
<212> RNA
<213> 人工序列
<220>
<223> I-OnuI LHE的工程化变体
<400> 94
ucgcgcagag aguccaucaa cccauggauu cugacugguu ucgcugaugc cgaaggaugc 60
uuccgacuag acauccgcaa cgcaaacucc augcguguaa aauacagaac uagacugucc 120
uucgaaaucg uacugcacaa caaggacaaa ucgauucugg agaauaucca gucgacuugg 180
aaggucggca aaaucacaaa cuacggcgac agaggagucc gccugagagu cggccguuuc 240
gaagauuuga aagugauuau cgaccacuuc gagaaauauc cgcugauaac acagaaauug 300
ggcgauuaca aguuguuuaa acaggcauuc agcgucaugg agaacaaaga acaucuuaag 360
gagaauggga uuaaggagcu cguacgaauc aaagcuaaga ugaauugggg ucucaaugac 420
gaauugaaaa aagcauuucc agagaacauu agcaaagagc gcccccuuau caauaagaac 480
auccccaauu ucaaauggcu ggcuggauuc acaucuggug acggcucguu caugguggaa 540
cuaaugaaga auaagaauaa cguuauugua cgugugcguc ugagauucuc aaucucccag 600
cacaucagag acaagaaccu gaugaauuca uugauaacau accuaggcug uggucguauc 660
guugagaaua acaaaucuga gcacaguugg cucgaauuca uuguaacaaa auucagcgau 720
aucaacgaca agaucauucc gguauuccag gaaaauacuc ugauuggcgu caaacucgag 780
gacuuugaag auuggugcaa gguugccaaa uugaucgaag agaagaaaca ccugaccgaa 840
uccgguuugg augagauuaa gaaaaucaag cugaacauga acaaaggucg u 891
<210> 95
<211> 303
<212> PRT
<213> 人工序列
<220>
<223> I-OnuI LHE的工程化变体
<220>
<221> MOD_RES
<222> (1)..(1)
<223> Xaa是任何氨基酸或不存在
<220>
<221> MOD_RES
<222> (2)..(2)
<223> Xaa是任何氨基酸或不存在
<220>
<221> MOD_RES
<222> (3)..(3)
<223> Xaa是任何氨基酸或不存在
<220>
<221> MOD_RES
<222> (4)..(4)
<223> Xaa是任何氨基酸或不存在
<220>
<221> MOD_RES
<222> (302)..(302)
<223> Xaa是任何氨基酸或不存在
<220>
<221> MOD_RES
<222> (303)..(303)
<223> Xaa是任何氨基酸或不存在
<400> 95
Xaa Xaa Xaa Xaa Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr
1 5 10 15
Gly Phe Ala Asp Ala Glu Gly Cys Phe Arg Leu Asp Ile Arg Asn Ala
20 25 30
Asn Ser Met Arg Val Lys Tyr Arg Thr Arg Leu Ser Phe Glu Ile Val
35 40 45
Leu His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp
50 55 60
Lys Val Gly Lys Ile Thr Asn Tyr Gly Asp Arg Gly Val Arg Leu Arg
65 70 75 80
Val Gly Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys
85 90 95
Tyr Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln
100 105 110
Ala Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile
115 120 125
Lys Glu Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly Leu Asn Asp
130 135 140
Glu Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Pro Leu
145 150 155 160
Ile Asn Lys Asn Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe Thr Ser
165 170 175
Gly Asp Gly Ser Phe Met Val Glu Leu Met Lys Asn Lys Asn Asn Val
180 185 190
Ile Val Arg Val Arg Leu Arg Phe Ser Ile Ser Gln His Ile Arg Asp
195 200 205
Lys Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Arg Ile
210 215 220
Val Glu Asn Asn Lys Ser Glu His Ser Trp Leu Glu Phe Ile Val Thr
225 230 235 240
Lys Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn
245 250 255
Thr Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val
260 265 270
Ala Lys Leu Ile Glu Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp
275 280 285
Glu Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Xaa Xaa
290 295 300
<210> 96
<211> 891
<212> DNA
<213> 人工序列
<220>
<223> I-OnuI LHE的工程化变体
<400> 96
tcgcgcagag agtccatcaa cccatggatt ctgactggtt tcgctgatgc cgaaggatgc 60
ttccgactag acatccgcaa cgcaaacgat ttaagagccg gatacagaac tagactggcc 120
ttcgaaatcg tactgcacaa caaggacaaa tcgattctgg agaatatcca gtcgacttgg 180
aaggtcggca caatctacaa cgcgggcgac aacgcagtca gactgcaagt cacacgtttc 240
gaagatttga aagtgattat cgaccacttc gagaaatatc cgctgataac acagaaattg 300
ggcgattaca agttgtttaa acaggcattc agcgtcatgg agaacaaaga acatcttaag 360
gagaatggga ttaaggagct cgtacgaatc aaagctaaga tgaattgggg tctcaatgac 420
gaattgaaaa aagcatttcc agagaacatt agcaaagagc gctcccttat caataagaac 480
attccgaatc tcaaatggct ggctggattc acatctggtg acggctcgtt cgtggtggaa 540
ctaaagaaga gaagaagccc cgtcaaggta ggagtgcggc tgcgattcag catcacccag 600
cacatcagag acaagaacct gatgaattca ttgataacat acctaggctg tggtcgtatc 660
gttgagaata acaaatctga gcacagttgg ctcgaattca ttgtaacaaa attcagcgat 720
atcaacgaca agatcattcc ggtattccag gaaaatactc tgattggcgt caaactcgag 780
gactttgaag attggtgcaa ggttgccaaa ttgatcgaag agaagaaaca cctgaccgaa 840
tccggtttgg atgagattaa gaaaatcaag ctgaacatga acaaaggtcg t 891
<210> 97
<211> 891
<212> RNA
<213> 人工序列
<220>
<223> I-OnuI LHE的工程化变体
<400> 97
ucgcgcagag aguccaucaa cccauggauu cugacugguu ucgcugaugc cgaaggaugc 60
uuccgacuag acauccgcaa cgcaaacgau uuaagagccg gauacagaac uagacuggcc 120
uucgaaaucg uacugcacaa caaggacaaa ucgauucugg agaauaucca gucgacuugg 180
aaggucggca caaucuacaa cgcgggcgac aacgcaguca gacugcaagu cacacguuuc 240
gaagauuuga aagugauuau cgaccacuuc gagaaauauc cgcugauaac acagaaauug 300
ggcgauuaca aguuguuuaa acaggcauuc agcgucaugg agaacaaaga acaucuuaag 360
gagaauggga uuaaggagcu cguacgaauc aaagcuaaga ugaauugggg ucucaaugac 420
gaauugaaaa aagcauuucc agagaacauu agcaaagagc gcucccuuau caauaagaac 480
auuccgaauc ucaaauggcu ggcuggauuc acaucuggug acggcucguu cgugguggaa 540
cuaaagaaga gaagaagccc cgucaaggua ggagugcggc ugcgauucag caucacccag 600
cacaucagag acaagaaccu gaugaauuca uugauaacau accuaggcug uggucguauc 660
guugagaaua acaaaucuga gcacaguugg cucgaauuca uuguaacaaa auucagcgau 720
aucaacgaca agaucauucc gguauuccag gaaaauacuc ugauuggcgu caaacucgag 780
gacuuugaag auuggugcaa gguugccaaa uugaucgaag agaagaaaca ccugaccgaa 840
uccgguuugg augagauuaa gaaaaucaag cugaacauga acaaaggucg u 891
<210> 98
<211> 303
<212> PRT
<213> 人工序列
<220>
<223> I-OnuI LHE的工程化变体
<220>
<221> MOD_RES
<222> (1)..(1)
<223> Xaa是任何氨基酸或不存在
<220>
<221> MOD_RES
<222> (2)..(2)
<223> Xaa是任何氨基酸或不存在
<220>
<221> MOD_RES
<222> (3)..(3)
<223> Xaa是任何氨基酸或不存在
<220>
<221> MOD_RES
<222> (4)..(4)
<223> Xaa是任何氨基酸或不存在
<220>
<221> MOD_RES
<222> (302)..(302)
<223> Xaa是任何氨基酸或不存在
<220>
<221> MOD_RES
<222> (303)..(303)
<223> Xaa是任何氨基酸或不存在
<400> 98
Xaa Xaa Xaa Xaa Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr
1 5 10 15
Gly Phe Ala Asp Ala Glu Gly Cys Phe Arg Leu Asp Ile Arg Asn Ala
20 25 30
Asn Asp Leu Arg Ala Gly Tyr Arg Thr Arg Leu Ala Phe Glu Ile Val
35 40 45
Leu His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp
50 55 60
Lys Val Gly Thr Ile Tyr Asn Ala Gly Asp Asn Ala Val Arg Leu Gln
65 70 75 80
Val Thr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys
85 90 95
Tyr Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln
100 105 110
Ala Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile
115 120 125
Lys Glu Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly Leu Asn Asp
130 135 140
Glu Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Ser Leu
145 150 155 160
Ile Asn Lys Asn Ile Pro Asn Leu Lys Trp Leu Ala Gly Phe Thr Ser
165 170 175
Gly Asp Gly Ser Phe Val Val Glu Leu Lys Lys Arg Arg Ser Pro Val
180 185 190
Lys Val Gly Val Arg Leu Arg Phe Ser Ile Thr Gln His Ile Arg Asp
195 200 205
Lys Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Arg Ile
210 215 220
Val Glu Asn Asn Lys Ser Glu His Ser Trp Leu Glu Phe Ile Val Thr
225 230 235 240
Lys Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn
245 250 255
Thr Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val
260 265 270
Ala Lys Leu Ile Glu Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp
275 280 285
Glu Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Xaa Xaa
290 295 300
<210> 99
<211> 443
<212> DNA
<213> 人工序列
<220>
<223> I-OnuI LHE的工程化变体
<400> 99
ttagcaggga gcgccccctt atcaataaga acattccgaa tgggaaatgg ctggctggat 60
tcacatctgg tgatggctcc ttcttcgtgc gcctaagaaa gtctaatgtt aatgctagag 120
tacgtgtgca actggtattc gagatctcac agcacatcag agacaagaac ctgatgaatt 180
cattgataac atacctaggc tgtggtcaca tctacgaggg aaacaaatct gagcgcagtt 240
ggctccaatt cagagtagaa aaattcagcg atatcaacga caagatcatt ccggtattcc 300
aggaaaatac tctgattggc atgaaactcg aggactttga agattggtgc aaggttgcca 360
aattgatcga agagaagaaa cacctgaccg aatccggttt ggatgagatt aagaaaatca 420
agctgaacat gaacaaaaga cgt 443
<210> 100
<211> 891
<212> RNA
<213> 人工序列
<220>
<223> I-OnuI LHE的工程化变体
<400> 100
ucucgcagag aguccaucaa cccauggacu cugacugguu ucgcugaugc cgaaggauca 60
uucgggcuaa gcauccucaa cagaaacaga gguacugcua gauaccacac ucgacuguca 120
uucacaauca ugcugcacaa caaggacaaa ucgauucugg agaauaucca gucgacuugg 180
aaggucggca gcauccucaa caauggcgac cacuacgucu cgcugguggu cuaccguuuc 240
gaagauuuga aagugauuau cgaccacuuc gagaaauauc cgcugauaac acagaaauug 300
ggcgauuaca aguuguuuaa acaggcauuc agcgucaugg agaacaaaga acaucuuaag 360
gagaauggga uuaaggagcu cguacgaauc aaagcuaaga ugaauugggg ucucaaugac 420
gaauugaaaa aagcauuucc agaggugauu agcagggagc gcccccuuau caauaagaac 480
auuccgaaug ggaaauggcu ggcuggauuc acaucuggug auggcuccuu cuucgugcgc 540
cuaagaaagu cuaauguuaa ugcuagagua cgugugcaac ugguauucga gaucucacag 600
cacaucagag acaagaaccu gaugaauuca uugauaacau accuaggcug uggucacauc 660
uacgagggaa acaaaucuga gcgcaguugg cuccaauuca gaguagaaaa auucagcgau 720
aucaacgaca agaucauucc gguauuccag gaaaauacuc ugauuggcau gaaacucgag 780
gacuuugaag auuggugcaa gguugccaaa uugaucgaag agaagaaaca ccugaccgaa 840
uccgguuugg augagauuaa gaaaaucaag cugaacauga acaaaagacg u 891
<210> 101
<211> 303
<212> PRT
<213> 人工序列
<220>
<223> I-OnuI LHE的工程化变体
<220>
<221> MOD_RES
<222> (1)..(1)
<223> Xaa是任何氨基酸或不存在
<220>
<221> MOD_RES
<222> (2)..(2)
<223> Xaa是任何氨基酸或不存在
<220>
<221> MOD_RES
<222> (3)..(3)
<223> Xaa是任何氨基酸或不存在
<220>
<221> MOD_RES
<222> (4)..(4)
<223> Xaa是任何氨基酸或不存在
<220>
<221> MOD_RES
<222> (302)..(302)
<223> Xaa是任何氨基酸或不存在
<220>
<221> MOD_RES
<222> (303)..(303)
<223> Xaa是任何氨基酸或不存在
<400> 101
Xaa Xaa Xaa Xaa Ser Arg Arg Glu Ser Ile Asn Pro Trp Thr Leu Thr
1 5 10 15
Gly Phe Ala Asp Ala Glu Gly Ser Phe Gly Leu Ser Ile Leu Asn Arg
20 25 30
Asn Arg Gly Thr Ala Arg Tyr His Thr Arg Leu Ser Phe Thr Ile Met
35 40 45
Leu His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp
50 55 60
Lys Val Gly Ser Ile Leu Asn Asn Gly Asp His Tyr Val Ser Leu Val
65 70 75 80
Val Tyr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys
85 90 95
Tyr Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln
100 105 110
Ala Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile
115 120 125
Lys Glu Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly Leu Asn Asp
130 135 140
Glu Leu Lys Lys Ala Phe Pro Glu Val Ile Ser Arg Glu Arg Pro Leu
145 150 155 160
Ile Asn Lys Asn Ile Pro Asn Gly Lys Trp Leu Ala Gly Phe Thr Ser
165 170 175
Gly Asp Gly Ser Phe Phe Val Arg Leu Arg Lys Ser Asn Val Asn Ala
180 185 190
Arg Val Arg Val Gln Leu Val Phe Glu Ile Ser Gln His Ile Arg Asp
195 200 205
Lys Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly His Ile
210 215 220
Tyr Glu Gly Asn Lys Ser Glu Arg Ser Trp Leu Gln Phe Arg Val Glu
225 230 235 240
Lys Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn
245 250 255
Thr Leu Ile Gly Met Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val
260 265 270
Ala Lys Leu Ile Glu Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp
275 280 285
Glu Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Arg Arg Xaa Xaa
290 295 300
<210> 102
<211> 303
<212> PRT
<213> 新榆枯萎病菌(Ophiostoma novo-ulmi)
<400> 102
Met Ala Tyr Met Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr
1 5 10 15
Gly Phe Ala Asp Ala Glu Gly Ser Phe Leu Leu Arg Ile Arg Asn Asn
20 25 30
Asn Lys Ser Ser Val Gly Tyr Ser Thr Glu Leu Gly Phe Gln Ile Thr
35 40 45
Leu His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp
50 55 60
Lys Val Gly Val Ile Ala Asn Ser Gly Asp Asn Ala Val Ser Leu Lys
65 70 75 80
Val Thr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys
85 90 95
Tyr Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Met Leu Phe Lys Gln
100 105 110
Ala Phe Cys Val Met Glu Asn Lys Glu His Leu Lys Ile Asn Gly Ile
115 120 125
Lys Glu Leu Val Arg Ile Lys Ala Lys Leu Asn Trp Gly Leu Thr Asp
130 135 140
Glu Leu Lys Lys Ala Phe Pro Glu Ile Ile Ser Lys Glu Arg Ser Leu
145 150 155 160
Ile Asn Lys Asn Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe Thr Ser
165 170 175
Gly Glu Gly Cys Phe Phe Val Asn Leu Ile Lys Ser Lys Ser Lys Leu
180 185 190
Gly Val Gln Val Gln Leu Val Phe Ser Ile Thr Gln His Ile Lys Asp
195 200 205
Lys Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Tyr Ile
210 215 220
Lys Glu Lys Asn Lys Ser Glu Phe Ser Trp Leu Asp Phe Val Val Thr
225 230 235 240
Lys Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn
245 250 255
Thr Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val
260 265 270
Ala Lys Leu Ile Glu Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp
275 280 285
Glu Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Val Phe
290 295 300
<210> 103
<211> 303
<212> PRT
<213> 新榆枯萎病菌(Ophiostoma novo-ulmi)
<400> 103
Met Ala Tyr Met Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr
1 5 10 15
Gly Phe Ala Asp Ala Glu Gly Ser Phe Leu Leu Arg Ile Arg Asn Asn
20 25 30
Asn Lys Ser Ser Val Gly Tyr Ser Thr Glu Leu Gly Phe Gln Ile Thr
35 40 45
Leu His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp
50 55 60
Lys Val Gly Val Ile Ala Asn Ser Gly Asp Asn Ala Val Ser Leu Lys
65 70 75 80
Val Thr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys
85 90 95
Tyr Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln
100 105 110
Ala Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile
115 120 125
Lys Glu Leu Val Arg Ile Lys Ala Lys Leu Asn Trp Gly Leu Thr Asp
130 135 140
Glu Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Ser Leu
145 150 155 160
Ile Asn Lys Asn Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe Thr Ser
165 170 175
Gly Glu Gly Cys Phe Phe Val Asn Leu Ile Lys Ser Lys Ser Lys Leu
180 185 190
Gly Val Gln Val Gln Leu Val Phe Ser Ile Thr Gln His Ile Lys Asp
195 200 205
Lys Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Tyr Ile
210 215 220
Lys Glu Lys Asn Lys Ser Glu Phe Ser Trp Leu Asp Phe Val Val Thr
225 230 235 240
Lys Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn
245 250 255
Thr Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val
260 265 270
Ala Lys Leu Ile Glu Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp
275 280 285
Glu Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Val Phe
290 295 300
<210> 104
<211> 303
<212> PRT
<213> 新榆枯萎病菌(Ophiostoma novo-ulmi)
<220>
<221> MOD_RES
<222> (1)..(1)
<223> 任何氨基酸或不存在
<220>
<221> MOD_RES
<222> (2)..(2)
<223> 任何氨基酸或不存在
<220>
<221> MOD_RES
<222> (3)..(3)
<223> 任何氨基酸或不存在
<400> 104
Xaa Xaa Xaa Met Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr
1 5 10 15
Gly Phe Ala Asp Ala Glu Gly Ser Phe Leu Leu Arg Ile Arg Asn Asn
20 25 30
Asn Lys Ser Ser Val Gly Tyr Ser Thr Glu Leu Gly Phe Gln Ile Thr
35 40 45
Leu His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp
50 55 60
Lys Val Gly Val Ile Ala Asn Ser Gly Asp Asn Ala Val Ser Leu Lys
65 70 75 80
Val Thr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys
85 90 95
Tyr Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln
100 105 110
Ala Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile
115 120 125
Lys Glu Leu Val Arg Ile Lys Ala Lys Leu Asn Trp Gly Leu Thr Asp
130 135 140
Glu Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Ser Leu
145 150 155 160
Ile Asn Lys Asn Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe Thr Ser
165 170 175
Gly Glu Gly Cys Phe Phe Val Asn Leu Ile Lys Ser Lys Ser Lys Leu
180 185 190
Gly Val Gln Val Gln Leu Val Phe Ser Ile Thr Gln His Ile Lys Asp
195 200 205
Lys Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Tyr Ile
210 215 220
Lys Glu Lys Asn Lys Ser Glu Phe Ser Trp Leu Asp Phe Val Val Thr
225 230 235 240
Lys Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn
245 250 255
Thr Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val
260 265 270
Ala Lys Leu Ile Glu Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp
275 280 285
Glu Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Val Phe
290 295 300
<210> 105
<211> 303
<212> PRT
<213> 新榆枯萎病菌(Ophiostoma novo-ulmi)
<220>
<221> MOD_RES
<222> (1)..(1)
<223> 任何氨基酸或不存在
<220>
<221> MOD_RES
<222> (2)..(2)
<223> 任何氨基酸或不存在
<220>
<221> MOD_RES
<222> (3)..(3)
<223> 任何氨基酸或不存在
<220>
<221> MOD_RES
<222> (4)..(4)
<223> 任何氨基酸或不存在
<220>
<221> MOD_RES
<222> (302)..(302)
<223> 任何氨基酸或不存在
<220>
<221> MOD_RES
<222> (303)..(303)
<223> 任何氨基酸或不存在
<400> 105
Xaa Xaa Xaa Xaa Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr
1 5 10 15
Gly Phe Ala Asp Ala Glu Gly Ser Phe Leu Leu Arg Ile Arg Asn Asn
20 25 30
Asn Lys Ser Ser Val Gly Tyr Ser Thr Glu Leu Gly Phe Gln Ile Thr
35 40 45
Leu His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp
50 55 60
Lys Val Gly Val Ile Ala Asn Ser Gly Asp Asn Ala Val Ser Leu Lys
65 70 75 80
Val Thr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys
85 90 95
Tyr Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln
100 105 110
Ala Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile
115 120 125
Lys Glu Leu Val Arg Ile Lys Ala Lys Leu Asn Trp Gly Leu Thr Asp
130 135 140
Glu Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Ser Leu
145 150 155 160
Ile Asn Lys Asn Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe Thr Ser
165 170 175
Gly Glu Gly Cys Phe Phe Val Asn Leu Ile Lys Ser Lys Ser Lys Leu
180 185 190
Gly Val Gln Val Gln Leu Val Phe Ser Ile Thr Gln His Ile Lys Asp
195 200 205
Lys Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Tyr Ile
210 215 220
Lys Glu Lys Asn Lys Ser Glu Phe Ser Trp Leu Asp Phe Val Val Thr
225 230 235 240
Lys Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn
245 250 255
Thr Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val
260 265 270
Ala Lys Leu Ile Glu Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp
275 280 285
Glu Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Xaa Xaa
290 295 300
<210> 106
<211> 303
<212> PRT
<213> 新榆枯萎病菌(Ophiostoma novo-ulmi)
<220>
<221> MOD_RES
<222> (1)..(1)
<223> 任何氨基酸或不存在
<220>
<221> MOD_RES
<222> (2)..(2)
<223> 任何氨基酸或不存在
<220>
<221> MOD_RES
<222> (3)..(3)
<223> 任何氨基酸或不存在
<220>
<221> MOD_RES
<222> (4)..(4)
<223> 任何氨基酸或不存在
<220>
<221> MOD_RES
<222> (5)..(5)
<223> 任何氨基酸或不存在
<220>
<221> MOD_RES
<222> (6)..(6)
<223> 任何氨基酸或不存在
<220>
<221> MOD_RES
<222> (7)..(7)
<223> 任何氨基酸或不存在
<220>
<221> MOD_RES
<222> (8)..(8)
<223> 任何氨基酸或不存在
<220>
<221> MOD_RES
<222> (302)..(302)
<223> 任何氨基酸或不存在
<220>
<221> MOD_RES
<222> (303)..(303)
<223> 任何氨基酸或不存在
<400> 106
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Ile Asn Pro Trp Ile Leu Thr
1 5 10 15
Gly Phe Ala Asp Ala Glu Gly Ser Phe Leu Leu Arg Ile Arg Asn Asn
20 25 30
Asn Lys Ser Ser Val Gly Tyr Ser Thr Glu Leu Gly Phe Gln Ile Thr
35 40 45
Leu His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp
50 55 60
Lys Val Gly Val Ile Ala Asn Ser Gly Asp Asn Ala Val Ser Leu Lys
65 70 75 80
Val Thr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys
85 90 95
Tyr Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln
100 105 110
Ala Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile
115 120 125
Lys Glu Leu Val Arg Ile Lys Ala Lys Leu Asn Trp Gly Leu Thr Asp
130 135 140
Glu Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Ser Leu
145 150 155 160
Ile Asn Lys Asn Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe Thr Ser
165 170 175
Gly Glu Gly Cys Phe Phe Val Asn Leu Ile Lys Ser Lys Ser Lys Leu
180 185 190
Gly Val Gln Val Gln Leu Val Phe Ser Ile Thr Gln His Ile Lys Asp
195 200 205
Lys Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Tyr Ile
210 215 220
Lys Glu Lys Asn Lys Ser Glu Phe Ser Trp Leu Asp Phe Val Val Thr
225 230 235 240
Lys Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn
245 250 255
Thr Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val
260 265 270
Ala Lys Leu Ile Glu Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp
275 280 285
Glu Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Xaa Xaa
290 295 300
<210> 107
<211> 660
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 107
atgctgcgga tcatagacac ggaaacctgt ggactccagg gtggtatcgt ggagatagcc 60
agtgtcgatg taattgatgg gaaaatcgta aacccgatga gccacctcgt ccgaccggat 120
aggccgataa gtccgcaggc tatggctata catagaatca cggaggctat ggtagctgac 180
aagccttgga tcgaggacgt tatcccacac tactatggga gcgaatggta tgtagcgcac 240
aacgcatcat ttgataggcg cgtgcttccc gaaatgcctg gggagtggat ttgcacaatg 300
aaactcgctc gccgactgtg gcctggtatc aagtatagta acatggcact ttacaagacc 360
cgcaaactca acgtacagac acctccgggt ctccaccacc atagggcact ctatgattgt 420
tacataaccg ctgctctcct tatagacata atgaatacct ccgggtggac cgccgaacaa 480
atggcagaca taacgggaag acctagtttg atgacaacct ttacatttgg aaagtatcgg 540
ggcaaggcgg tgagcgatgt ggccgaaaga gatcccggtt atcttagatg gctcttcaat 600
aacttggatt ctatgtcacc tgaacttcgc cttaccctga agcattacct tgagaatacc 660
<210> 108
<211> 660
<212> RNA
<213> 大肠杆菌(Escherichia coli)
<400> 108
augcugcgga ucauagacac ggaaaccugu ggacuccagg gugguaucgu ggagauagcc 60
agugucgaug uaauugaugg gaaaaucgua aacccgauga gccaccucgu ccgaccggau 120
aggccgauaa guccgcaggc uauggcuaua cauagaauca cggaggcuau gguagcugac 180
aagccuugga ucgaggacgu uaucccacac uacuauggga gcgaauggua uguagcgcac 240
aacgcaucau uugauaggcg cgugcuuccc gaaaugccug gggaguggau uugcacaaug 300
aaacucgcuc gccgacugug gccugguauc aaguauagua acauggcacu uuacaagacc 360
cgcaaacuca acguacagac accuccgggu cuccaccacc auagggcacu cuaugauugu 420
uacauaaccg cugcucuccu uauagacaua augaauaccu ccggguggac cgccgaacaa 480
auggcagaca uaacgggaag accuaguuug augacaaccu uuacauuugg aaaguaucgg 540
ggcaaggcgg ugagcgaugu ggccgaaaga gaucccgguu aucuuagaug gcucuucaau 600
aacuuggauu cuaugucacc ugaacuucgc cuuacccuga agcauuaccu ugagaauacc 660
<210> 109
<211> 220
<212> PRT
<213> 大肠杆菌(Escherichia coli)
<400> 109
Met Leu Arg Ile Ile Asp Thr Glu Thr Cys Gly Leu Gln Gly Gly Ile
1 5 10 15
Val Glu Ile Ala Ser Val Asp Val Ile Asp Gly Lys Ile Val Asn Pro
20 25 30
Met Ser His Leu Val Arg Pro Asp Arg Pro Ile Ser Pro Gln Ala Met
35 40 45
Ala Ile His Arg Ile Thr Glu Ala Met Val Ala Asp Lys Pro Trp Ile
50 55 60
Glu Asp Val Ile Pro His Tyr Tyr Gly Ser Glu Trp Tyr Val Ala His
65 70 75 80
Asn Ala Ser Phe Asp Arg Arg Val Leu Pro Glu Met Pro Gly Glu Trp
85 90 95
Ile Cys Thr Met Lys Leu Ala Arg Arg Leu Trp Pro Gly Ile Lys Tyr
100 105 110
Ser Asn Met Ala Leu Tyr Lys Thr Arg Lys Leu Asn Val Gln Thr Pro
115 120 125
Pro Gly Leu His His His Arg Ala Leu Tyr Asp Cys Tyr Ile Thr Ala
130 135 140
Ala Leu Leu Ile Asp Ile Met Asn Thr Ser Gly Trp Thr Ala Glu Gln
145 150 155 160
Met Ala Asp Ile Thr Gly Arg Pro Ser Leu Met Thr Thr Phe Thr Phe
165 170 175
Gly Lys Tyr Arg Gly Lys Ala Val Ser Asp Val Ala Glu Arg Asp Pro
180 185 190
Gly Tyr Leu Arg Trp Leu Phe Asn Asn Leu Asp Ser Met Ser Pro Glu
195 200 205
Leu Arg Leu Thr Leu Lys His Tyr Leu Glu Asn Thr
210 215 220
<210> 110
<211> 1425
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 110
atgaacgatg gcaagcagca aagtacattc cttttccacg actacgaaac ctttggaacc 60
caccccgctc tggatcggcc agcacagttc gctgccataa gaaccgacaa cgagttcaat 120
gtgatcggtg aacccgaggt gttttactgc aaacctgcgg acgattatct gcctcaaccg 180
ggagccgttc tgattacggg aattaccccc caagaagcgc gtgccaaggg cgaaaatgaa 240
gctgcatttg ccgctagaat ccatagcctg tttactgttc cgaagacgtg tatcttggga 300
tacaacaatg tacgctttga tgacgaagta actcgtaacg tgttctatcg gaacttctat 360
gacccttacg cctggagttg gcagcacgac aattctcgat gggatcttct ggatgtgatg 420
agagcctgct acgctctcag gccagagggc attaactggc ctgagaacga cgatgggctt 480
ccgtcatttc gccttgagca tcttaccaaa gcgaatggca tcgaacactc caatgcacac 540
gacgctatgg ctgatgtcta cgcgactatt gcaatggcga aattggtcaa gacaagacag 600
ccgaggttgt ttgactacct gtttacgcat cgaaacaaac ataagctcat ggcactgata 660
gatgtccctc aaatgaagcc cctcgttcac gtcagcggta tgttcggagc gtggagagga 720
aacacgtcct gggttgctcc tctggcatgg catccagaga acagaaacgc ggtaatcatg 780
gtcgatctcg caggagacat tagcccactt ctcgaattgg attccgacac cttgcgagaa 840
cgcctttaca cagcgaaaac tgatttgggt gataacgctg ccgtacctgt caaattggta 900
catatcaaca aatgtccggt gctcgcgcaa gccaacaccc tgagacctga agacgcggat 960
agactgggga taaaccggca gcactgtttg gataatctca agatactcag ggagaatccg 1020
caggtgcgcg aaaaagtcgt agccatcttt gcggaggcag aaccatttac gccgtctgac 1080
aatgtagatg ctcagttata caatggcttc tttagtgacg ctgaccgagc tgcgatgaag 1140
attgtgctag agactgaacc gagaaacctc tctgcattgg acatcacttt tgtcgataag 1200
cgaatagaga agttgctgtt caactatcgt gcccgaaact ttccagggac actcgattac 1260
gcagagcaac aaaggtggct tgagcatcgc aggcaggtgt ttaccccaga gttcttgcag 1320
ggttacgctg aggagattca aatgttggca cagcagtatg cagatgataa ggagaaagtc 1380
gcgttgctga aggctctgtg gcaatacgcc gaagagatag tgtga 1425
<210> 111
<211> 1425
<212> RNA
<213> 大肠杆菌(Escherichia coli)
<400> 111
augaacgaug gcaagcagca aaguacauuc cuuuuccacg acuacgaaac cuuuggaacc 60
caccccgcuc uggaucggcc agcacaguuc gcugccauaa gaaccgacaa cgaguucaau 120
gugaucggug aacccgaggu guuuuacugc aaaccugcgg acgauuaucu gccucaaccg 180
ggagccguuc ugauuacggg aauuaccccc caagaagcgc gugccaaggg cgaaaaugaa 240
gcugcauuug ccgcuagaau ccauagccug uuuacuguuc cgaagacgug uaucuuggga 300
uacaacaaug uacgcuuuga ugacgaagua acucguaacg uguucuaucg gaacuucuau 360
gacccuuacg ccuggaguug gcagcacgac aauucucgau gggaucuucu ggaugugaug 420
agagccugcu acgcucucag gccagagggc auuaacuggc cugagaacga cgaugggcuu 480
ccgucauuuc gccuugagca ucuuaccaaa gcgaauggca ucgaacacuc caaugcacac 540
gacgcuaugg cugaugucua cgcgacuauu gcaauggcga aauuggucaa gacaagacag 600
ccgagguugu uugacuaccu guuuacgcau cgaaacaaac auaagcucau ggcacugaua 660
gaugucccuc aaaugaagcc ccucguucac gucagcggua uguucggagc guggagagga 720
aacacguccu ggguugcucc ucuggcaugg cauccagaga acagaaacgc gguaaucaug 780
gucgaucucg caggagacau uagcccacuu cucgaauugg auuccgacac cuugcgagaa 840
cgccuuuaca cagcgaaaac ugauuugggu gauaacgcug ccguaccugu caaauuggua 900
cauaucaaca aauguccggu gcucgcgcaa gccaacaccc ugagaccuga agacgcggau 960
agacugggga uaaaccggca gcacuguuug gauaaucuca agauacucag ggagaauccg 1020
caggugcgcg aaaaagucgu agccaucuuu gcggaggcag aaccauuuac gccgucugac 1080
aauguagaug cucaguuaua caauggcuuc uuuagugacg cugaccgagc ugcgaugaag 1140
auugugcuag agacugaacc gagaaaccuc ucugcauugg acaucacuuu ugucgauaag 1200
cgaauagaga aguugcuguu caacuaucgu gcccgaaacu uuccagggac acucgauuac 1260
gcagagcaac aaagguggcu ugagcaucgc aggcaggugu uuaccccaga guucuugcag 1320
gguuacgcug aggagauuca aauguuggca cagcaguaug cagaugauaa ggagaaaguc 1380
gcguugcuga aggcucugug gcaauacgcc gaagagauag uguga 1425
<210> 112
<211> 474
<212> PRT
<213> 大肠杆菌(Escherichia coli)
<400> 112
Met Asn Asp Gly Lys Gln Gln Ser Thr Phe Leu Phe His Asp Tyr Glu
1 5 10 15
Thr Phe Gly Thr His Pro Ala Leu Asp Arg Pro Ala Gln Phe Ala Ala
20 25 30
Ile Arg Thr Asp Asn Glu Phe Asn Val Ile Gly Glu Pro Glu Val Phe
35 40 45
Tyr Cys Lys Pro Ala Asp Asp Tyr Leu Pro Gln Pro Gly Ala Val Leu
50 55 60
Ile Thr Gly Ile Thr Pro Gln Glu Ala Arg Ala Lys Gly Glu Asn Glu
65 70 75 80
Ala Ala Phe Ala Ala Arg Ile His Ser Leu Phe Thr Val Pro Lys Thr
85 90 95
Cys Ile Leu Gly Tyr Asn Asn Val Arg Phe Asp Asp Glu Val Thr Arg
100 105 110
Asn Val Phe Tyr Arg Asn Phe Tyr Asp Pro Tyr Ala Trp Ser Trp Gln
115 120 125
His Asp Asn Ser Arg Trp Asp Leu Leu Asp Val Met Arg Ala Cys Tyr
130 135 140
Ala Leu Arg Pro Glu Gly Ile Asn Trp Pro Glu Asn Asp Asp Gly Leu
145 150 155 160
Pro Ser Phe Arg Leu Glu His Leu Thr Lys Ala Asn Gly Ile Glu His
165 170 175
Ser Asn Ala His Asp Ala Met Ala Asp Val Tyr Ala Thr Ile Ala Met
180 185 190
Ala Lys Leu Val Lys Thr Arg Gln Pro Arg Leu Phe Asp Tyr Leu Phe
195 200 205
Thr His Arg Asn Lys His Lys Leu Met Ala Leu Ile Asp Val Pro Gln
210 215 220
Met Lys Pro Leu Val His Val Ser Gly Met Phe Gly Ala Trp Arg Gly
225 230 235 240
Asn Thr Ser Trp Val Ala Pro Leu Ala Trp His Pro Glu Asn Arg Asn
245 250 255
Ala Val Ile Met Val Asp Leu Ala Gly Asp Ile Ser Pro Leu Leu Glu
260 265 270
Leu Asp Ser Asp Thr Leu Arg Glu Arg Leu Tyr Thr Ala Lys Thr Asp
275 280 285
Leu Gly Asp Asn Ala Ala Val Pro Val Lys Leu Val His Ile Asn Lys
290 295 300
Cys Pro Val Leu Ala Gln Ala Asn Thr Leu Arg Pro Glu Asp Ala Asp
305 310 315 320
Arg Leu Gly Ile Asn Arg Gln His Cys Leu Asp Asn Leu Lys Ile Leu
325 330 335
Arg Glu Asn Pro Gln Val Arg Glu Lys Val Val Ala Ile Phe Ala Glu
340 345 350
Ala Glu Pro Phe Thr Pro Ser Asp Asn Val Asp Ala Gln Leu Tyr Asn
355 360 365
Gly Phe Phe Ser Asp Ala Asp Arg Ala Ala Met Lys Ile Val Leu Glu
370 375 380
Thr Glu Pro Arg Asn Leu Ser Ala Leu Asp Ile Thr Phe Val Asp Lys
385 390 395 400
Arg Ile Glu Lys Leu Leu Phe Asn Tyr Arg Ala Arg Asn Phe Pro Gly
405 410 415
Thr Leu Asp Tyr Ala Glu Gln Gln Arg Trp Leu Glu His Arg Arg Gln
420 425 430
Val Phe Thr Pro Glu Phe Leu Gln Gly Tyr Ala Glu Glu Ile Gln Met
435 440 445
Leu Ala Gln Gln Tyr Ala Asp Asp Lys Glu Lys Val Ala Leu Leu Lys
450 455 460
Ala Leu Trp Gln Tyr Ala Glu Glu Ile Val
465 470
<210> 113
<211> 3
<212> PRT
<213> 人工序列
<220>
<223> 示例性连接子序列
<400> 113
Gly Gly Gly
1
<210> 114
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 示例性连接子序列
<400> 114
Asp Gly Gly Gly Ser
1 5
<210> 115
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 示例性连接子序列
<400> 115
Thr Gly Glu Lys Pro
1 5
<210> 116
<211> 4
<212> PRT
<213> 人工序列
<220>
<223> 示例性连接子序列
<400> 116
Gly Gly Arg Arg
1
<210> 117
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 示例性连接子序列
<400> 117
Gly Gly Gly Gly Ser
1 5
<210> 118
<211> 14
<212> PRT
<213> 人工序列
<220>
<223> 示例性连接子序列
<400> 118
Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Val Asp
1 5 10
<210> 119
<211> 18
<212> PRT
<213> 人工序列
<220>
<223> 示例性连接子序列
<400> 119
Lys Glu Ser Gly Ser Val Ser Ser Glu Gln Leu Ala Gln Phe Arg Ser
1 5 10 15
Leu Asp
<210> 120
<211> 8
<212> PRT
<213> 人工序列
<220>
<223> 示例性连接子序列
<400> 120
Gly Gly Arg Arg Gly Gly Gly Ser
1 5
<210> 121
<211> 9
<212> PRT
<213> 人工序列
<220>
<223> 示例性连接子序列
<400> 121
Leu Arg Gln Arg Asp Gly Glu Arg Pro
1 5
<210> 122
<211> 12
<212> PRT
<213> 人工序列
<220>
<223> 示例性连接子序列
<400> 122
Leu Arg Gln Lys Asp Gly Gly Gly Ser Glu Arg Pro
1 5 10
<210> 123
<211> 16
<212> PRT
<213> 人工序列
<220>
<223> 示例性连接子序列
<400> 123
Leu Arg Gln Lys Asp Gly Gly Gly Ser Gly Gly Gly Ser Glu Arg Pro
1 5 10 15
<210> 124
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> TEV蛋白酶切割序列
<220>
<221> misc_feature
<222> (2)..(3)
<223> Xaa是任何氨基酸
<220>
<221> misc_feature
<222> (5)..(5)
<223> Xaa是任何氨基酸
<220>
<221> MISC_FEATURE
<222> (7)..(7)
<223> Xaa = Gly或Ser
<400> 124
Glu Xaa Xaa Tyr Xaa Gln Xaa
1 5
<210> 125
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> TEV蛋白酶切割序列
<400> 125
Glu Asn Leu Tyr Phe Gln Gly
1 5
<210> 126
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> TEV蛋白酶切割序列
<400> 126
Glu Asn Leu Tyr Phe Gln Ser
1 5
<210> 127
<211> 22
<212> PRT
<213> 人工序列
<220>
<223> 包括2A位点的自切割多肽
<400> 127
Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val
1 5 10 15
Glu Glu Asn Pro Gly Pro
20
<210> 128
<211> 19
<212> PRT
<213> 人工序列
<220>
<223> 包括2A位点的自切割多肽
<400> 128
Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn
1 5 10 15
Pro Gly Pro
<210> 129
<211> 14
<212> PRT
<213> 人工序列
<220>
<223> 包括2A位点的自切割多肽
<400> 129
Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro
1 5 10
<210> 130
<211> 21
<212> PRT
<213> 人工序列
<220>
<223> 包括2A位点的自切割多肽
<400> 130
Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu
1 5 10 15
Glu Asn Pro Gly Pro
20
<210> 131
<211> 18
<212> PRT
<213> 人工序列
<220>
<223> 包括2A位点的自切割多肽
<400> 131
Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro
1 5 10 15
Gly Pro
<210> 132
<211> 13
<212> PRT
<213> 人工序列
<220>
<223> 包括2A位点的自切割多肽
<400> 132
Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro
1 5 10
<210> 133
<211> 23
<212> PRT
<213> 人工序列
<220>
<223> 包括2A位点的自切割多肽
<400> 133
Gly Ser Gly Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp
1 5 10 15
Val Glu Ser Asn Pro Gly Pro
20
<210> 134
<211> 20
<212> PRT
<213> 人工序列
<220>
<223> 包括2A位点的自切割多肽
<400> 134
Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp Val Glu Ser
1 5 10 15
Asn Pro Gly Pro
20
<210> 135
<211> 14
<212> PRT
<213> 人工序列
<220>
<223> 包括2A位点的自切割多肽
<400> 135
Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly Pro
1 5 10
<210> 136
<211> 25
<212> PRT
<213> 人工序列
<220>
<223> 包括2A位点的自切割多肽
<400> 136
Gly Ser Gly Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala
1 5 10 15
Gly Asp Val Glu Ser Asn Pro Gly Pro
20 25
<210> 137
<211> 22
<212> PRT
<213> 人工序列
<220>
<223> 包括2A位点的自切割多肽
<400> 137
Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val
1 5 10 15
Glu Ser Asn Pro Gly Pro
20
<210> 138
<211> 14
<212> PRT
<213> 人工序列
<220>
<223> 包括2A位点的自切割多肽
<400> 138
Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly Pro
1 5 10
<210> 139
<211> 19
<212> PRT
<213> 人工序列
<220>
<223> 包括2A位点的自切割多肽
<400> 139
Leu Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn
1 5 10 15
Pro Gly Pro
<210> 140
<211> 19
<212> PRT
<213> 人工序列
<220>
<223> 包括2A位点的自切割多肽
<400> 140
Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn
1 5 10 15
Pro Gly Pro
<210> 141
<211> 14
<212> PRT
<213> 人工序列
<220>
<223> 包括2A位点的自切割多肽
<400> 141
Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly Pro
1 5 10
<210> 142
<211> 17
<212> PRT
<213> 人工序列
<220>
<223> 包括2A位点的自切割多肽
<400> 142
Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly
1 5 10 15
Pro
<210> 143
<211> 20
<212> PRT
<213> 人工序列
<220>
<223> 包括2A位点的自切割多肽
<400> 143
Gln Leu Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser
1 5 10 15
Asn Pro Gly Pro
20
<210> 144
<211> 24
<212> PRT
<213> 人工序列
<220>
<223> 包括2A位点的自切割多肽
<400> 144
Ala Pro Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly
1 5 10 15
Asp Val Glu Ser Asn Pro Gly Pro
20
<210> 145
<211> 40
<212> PRT
<213> 人工序列
<220>
<223> 包括2A位点的自切割多肽
<400> 145
Val Thr Glu Leu Leu Tyr Arg Met Lys Arg Ala Glu Thr Tyr Cys Pro
1 5 10 15
Arg Pro Leu Leu Ala Ile His Pro Thr Glu Ala Arg His Lys Gln Lys
20 25 30
Ile Val Ala Pro Val Lys Gln Thr
35 40
<210> 146
<211> 18
<212> PRT
<213> 人工序列
<220>
<223> 包括2A位点的自切割多肽
<400> 146
Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro
1 5 10 15
Gly Pro
<210> 147
<211> 40
<212> PRT
<213> 人工序列
<220>
<223> 包括2A位点的自切割多肽
<400> 147
Leu Leu Ala Ile His Pro Thr Glu Ala Arg His Lys Gln Lys Ile Val
1 5 10 15
Ala Pro Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly
20 25 30
Asp Val Glu Ser Asn Pro Gly Pro
35 40
<210> 148
<211> 33
<212> PRT
<213> 人工序列
<220>
<223> 包括2A位点的自切割多肽
<400> 148
Glu Ala Arg His Lys Gln Lys Ile Val Ala Pro Val Lys Gln Thr Leu
1 5 10 15
Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly
20 25 30
Pro
<210> 149
<211> 10
<212> DNA
<213> 未知
<220>
<223> Kozak序列
<400> 149
gccrccatgg 10
<210> 150
<211> 10
<212> PRT
<213> 人工序列
<220>
<223> 示例性连接子序列
<400> 150
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10
<210> 151
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 示例性连接子序列
<400> 151
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
<210> 152
<211> 20
<212> PRT
<213> 人工序列
<220>
<223> 示例性连接子序列
<400> 152
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
1 5 10 15
Gly Gly Gly Ser
20
<210> 153
<211> 25
<212> PRT
<213> 人工序列
<220>
<223> 示例性连接子序列
<400> 153
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
1 5 10 15
Gly Gly Gly Ser Gly Gly Gly Gly Ser
20 25
<210> 154
<211> 41
<212> DNA
<213> 人工序列
<220>
<223> 在实验室中制备-PD-1 megaTAL多核苷酸区段
<400> 154
ctggtggggc tgctccaggc atgcagatcc cacaggcgcc c 41
<210> 155
<211> 38
<212> DNA
<213> 人工序列
<220>
<223> 在实验室中制备-具有缺失的PD-1 megaTAL多核苷酸区段
<400> 155
ctggtggggc tgctccaggc atgcagccac aggcgccc 38
<210> 156
<211> 37
<212> DNA
<213> 人工序列
<220>
<223> 在实验室中制备-具有缺失的PD-1 megaTAL多核苷酸区段
<400> 156
ctggtggggc tgctccaggc atgcagcaca ggcgccc 37
<210> 157
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> 在实验室中制备-具有缺失的PD-1 megaTAL多核苷酸区段
<400> 157
ctggtggggc tgctccaggc atgcacacag gcgccc 36
<210> 158
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 在实验室中制备-具有缺失的PD-1 megaTAL多核苷酸区段
<400> 158
ctggtggggc tgctccacag gcgccc 26
<210> 159
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 在实验室中制备-PD-1 megaTAL多肽区段
<400> 159
Met Gln Ile Pro Gln Ala Pro
1 5
<210> 160
<211> 6
<212> PRT
<213> 人工序列
<220>
<223> 在实验室中制备-具有缺失的PD-1 megaTAL多肽区段
<400> 160
Met Gln Pro Gln Ala Pro
1 5
<210> 161
<211> 6
<212> PRT
<213> 人工序列
<220>
<223> 在实验室中制备-具有缺失的PD-1 megaTAL多肽区段
<400> 161
Met Gln His Arg Arg Pro
1 5
<210> 162
<211> 6
<212> PRT
<213> 人工序列
<220>
<223> 在实验室中制备-具有缺失的PD-1 megaTAL多肽区段
<400> 162
Met His Thr Gly Ala Leu
1 5
<210> 163
<211> 29
<212> DNA
<213> 智人(Homo sapiens)
<400> 163
gtggggtatg tgtgtgtccg gggctgccg 29
<210> 164
<211> 21
<212> DNA
<213> 智人(Homo sapiens)
<400> 164
aacaaaagcg gctctgagtc t 21
Claims (124)
1.一种融合多肽,其包括DNA结合结构域和归巢核酸内切酶(HE)变体,所述HE变体结合并且切割细胞中的选定双链DNA(dsDNA)靶位点;连接子结构域;以及核酸外切酶或其生物活性片段。
2.根据权利要求1所述的融合多肽,其中所述核酸外切酶是Trex2、ExoI或ExoX或其生物活性片段。
3.根据权利要求1所述的融合多肽,其中所述核酸外切酶是ExoX或其生物活性片段。
4.根据权利要求1所述的融合多肽,其中所述核酸外切酶是ExoI或其生物活性片段。
5.根据权利要求1至4中任一项所述的融合多肽,其中所述归巢核酸内切酶是工程化的归巢核酸内切酶。
6.根据权利要求1至5中任一项所述的融合多肽,其中所述选定dsDNA靶位点是非天然归巢核酸内切酶靶位点。
7.根据权利要求1至6中任一项所述的融合多肽,其中所述DNA结合结构域结合所述核酸内切酶dsDNA靶位点上游的dsDNA靶位点。
8.根据权利要求1至7中任一项所述的融合多肽,其中所述DNA结合结构域包括TALEDNA结合结构域。
9.根据权利要求1至8中任一项所述的融合多肽,其中所述TALE DNA结构域包括约9.5个TALE重复单元至约15.5个TALE重复单元。
10.根据权利要求1至9中任一项所述的融合多肽,其中所述TALE DNA结构域包括11.5个TALE重复单元或12.5个TALE重复单元。
11.根据权利要求1至10中任一项所述的融合多肽,其中所述DNA结合结构域包括锌指DNA结合结构域。
12.根据权利要求11所述的融合多肽,其中所述锌指DNA结合结构域包括2个、3个、4个、5个、6个、7个或8个锌指基序。
13.根据权利要求1至12中任一项所述的融合多肽,其中所述连接子结构域是肽连接子。
14.根据权利要求13所述的融合多肽,其中所述肽连接子是自切割肽连接子。
15.根据权利要求13或权利要求14所述的融合多肽,其中所述肽连接子包括约4个至约30个氨基酸。
16.根据权利要求13至15中任一项所述的融合多肽,其中所述肽连接子包括约10个至约16个氨基酸。
17.根据权利要求13至16中任一项所述的融合多肽,其中所述肽连接子包括约12个氨基酸。
18.根据权利要求13至17中任一项所述的融合多肽,其中所述肽连接子是(GGGGS)1-4连接子(SEQ ID NO:117和150-152)。
19.根据权利要求13至18中任一项所述的融合多肽,其中所述肽连接子包括(GGGGS)2连接子(SEQ ID NO:150)。
20.根据权利要求1至19中任一项所述的融合多肽,其中所述HE变体是LAGLIDADG归巢核酸内切酶(LHE)变体。
21.根据权利要求1至20中任一项所述的融合多肽,其中与对应的野生型HE相比,所述HE变体缺少1个、2个、3个、4个、5个、6个、7个或8个N末端氨基酸。
22.根据权利要求1至21中任一项所述的融合多肽,其中与对应的野生型HE相比,所述HE变体缺少4个N末端氨基酸。
23.根据权利要求1至21中任一项所述的融合多肽,其中与对应的野生型HE相比,所述HE变体缺少8个N末端氨基酸。
24.根据权利要求1至23中任一项所述的融合多肽,其中与对应的野生型HE相比,所述HE变体缺少1个、2个、3个、4个或5个C末端氨基酸。
25.根据权利要求1至24中任一项所述的融合多肽,其中与对应的野生型HE相比,所述HE变体缺少C末端氨基酸。
26.根据权利要求1至24中任一项所述的融合多肽,其中与对应的野生型HE相比,所述HE变体缺少2个C末端氨基酸。
27.根据权利要求1至26中任一项所述的融合多肽,其中所述HE变体是选自由以下组成的组的LHE的变体:I-AabMI、I-AaeMI、I-AniI、I-ApaMI、I-CapIII、I-CapIV、I-CkaMI、I-CpaMI、I-CpaMII、I-CpaMIII、I-CpaMIV、I-CpaMV、I-CpaV、I-CraMI、I-EjeMI、I-GpeMI、I-GpiI、I-GzeMI、I-GzeMII、I-GzeMIII、I-HjeMI、I-LtrII、I-LtrI、I-LtrWI、I-MpeMI、I-MveMI、I-NcrII、I-Ncrl、I-NcrMI、I-OheMI、I-OnuI、I-OsoMI、I-OsoMII、I-OsoMIII、I-OsoMIV、I-PanMI、I-PanMII、I-PanMIII、I-PnoMI、I-SceI、I-ScuMI、I-SmaMI、I-SscMI和I-Vdi141I。
28.根据权利要求1至27中任一项所述的融合多肽,其中所述HE变体是选自由以下组成的组的LHE的变体:I-CpaMI、I-HjeMI、I-OnuI、I-PanMI和I-SmaMI。
29.根据权利要求1至28中任一项所述的融合多肽,其中所述HE变体是I-OnuI LHE变体。
30.根据权利要求1至29中任一项所述的融合多肽,其中所述HE靶位点位于免疫系统检查点基因、珠蛋白基因、编码有助于抑制γ-珠蛋白基因表达和HbF的多肽的基因或免疫抑制信号传导基因内。
31.根据权利要求1至29中任一项所述的融合多肽,其中所述HE靶位点位于选自由以下组成的组的基因内:程序性细胞死亡蛋白1(PD-1;PDCD1)、淋巴细胞激活基因3蛋白(LAG-3)、T细胞免疫球蛋白结构域和粘蛋白结构域蛋白3(TIM-3)、细胞毒性T淋巴细胞抗原-4(CTLA-4)、带T淋巴细胞弱化因子(BTLA)、T细胞免疫球蛋白和基于免疫受体酪氨酸的抑制基序结构域(TIGIT)、T细胞激活的V结构域Ig抑制因子(VISTA)和杀伤细胞免疫球蛋白样受体(KIR)、CCR5、TRAC(TCRα)、TCRβ、IL10Rα、IL10Rβ、TGFBR1、TGFBR2、CBL-B、PCSK9、AHR、BTK、α-珠蛋白、β-珠蛋白、γ-珠蛋白、BCL11A、KLF1、SOX6、GATA1、LSD1、α叶酸受体(FRα)、αvβ6整合素、B细胞成熟抗原(BCMA)、B7-H3(CD276)、B7-H6、碳酸酐酶IX(CAIX)、CD16、CD19、CD20、CD22、CD30、CD33、CD37、CD38、CD44、CD44v6、CD44v7/8、CD70、CD79a、CD79b、CD123、CD133、CD138、CD171、癌胚抗原(CEA)、C型凝集素样分子-1(CLL-1)、CD2子集1(CS-1)、硫酸软骨素蛋白聚糖4(CSPG4)、皮肤T细胞淋巴瘤相关抗原1(CTAGE1)、表皮生长因子受体(EGFR)、表皮生长因子受体变体III(EGFRvIII)、上皮糖蛋白2(EGP2)、上皮糖蛋白40(EGP40)、上皮细胞粘附分子(EPCAM)、肝配蛋白A型受体2(EPHA2)、成纤维细胞激活蛋白(FAP)、Fc受体样5(FCRL5)、胎儿乙酰胆碱酯酶受体(AchR)、神经节苷脂G2(GD2)、神经节苷脂G3(GD3)、磷脂酰肌醇蛋白聚糖-3(GPC3)、包含ErbB2(HER2)的EGFR家族、IL-11Rα、IL-13Rα2、κ、癌/睾丸抗原2(LAGE-1A)、λ、路易斯-Y(Lewis-Y,LeY)、L1细胞粘附分子(L1-CAM)、黑色素瘤抗原基因(MAGE)-A1、MAGE-A3、MAGE-A4、MAGE-A6、MAGEA10、T细胞识别的黑色素瘤抗原1(MelanA或MART1)、间皮素(MSLN)、MUC1、MUC16、MHCI类链相关蛋白A(MICA)、MHCI类链相关蛋白B(MICB)、神经细胞粘附分子(NCAM)、癌/睾丸抗原1(NY-ESO-1)、聚唾液酸;胎盘特异性1(PLAC1)、黑色素瘤中优先表达的抗原(PRAME)、前列腺干细胞抗原(PSCA)、前列腺特异性膜抗原(PSMA)、受体酪氨酸激酶样孤儿受体1(ROR1)、滑膜肉瘤、X断点2(SSX2)、存活蛋白、肿瘤相关糖蛋白72(TAG72)、肿瘤内皮标志物1(TEM1/CD248)、肿瘤内皮标志物7相关(TEM7R)、TEM5、TEM8、滋养层糖蛋白(TPBG)、UL16结合蛋白(ULBP)1、ULBP2、ULBP3、ULBP4、ULBP5、ULBP6、血管内皮生长因子受体2(VEGFR2)和威尔姆斯瘤1(WT-1)基因。
32.根据权利要求1至29中任一项所述的融合多肽,其中所述HE靶位点位于选自由以下组成的组的基因内:程序性细胞死亡蛋白1(PD-1;PDCD1)、淋巴细胞激活基因3蛋白(LAG-3)、T细胞免疫球蛋白结构域和粘蛋白结构域蛋白3(TIM-3)、细胞毒性T淋巴细胞抗原-4(CTLA-4)、带T淋巴细胞弱化因子(BTLA)、T细胞免疫球蛋白和基于免疫受体酪氨酸的抑制基序结构域(TIGIT)、T细胞激活的V结构域Ig抑制因子(VISTA)和杀伤细胞免疫球蛋白样受体(KIR)、CCR5、TRAC(TCRα)、IL10Rα、TGFBR2、CBL-B、PCSK9、AHR、BTK、α-珠蛋白、β-珠蛋白、γ-珠蛋白和BCL11A基因。
33.根据权利要求1至29中任一项所述的融合多肽,其中所述HE靶位点位于TRAC(TCRα)基因、CBL-B基因或PDCD1(PD-1)基因内。
34.根据权利要求33所述的融合多肽,其中所述TCRα基因靶位点包括SEQ ID NO:1中所示的氨基酸序列,所述CBL-B基因靶位点包括SEQ ID NO:2中所示的氨基酸序列,或者所述PD-1基因靶位点包括SEQ ID NO:3中所示的氨基酸序列。
35.根据权利要求1至34中任一项所述的融合多肽,其中所述DNA结合结构域包括具有如SEQ ID NO:4中所示的靶位点的TALE DNA结合结构域。
36.根据权利要求3至35中任一项所述的融合多肽,其中所述ExoX或其生物活性片段包括与如SEQ ID NO:109中所示的氨基酸序列具有至少85%、90%、95%、96%、97%、98%或99%同一性的氨基酸氨基酸序列。
37.根据权利要求36所述的融合多肽,其中所述ExoX或其生物活性片段包括如SEQ IDNO:109中所示的氨基酸氨基酸序列。
38.根据权利要求36所述的融合多肽,其包括与如SEQ ID NO:46、64、73和82中的任一个中所示的氨基酸序列具有至少85%、90%、95%、96%、97%、98%或99%同一性的氨基酸序列。
39.根据权利要求36所述的融合多肽,其包括如SEQ ID NO:46、64、73和82中的任一个中所示的氨基酸序列。
40.根据权利要求4至35中任一项所述的融合多肽,其中所述ExoI或其生物活性片段包括与如SEQ ID NO:112中所示的氨基酸序列具有至少85%、90%、95%、96%、97%、98%或99%同一性的氨基酸氨基酸序列。
41.根据权利要求40所述的融合多肽,其中所述ExoI或其生物活性片段包括如SEQ IDNO:112中所示的氨基酸氨基酸序列。
42.根据权利要求40所述的融合多肽,其包括与如SEQ ID NO:43中所示的氨基酸序列具有至少85%、90%、95%、96%、97%、98%或99%同一性的氨基酸序列。
43.根据权利要求40所述的融合多肽,其包括如SEQ ID NO:43中所示的氨基酸序列。
44.一种多核苷酸,其编码根据权利要求1至43中任一项所述的融合多肽。
45.根据权利要求44所述的多核苷酸,其中所述多核苷酸包括与如SEQ ID NO:44、62、71和80中的任一个中所示的多核苷酸序列具有至少85%、90%、95%、96%、97%、98%或99%同一性的核苷酸序列。
46.根据权利要求44所述的多核苷酸,其中所述多核苷酸包括如SEQ ID NO:44、62、71和80中的任一个中所示的多核苷酸序列。
47.一种编码根据权利要求1至43中任一项所述的多肽的mRNA。
48.根据权利要求47所述的mRNA,其中所述mRNA包括与如SEQ ID NO:45、63、72和81中的任一个中所示的RNA序列具有至少85%、90%、95%、96%、97%、98%或99%同一性的RNA序列。
49.根据权利要求48所述的mRNA,其中所述mRNA包括如SEQ ID NO:45、63、72和81中的任一个中所示的RNA序列。
50.根据权利要求47所述的mRNA,其中所述mRNA包括与如SEQ ID NO:42中所示的RNA序列具有至少85%、90%、95%、96%、97%、98%或99%同一性的RNA序列。
51.根据权利要求50所述的mRNA,其中所述mRNA包括如SEQ ID NO:42中的任一个中所示的RNA序列。
52.一种载体,其编码根据权利要求1至43中任一项所述的多肽。
53.一种载体,其包括根据权利要求44至46中任一项所述的多核苷酸。
54.一种细胞,其包括根据权利要求1至43中任一项所述的多肽。
55.一种细胞,其包括根据权利要求44至46中任一项所述的多核苷酸。
56.一种细胞,其包括根据权利要求47至51中任一项所述的mRNA。
57.一种细胞,其包括根据权利要求52或权利要求53所述的载体。
58.根据权利要求54至57中任一项所述的细胞,其包括一种或多种基因组修饰。
59.根据权利要求54至58中任一项所述的细胞,其中所述细胞是造血细胞。
60.根据权利要求54至59中任一项所述的细胞,其中所述细胞是造血干细胞或祖细胞。
61.根据权利要求54至60中任一项所述的细胞,其中所述细胞是CD34+细胞。
62.根据权利要求54至61中任一项所述的细胞,其中所述细胞是CD133+细胞。
63.根据权利要求54至59中任一项所述的细胞,其中所述细胞是免疫效应细胞。
64.根据权利要求63所述的细胞,其中所述免疫效应细胞是细胞毒性T淋巴细胞(CTL)、肿瘤浸润淋巴细胞(TIL)或辅助T细胞。
65.根据权利要求63所述的细胞,其中所述免疫效应细胞是T细胞。
66.根据权利要求63所述的细胞,其中所述免疫效应细胞是αβT细胞、γδT细胞、自然杀伤(NK)细胞或自然杀伤T(NKT)细胞。
67.一种细胞群体,其包括根据权利要求54至66中任一项所述的细胞。
68.一种组合物,其包括根据权利要求1至43中任一项所述的融合多肽、根据权利要求权利要求44至46中任一项所述的多核苷酸、根据权利要求47至51中任一项所述的mRNA、根据权利要求52或权利要求53所述的载体、根据权利要求54至66中任一项所述的细胞或根据权利要求67所述的细胞群体。
69.根据权利要求68所述的组合物,其进一步包括药学上可接受的载体。
70.一种定点诱变方法,所述方法包括:
a)选择双链DNA(dsDNA)靶位点;以及
b)向细胞中引入根据权利要求1至43中任一项所述的融合多肽、根据权利要求44至46中任一项所述的多核苷酸、根据权利要求47至51中任一项所述的mRNA或根据权利要求52或权利要求53所述的载体;
其中融合肽产生定向偏向的缺失,所述定向偏向的缺失的缺失中心靠近所述细胞中的选定dsDNA靶切割位点。
71.根据权利要求70所述的方法,其中大于50%、大于51%、大于52%、大于53%、大于54%、大于55%、大于56%、大于57%、大于58%、大于59%或大于60%的所述定向偏向的缺失的缺失中心位置位于HE靶位点中心位置的一侧上。
72.根据权利要求70所述的方法,其中大于50%的所述定向偏向的缺失的缺失中心位置位于所述HE靶位点中心位置的一侧上。
73.根据权利要求70所述的方法,其中大于55%的所述定向偏向的缺失的缺失中心位置位于所述HE靶位点中心位置的一侧上。
74.根据权利要求70所述的方法,其中大于60%的所述定向偏向的缺失的缺失中心位置位于所述HE靶位点中心位置的一侧上。
75.根据权利要求70所述的方法,其中大于65%的所述定向偏向的缺失的缺失中心位置位于所述HE靶位点中心位置的一侧上。
76.根据权利要求70所述的方法,其中大于70%的所述定向偏向的缺失的缺失中心位置位于所述HE靶位点中心位置的一侧上。
77.根据权利要求70所述的方法,其中大于75%的所述定向偏向的缺失的缺失中心位置位于所述HE靶位点中心位置的一侧上。
78.根据权利要求70所述的方法,其中大于80%的所述定向偏向的缺失的缺失中心位置位于所述HE靶位点中心位置的一侧上。
79.根据权利要求70至78中任一项所述的方法,其中相对于所述HE靶位点中心位置,所述缺失中心位置与所述DNA结合结构域靶位点位于同一侧上。
80.根据权利要求70至79中任一项所述的方法,其中所述缺失中心位置位于所述HE靶位点中心位置的5'端。
81.根据权利要求70至80中任一项所述的方法,其中至少50%、至少51%、至少52%、至少53%、至少54%、至少55%、至少56%、至少57%、至少58%、至少59%或至少60%的缺失的缺失中心距所述HE靶位点中心位置大于4个核苷酸。
82.根据权利要求70至81中任一项所述的方法,其中至少50%的缺失的缺失中心距所述HE靶位点中心位置大于4个核苷酸。
83.根据权利要求70至81中任一项所述的方法,其中至少55%的缺失的缺失中心距所述HE靶位点中心位置大于4个核苷酸。
84.根据权利要求70至81中任一项所述的方法,其中至少65%的缺失的缺失中心距所述HE靶位点中心位置大于4个核苷酸。
85.根据权利要求70至81中任一项所述的方法,其中至少70%的缺失的缺失中心距所述HE靶位点中心位置大于4个核苷酸。
86.根据权利要求70至81中任一项所述的方法,其中至少75%的缺失的缺失中心距所述HE靶位点中心位置大于4个核苷酸。
87.根据权利要求70至81中任一项所述的方法,其中至少80%的缺失的缺失中心距所述HE靶位点中心位置大于4个核苷酸。
88.根据权利要求70至87中任一项所述的方法,其中至少10%、至少11%、至少12%、至少13%、至少14%、至少15%、至少16%、至少17%、至少18%、至少19%或至少20%的缺失的缺失中心距所述HE靶位点中心位置大于8个核苷酸。
89.根据权利要求70至87中任一项所述的方法,其中至少10%的缺失的缺失中心距所述HE靶位点中心位置大于8个核苷酸。
90.根据权利要求70至87中任一项所述的方法,其中至少15%的缺失的缺失中心距所述HE靶位点中心位置大于8个核苷酸。
91.根据权利要求70至87中任一项所述的方法,其中至少20%的缺失的缺失中心距所述HE靶位点中心位置大于8个核苷酸。
92.根据权利要求70至87中任一项所述的方法,其中至少25%的缺失的缺失中心距所述HE靶位点中心位置大于8个核苷酸。
93.根据权利要求70至87中任一项所述的方法,其中至少30%的缺失的缺失中心距所述HE靶位点中心位置大于8个核苷酸。
94.根据权利要求70至87中任一项所述的方法,其中至少35%的缺失的缺失中心距所述HE靶位点中心位置大于8个核苷酸。
95.根据权利要求70至94中任一项所述的方法,其中至少50%、至少51%、至少52%、至少53%、至少54%、至少55%、至少56%、至少57%、至少58%、至少50%、至少59%或至少60%的缺失的长度为6bp或更长。
96.根据权利要求70至94中任一项所述的方法,其中至少50%的缺失的长度为6bp或更长。
97.根据权利要求70至94中任一项所述的方法,其中至少55%的缺失的长度为6bp或更长。
98.根据权利要求70至94中任一项所述的方法,其中至少60%的缺失的长度为6bp或更长。
99.根据权利要求70至94中任一项所述的方法,其中至少65%的缺失的长度为6bp或更长。
100.根据权利要求70至94中任一项所述的方法,其中至少70%的缺失的长度为6bp或更长。
101.根据权利要求70至94中任一项所述的方法,其中至少75%的缺失的长度为6bp或更长。
102.根据权利要求70至94中任一项所述的方法,其中至少80%的缺失的长度为6bp或更长。
103.根据权利要求70至102中任一项所述的方法,其中至少30%、至少31%、至少32%、至少33%、至少34%、至少35%、至少36%、至少37%、至少38%、至少39%或至少40%的缺失的长度为12bp或更长。
104.根据权利要求70至102中任一项所述的方法,其中至少35%的缺失的长度为12bp或更长。
105.根据权利要求70至102中任一项所述的方法,其中至少40%的缺失的长度为12bp或更长。
106.根据权利要求70至102中任一项所述的方法,其中至少45%的缺失的长度为12bp或更长。
107.根据权利要求70至102中任一项所述的方法,其中至少50%的缺失的长度为12bp或更长。
108.根据权利要求70至102中任一项所述的方法,其中至少55%的缺失的长度为12bp或更长。
109.根据权利要求70至102中任一项所述的方法,其中至少60%的缺失的长度为12bp或更长。
110.根据权利要求70至109中任一项所述的方法,其中所述定向偏向的缺失的长度包括约10个、约11个、约12个、约13个、约14个、约15个、约16个、约17个、约18个、约19个或约20个核苷酸。
111.根据权利要求70至110中任一项所述的方法,其中所述缺失延伸到所述DNA结合结构域靶位点中。
112.根据权利要求70至110中任一项所述的方法,其中所述缺失中心位置位于所述DNA结合结构域靶位点内。
113.根据权利要求70至112中任一项所述的方法,其中所述方法进一步包括向所述细胞中引入末端加工酶或其生物活性片段。
114.根据权利要求70至113中任一项所述的方法,其中所述末端加工酶或其生物活性片段选自由以下组成的组:Trex2、Trex1、不含跨膜结构域的Trex1、Apollo、Artemis、DNA2、ExoI、ExoT、ExoIII、ExoX、Fen1、Fan1、MreII、Rad2、Rad9、TdT(末端脱氧核苷酸转移酶)、PNKP、RecE、RecJ、RecQ、λ核酸外切酶、Sox、牛痘DNA聚合酶、核酸外切酶I、核酸外切酶III、核酸外切酶VII、NDK1、NDK5、NDK7、NDK8、WRN、T7-核酸外切酶基因6、禽类成髓细胞瘤病毒整合蛋白(IN)、Bloom、热敏磷酸酶(Antarctic Phosphatase)、碱性磷酸酶、多核苷酸激酶(PNK)、ApeI、绿豆核酸酶(Mung Bean nuclease)、Hex1、TTRAP(TDP2)、Sgs1、Sae2、CUP、Polμ、Polλ、MUS81、EME1、EME2、SLX1、SLX4以及UL-12。
115.根据权利要求114所述的方法,其中所述末端加工酶是核酸外切酶或其生物活性片段。
116.根据权利要求115所述的方法,其中所述核酸外切酶是Trex2或其生物活性片段。
117.根据权利要求70至116中任一项所述的方法,其中所述方法是体外方法。
118.根据权利要求70至116中任一项所述的方法,其中所述方法是离体方法。
119.根据权利要求70至116中任一项所述的方法,其中所述方法是体内方法。
120.一种治疗、预防或改善疾病或与其相关的病状的至少一种症状的方法,所述方法包括:从受试者采集细胞群体;根据权利要求70至119中任一项所述的方法编辑所述细胞群体;以及向所述受试者施用经编辑的细胞群体。
121.一种根据权利要求54至66中任一项所述的细胞用于治疗、预防或改善疾病或与其相关的病状的至少一种症状的用途。
122.一种根据权利要求67所述的细胞群体用于治疗、预防或改善疾病或与其相关的病状的至少一种症状的用途。
123.一种根据权利要求68或69所述的组合物用于治疗、预防或改善疾病或与其相关的病状的至少一种症状的用途。
124.根据权利要求120至123中任一项所述的方法或用途,其中所述疾病或病状是免疫病症或癌症。
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