申请/专利权人：许景焜;林隆志;许益华

申请日：2017-12-20

公开（公告）日：2024-04-30

公开（公告）号：CN110312803B

主分类号：C12N15/73

分类号：C12N15/73;C12N15/66;C12N15/90

优先权：["20161221 US 62/437,347"]

专利状态码：有效-授权

法律状态：2024.04.30#授权;2019.11.01#实质审查的生效;2019.10.08#公开

摘要：本公开提供一种用于在体外、离体或体内同时靶向核酸序列，并在细胞中提供内含子选择intronselection的组合物。该组合物包括一或多个核酸分子，每个核酸分子包含两端与封端序列cappingsequence相接的人工核酸序列，以及核酸外切酶λ‑核酸重组酶β蛋白质，或包含编码该核酸外切酶λ‑核酸重组酶β蛋白质的核酸序列的线性或环状载体，其中每个封端序列与位于该目标核酸序列中的区域为同源homologous，且该人工核酸序列为内含子序列。本公开还提供一种借由将该组合物引入细胞中以编辑目标核酸序列的方法。

主权项：1.一种用于编辑真核细胞中目标核酸序列的组合物，包括：a一或多个核酸分子，所述核酸分子各自包含两端与封端序列相接的人工核酸序列，其中，所述封端序列各自与位于所述目标核酸序列中的区域为同源，其中，所述核酸分子是双链DNA，以及其中，所述人工核酸序列是包括剪接供体位点、剪接受体位点、分支位点及选择标记的内含子序列，以及b核酸外切酶λ-核酸重组酶β蛋白质，或包含编码所述核酸外切酶λ-核酸重组酶β蛋白质的核酸序列的线性或环状载体，其中，所述载体包括可操作地连接至所述编码核酸外切酶λ-核酸重组酶β蛋白质的核酸序列的启动子。

全文数据：编辑核酸序列的组合物及方法技术领域本公开涉及一种在体外、离体或体内用于编辑细胞中目标基因组基因座genomiclocus的组合物及方法，更特定而言，涉及在真核细胞中用于重组工程、基因组修饰、基因敲入或基因敲除的组合物及方法。背景技术细菌基因组编辑已被充分地确立及应用于敲入敲除knockinknockout细菌基因，例如成簇规律间隔短回文重复序列clusteredregularlyinterspacedshortpalindromicrepeats,CRISPR相关蛋白质CRISPRCas、cre-lox系统及λ-Red系统。数十年来，cre-lox系统已应用于真核系统的基因修饰。源自噬菌体的cre-lox系统主要用于经基因修饰的动物。对于借由cre蛋白质识别及同源重组的外源片段及目标生物体，需要一个特殊的lox序列。植入片段与选择标记及或外源基因组合，并经lox序列双封端bi-capping的线性化DNA。为了重组工程，应事先在目标生物体基因组上创建特殊序列lox。CRISPRCas系统则完全不同于cre-lox系统，其衍生自细菌针对噬菌体的自我防卫及免疫记忆，如lox的特别序列是不必要的。用于CRISPRCas的机器为Cas蛋白质与引导RNAguidantRNA,gRNA所构成的核糖蛋白质复合物。该引导RNA携带短的同源区域大约17bp至24bp以专一性地靶向基因组。用于I型及II型CRISPR的核心Cas蛋白质Cas3及Cas9作为核糖酶，用以切割目标序列附近的DNA。此种机制被细菌及古细菌广泛采用。由于Cas-gRNA复合物的组合物，使衍生自聚球藻Synechococcussp.的II型CRISPR较为简单且更容易在其他生物系统中进行编辑。目前，cre-lox及CRISPRCas系统是在真核生物中进行基因组编辑的主要工具。但基于其机制的限制，使得遗传修饰既耗时且效率甚低。在基因转植动物中建立cre-lox系统以及在目标生物体上创建特殊序列lox是如此地困难，以致在大多数生物体中，位于预计位点的lox序列并不常见。相对地，CRISPRCas较为优越，并采用使用者设计的序列。CRISPRCas的gRNA靶向使用者设计的特定区域。但gRNA的序列太短而不能在整个基因组上定位，其将导致大量的非专一性靶向即所谓的脱靶效应off-targeteffect。再者，CRISPR在基因组编辑中的作用是通过引入由核心Cas蛋白质引起的双链断裂double-strandbreaking,DSB，以及借由非同源末端连接non-homologousendjoining,NHEJ整合外源片段而完成。但NHEJ的效率甚低，且经基因修饰的细胞无法直接有效地被选择，以致进一步限制了CRISPRCas的应用。λ-Red系统最初是在大肠杆菌E.coli的recA基因研究中被发现。于recA缺陷的大肠杆菌菌株中，在λ噬菌体中编码的系统显示重组效率增加了约100倍，而recA+的大肠杆菌菌株，其重组效率则显示下降。因此，该系统命名为“Red”Recombinationdefective重组缺陷，以区别于大肠杆菌宿主中的重组系统。该λ-Red系统包括α、β及γ蛋白质，其分别作用为核酸外切酶、单链DNAssDNA退火annealing蛋白质及RecBCD复合物的抑制剂。已知λ-Red重组包括三个主要步骤：1α-蛋白质从5’到3’分解双链DNAdsDNA；2β-蛋白质结合dsDNA的粘端stickyend；3借由RecA的侵入将单链DNAssDNA与目标序列退火。λ-Red不仅能够构建包含选择标记及或外源基因的大DNA片段，亦可借由在一或两个不同区域处同源的两个序列封盖该片段的两端而加以高度特定及重组。λ-Red重组工程已在大肠杆菌中用于单基因敲入敲除的可治愈质粒curableplasmid中构建。最近，其亦用于枯草芽孢杆菌B.subtilis的基因组编辑。另外，亦从其他噬菌体中发现类似的系统，并证实其在原核生物中的重组效率。但作为跨物种或多生物系统的基因组编辑的迫切要求，利用λ-Red及其相关噬菌体重组系统在真核细胞及生物体包括植物、动物及人类中提供更直接、有效及精确的基因组编辑方法仍是一个重要课题。发明内容鉴于上述课题，本公开提供一种在体外、离体或体内用于编辑细胞中的目标核酸序列的组合物，其包括：一或多个核酸分子，每个核酸分子包含两端与封端序列cappingsequence相接的人工核酸序列；以及核酸外切酶λ-核酸重组酶β蛋白质，或包含编码该核酸外切酶λ-核酸重组酶β蛋白质的核酸序列的载体。于本公开的一实施方式中，每个封端序列与目标核酸序列中的区域为同源homologous。目标核酸序列可为外显子exon或内含子intron，取决于目的，如敲入、敲除、框内插入in-frameinsertion、基因组修饰或重组工程。再者，人工核酸序列可为内含子序列。此外，该人工核酸序列可包含选择标记selectionmarker，其允许直接、有效且精确地选择基因组中的整合体integrant。另外，该载体可进一步包括可操作地连接至编码核酸外切酶λ-核酸重组酶β蛋白质的核酸序列的启动子，及选自于由编码核酸外切酶的核酸序列、编码抗RecBCD蛋白质的核酸序列及报道基因reportergene所组成的组中的至少一者。于本公开的一实施方式中，该组合物进一步包括核酸外切酶及抗RecBCD蛋白质中的至少一者。于本公开的另一实施方式中，该组合物与选自锌指核酸酶zinc-fingernucleases,ZFNs系统、类转录活化因子效应物核酸酶transcriptionactivator-likeeffectornucleases,TALENs系统及成簇规律间隔短回文重复序列CRISPRCas系统中的至少一者组合使用。根据另一实施方式，本公开进一步提供一种在体外、离体或体内用于编辑细胞中的目标核酸序列的方法。该方法包括：将上述组合物引入细胞中，以诱导于该目标部位中发生遗传改变。该方法进一步包括在适于诱导核酸分子及目标核酸序列之间的同源重组的条件下培养该细胞。于本公开的一实施方式中，引入细胞中或由细胞中的载体编码的核酸外切酶λ-核酸重组酶β蛋白质与核酸分子结合，并进一步促进核酸分子的封端序列与目标核酸序列中的区域之间的退火annealing，借以在细胞中形成重组体recombinant。此外，作为内含子序列的人工核酸序列将在目标核酸序列的RNA产物成熟期间借由RNA剪接而被除去，同时于细胞中发生遗传改变。于本公开的一实施方式中，可基于选择标记检测及克隆具有遗传改变的细胞。综上所述，本公开提供一种用于在体外、离体或体内同时靶向核酸序列，并在细胞中提供内含子选择包括选择标记的内含子的组合物。另外，本公开提供一种用于编辑细胞中的目标部位以进行诸如重组工程、基因组修饰、基因敲入及基因敲除的更直接、有效且精确的方法。此外，该细胞可为真核细胞，包括但不限于哺乳动物细胞，例如人类细胞及人类诱导性多能干细胞。附图说明本公开可借由参考附图并阅读下述实施例的详细描述，而更全面地理解的。图1A显示包含两端与封端序列相接的人工核酸序列的核酸分子的配置。图1B显示质粒pAB-mCherry的构建。图2A至图2C显示借由荧光显微镜术图2A或流式细胞测量术图2B观察到的使用GJB2-EX-AF及质粒pAB-mCherry以不同比率共转染人类HEK293T细胞48小时的结果图，并基于借由流式细胞测量术评估的EGFP阳性细胞数加以定量图2C。数据表示为平均值±sem，*pA突变。图4B是使用质粒pAB-mCherry及GJB2-EX35-AF或GJB2-EX109-AF共转染人类HEK293T细胞48小时的结果图。观察到具有eGFP绿色荧光的HEK293T细胞。比例尺：100μm。图4C显示源自不同组转染的HEK293T细胞的DNA，对EGFP基因进行PCR扩增的电泳结果，其中泳道M、1、2及3分别表示kb梯标、c.35delG组、媒液对照组及c.109GA组。图4D及图4E显示源自不同组转染的HEK293T细胞的DNA的GJB2基因的外显子2测序结果。图5A及图5B显示借由流式细胞测量术分析本公开的方法及CRISPRCas9nD10A系统图5A的GJB2基因c.35的基因编辑效率比较，并使用条形图表示图5B，其中a为媒介vehicle对照组，b为GJB2-EX35-AF组，c为GJB2-EX35-AF+质粒pAB组，以及d为GJB2-EX35-AF+CRISPRCas9nD10AR+L组。数据表示为平均值±sem，**pA的点突变，并使用DpnI消除原始质粒的模板，以获得质粒pQE-GJB2-EX35AF及pQE-GJB2-EX109AF。正常或含突变c.35delG或c.109GA的GJB2基因的外显子2分别如SEQIDNOs.3至5所示。为富集线性化的GJB2-EX-AF、GJB2-EX35-AF及GJB2-EX109-AF，源自质粒pQE-GJB2-EXAF、pQE-GJB2-EX35AF及pQE-GJB2-EX109AF的PCR扩增借由用于克隆GJB2基因的外显子1及2图4A的引物进行。GJB2-EX-AF、GJB2-EX35-AF及GJB2-EX109-AF的序列分别如SEQIDNOs.6至8所示。下述表1表示用于实施例1及2的引物序列。表1实施例3：质粒pAB-mCherry及GJB2-EX-AF至人类HEK293T细胞及iPSC细胞的共转染使用不同比例的pAB-mCherry及GJB2-EX-AF转染至人类HEK293T及iPSC细胞，并在6孔板中培养48小时。借由荧光显微镜术观察结果。再者，借由流式细胞测量术分析重组效率。荧光显微镜术的观察如图2A所示，大部分HEK293T细胞表达eGFP，其由包埋于GJB2-EX-AF人工内含子的EGFP所编码，并以CMV启动子驱动。再者，还观察到两个独立人类iPSC植株中eGFP的表达图3。因此，此种结果证实于GJB2-EX-AF与人类HEK293T细胞及iPS细胞中的目标部位之间发生重组。流式细胞测量术的分析使用流式细胞测量术进行定量分析以进一步评估重组效率。具体而言，所有的分析均使用SH800细胞分选系统Sony，其由马偕纪念医院台湾新北市的流式细胞仪核心设备提供。使用SH800软件Sony进行数据分析。根据前向角及侧向散射光圈选gating，将坏死细胞及碎片排除在分析之外。尽可能收集10,000个圈选事件进行分析。表2表示图2B及图2C显示不同比例的pAB-mCherry与GJB2-EX-AF的人类HEK293T细胞中的重组效率。表2此外，将经1μg的pAB-mCherry及0.12μg的GJB2-EX-AF转染的人类HEK293T细胞中的EGFP+mCherry+细胞分选，以验证GJB2-EX-AF整合入GJB2基因部位，并借由RT-PCR分析内源性GJB2mRNA的表达。如图2D所示，结果显示pAB重组工程系统并不会干扰HEK293T细胞的内源性GJB2基因mRNA的表达。实施例4：将GJB2-EX35-AF及GJB2-EX109-AF与质粒pAB-mCherry共转染至人类HEK293T细胞如实施例2所述，GJB2-EX35-AF及GJB2-EX109-AF设计为在GJB2基因的外显子2中分别含有c.35delG或c.109GA突变。于GJB2基因的c.35delG及c.109GA突变在高加索人的耳聋突变中占大多数，故已确定其等为听力损伤的重要遗传原因。将GJB2-EX35-AF及GJB2-EX109-AF分别与pAB-mCherry一起转染至HEK293T细胞48小时线性DNA：载体＝0.12μg：1μg，并借由荧光显微镜观察结果。如图4B所示，一些HEK293T细胞表达eGFP，其在GJB2-EX35-AF及GJB2-EX109-AF的人工内含子中编码，并借由CMV启动子驱动。此外，借由凝胶电泳确认源自经转染的HEK293T细胞的基因组DNA的PCR扩增EGFP基因。如图4C所示，泳道2是不含DNA样品的媒介对照组，且仅泳道1及3即c.35delG及c.109GA突变显示EGFP条带，其证明基因组编辑。此外，经转染的HEK293T细胞的基因组DNA借由DNA测序进一步证实。如图4D及图4E所示，测序结果显示GJB2基因的遗传改变确实发生在使用GJB2-EX35-AF或GJB2-EX109-AF转染的HEK293T细胞中。因此，此种结果证明于GJB2-EX35-AF及GJB2-EX109-AF与人类HEK293T细胞中的目标部位之间发生重组。另外，此种结果显示，本公开导致人类细胞中从突变型外显子至正常外显子的目标置换。类似地，可以理解的是，透过本公开的使用，可将特定疾病中的突变外显子替换为正常外显子。即证实本公开有用于基因组编辑及基因治疗。实施例5：pAB重组工程系统与CRISPRCas9nD10A的基因靶向效率的比较产生c.35突变的质粒经设计及构建成pCRISPRCas9nD10ALSEQIDNO.27及pCRISPRCas9nD10ARSEQIDNO.28，其由ColdSpringBiotechCorp台湾生产。合成pCRISPRCas9nD10AR及pCRISPRCas9nD10AL的gRNA，其序列分别如SEQIDNO.29及SEQIDNO.30所示。GJB2基因的c.35delG借由Cas9蛋白质与gRNA进行，其从质粒pCRISPRCas9nD10AR表达。亦使用NdeI降解启动子区域，以去活化gRNA及cas9基因的表达，借以构建作为阴性对照的无能力质粒pCRISPRCas9nD10ADNSEQIDNO.31。将人类HEK293T细胞与媒介vehicle处理以作为对照组，或与GJB2-EX35-AF及pAB或CRISPRCas9nD10AR+L共转染48小时。然后，使用流式细胞测量术观察并分析表达GFP的HEK293T细胞。进一步分析及分选GFP+细胞，以评估基因靶向的效率。如图5A及图5B所示，人类HEK293T细胞经a媒介、bGJB2-EX35-AF、cGJB2-EX35-AF及质粒pAB、或dGJB2-EX35-AF及CRISPRCas9nD10AR+L处理，其中pAB对于GJB2基因c.35delG突变的基因编辑效率为52.4％，显著高于CRISPRCas9nD10A的42.3％。根据上述实施例可知，本公开显示人工内含子EGFP报道基因与HEK293T细胞中的目标GJB2基因组基因座之间发生λ-Red重组。借由pAB重组工程系统在HEK293T细胞中成功编辑GJB2基因的耳聋基因突变c.35delG及c.109GA，并由dsDNAEGFP报道基因监测。上述数据表明，通过pAB重组工程系统可实现于HEK293T细胞中从野生型基因组序列至经设计的突变基因组序列的目标置换。因此，本公开的pAB重组工程系统借由利用dsDNAEGFP报道基因及FACS系统的结合而提供用于人类基因组编辑的高效且易于选择的平台。其可应用于体外及体内创建人类疾病模型，以促进发现疾病机制及药物开发。总而言之，本公开的pAB重组工程系统不仅在基础科学领域中，且在临床及再生医学领域中皆能促进精确及高效的人类基因组靶向编辑。上述为详细描述本公开的示例性实施例。但应理解，本公开的范围不限于所公开的实施例。反之，其意在涵盖各种修改及类似的重新排列。是以，权利要求应赋予最广泛的解释，以涵盖所有此种修改及类似的安排。序列表林隆志（Lin,Lung-Jr）许益华（Hsu,Yi-Hua）许景焜（Hsu,Ethan）编辑核酸序列的组合物及方法29649USUS62437,3472016-12-2141PatentInversion3.519536DNA人工序列pAB-mCherry1gacggatcgggagatctcccgatcccctatggtgcactctcagtacaatctgctctgatg60ccgcatagttaagccagtatctgctccctgcttgtgtgttggaggtcgctgagtagtgcg120cgagcaaaatttaagctacaacaaggcaaggcttgaccgacaattgcatgaagaatctgc180ttagggttaggcgttttgcgctgcttcgcgatgtacgggccagatatacgcgttgacatt240gattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatata300tggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacc360cccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttcc420attgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgt480atcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcatt540atgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtca600tcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttg660actcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcacc720aaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcg780gtaggcgtgtacggtgggaggtctatataagcagagctctctggctaactagagaaccca840ctgcttactggcttatcgaaattaatacgactcactatagggagacccaagctggctagc900gtttaaacttaagcttggtaccgagctcgccaccatgagtactgcactcgcaacgctggc960tgggaagctggctgaacgtgtcggcatggattctgtcgacccacaggaactgatcaccac1020tcttcgccagacggcatttaaaggtgatgccagcgatgcgcagttcatcgcattactgat1080cgttgccaaccagtacggccttaatccgtggacgaaagaaatttacgcctttcctgataa1140gcagaatggcatcgttccggtggtgggcgttgatggctggtcccgcatcatcaatgaaaa1200ccagcagtttgatggcatggactttgagcaggacaatgaatcctgtacatgccggattta1260ccgcaaggaccgtaatcatccgatctgcgttaccgaatggatggatgaatgccgccgcga1320accattcaaaactcgcgaaggcagagaaatcacggggccgtggcagtcgcatcccaaacg1380gatgttacgtcataaagccatgattcagtgtgcccgtctggccttcggatttgctggtat1440ctatgacaaggatgaagccgagcgcattgtcgaaaatactgcatacactgcagaacgtca1500gccggaacgcgacatcactccggttaacgatgaaaccatgcaggagattaacactctgct1560gatcgccctggataaaacatgggatgacgacttattgccgctctgttcccagatatttcg1620ccgcgacattcgtgcatcgtcagaactgacacaggccgaagcagtaaaagctcttggatt1680cctgaaacagaaagccgcagagcagaaggtggcagcatgatagtgagatccgcggccgca1740tagataactgatccagtgtgctggaattaattcgctgtctgcgagggccagctgttgggg1800tgagtactccctctcaaaagcgggcatgacttctgcgctaagattgtcagtttccaaaaa1860cgaggaggatttgatattcacctggcccgcggtgatgcctttgagggtggccgcgtccat1920ctggtcagaaaagacaatctttttgttgtcaagcttgaggtgtggcaggcttgagatctg1980gccatacacttgagtgacaatgacatccactttgcctttctctccacaggtgtccactcc2040caggtccaactgcaggtcgagcatgcatctagggcggccaattccgcccctctccccccc2100acccctctccctcccccccccctaacgttactggccgaagccgcttggaataaggccggt2160gtgcgtttgtctatatgttattttccaccatattgccgtcttttggcaatgtgagggccc2220ggaaacctggccctgtcttcttgacgagcattcctaggggtctttcccctctcgccaaag2280gaatgcaaggtctgttgaatgtcgtgaaggaagcagttcctctggaagcttcttgaagac2340aaacaacgtctgtagcgaccctttgcaggcagcggaaccccccacctggcgacaggtgcc2400tctgcggccaaaagccacgtgtataagatacacctgcaaaggcggcacaaccccagtgcc2460acgttgtgagttggatagttgtggaaagagtcaaatggctctcctcaagcgtattcaaca2520aggggctgaaggatgcccagaaggtaccccattgtatgggatctgatctggggcctcggt2580gcacatgctttacatgtgtttagtcgaggttaaaaaaacgtctaggccccccgaaccacg2640gggacgtggttttcctttgaaaaacacgatgataagcttgccacaacccacaaggagacg2700accttccgccaccatgacaccggacattatcctgcagcgtaccgggatcgatgtgagagc2760tgtcgaacagggggatgatgcgtggcacaaattacggctcggcgtcatcaccgcttcaga2820agttcacaacgtgatagcaaaaccccgctccggaaagaagtggcctgacatgaaaatgtc2880ctacttccacaccctgcttgctgaggtttgcaccggtgtggctccggaagttaacgctaa2940agcactggcctggggaaaacagtacgagaacgacgccagaaccctgtttgaattcacttc3000cggcgtgaatgttactgaatccccgatcatctatcgcgacgaaagtatgcgtaccgcctg3060ctctcccgatggtttatgcagtgacggcaacggccttgaactgaaatgcccgtttacctc3120ccgggatttcatgaagttccggctcggtggtttcgaggccataaagtcagcttacatggc3180ccaggtgcagtacagcatgtgggtgacgcgaaaaa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catgcacgtggcctaccggagacat322ProSerGlyHisAlaArgGlyLeuProGluThr10010563815DNA人工序列GJB2-EX-AFprim_转录1..38156ggggtgcggttaaaaggcgccacggcgggagacaggtgttgcggccccgcagcgcccgcg60cgctcctctccccgactcggagcccctcggcggcgcccggcccaggacccgcctaggagc120gcaggagccccagcgcagagaccccaacgccgagacccccgccccggccccgccgcgctt180cctcccgacgcaggtaggtaagttaggcagggatattcaccattatcgtttcagacccac240ctcccaaccccgaggggacccgacaggcccgaaggaatagaagaagaaggtggagagaga300gacagagacagatccattcgattagtgaacggatctcgacggtatcgatactagtattat360gcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatc420gctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgac480tcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaa540aatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggt600aggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccgtcagatcgcc660tggagacgccatccacgctgttttgacctccatagaagattctagaaccatggctagcgt720gagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcga780cgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaa840gctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgt900gaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagca960cgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaa1020ggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaa1080ccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagct1140ggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcat1200caaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgacca1260ctaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacct1320gagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgct1380ggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtgatccgt1440tcaactagcagaccgtttaaacaattcaagcttttttcaattctcgacctcgagacaaat1500ggcagtattcatccacaattttaaaagaaaaggggggattggggggtacagtgcagggga1560aagaatagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattac1620aaaaattcaaaattttcgggtttattacagggacagcagagatccagtttggccgcggct1680cgagggggttggggttgcgccttttccaaggcagccctgggtttgcgcagggacgcggct1740gctctgggcgtggttccgggaaacgcagcggcgccgaccctgggtctcgcacattcttca1800cgtccgttcgcagcgtcacccggatcttcgccgctacccttgtgggccccccggcgacgc1860ttcctgctccgcccctaagtcgggaaggttccttgcggttcgcggcgtgccggacgtgac1920aaacggaagccgcacgtctcactagtaccctcgcagacggacagcgccagggagcaatgg1980cagcgcgccgaccgcgatgggctgtggccaatagcggctgctcagcagggcgcgccgaga2040gcagcggccgggaaggggcggtgcgggaggcggggtgtggggcggtagtgtgggccctgt2100tcctgcccgcgcggtgttccgcattctgcaagcctccggagcgcacgtcggcagtcggct2160ccctcgttgaccgaatcaccgacctctctccccagggggatccaccggagcttaccatga2220ccgagtacaagcccacggtgcgcctcgccacccgcgacgacgtccccagggcggtacgca2280ccctcgccgccgcgttcgccgactaccccgccacgcgccacaccgtcgatccggaccgcc2340acatcgagcgggtcaccgagctgcaagaactcttcctcacgcgcgtcgggctcgacatcg2400gcaaggtgtgggtcgcggacgacggcgccgcggtggcggtctggaccacgccggagagcg2460tcgaagcgggggcggtgttcgccgagatcggcccgcgcatggccgagttgagcggttccc2520ggctggccgcgcagcaacagatggaaggcctcctggcgccgcaccggcccaaggagcccg2580cgtggttcctggccaccgtcggcgtctcgcccgaccaccagggcaagggtctgggcagcg2640ccgtcgtgctccccggagtggaggcggccgagcgcgccggggtgcccgccttcctggaga2700cctccgcgccccgcaacctccccttctacgagcggctcggcttcaccgtcaccgccgacg2760tcgaggtgcccgaaggaccgcgcacctggtgcatgacccgcaagcccggtgcctgacgcc2820cgccccacgacccgcagcgcccgaccgaaaggagcgcacgaccccatgcatcgttaagag2880ctcggtacctttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaa2940agaaaaggggggactggaagggctaattcactcccaacgaagacaagatctgctttttgc3000ttgtactgggtctctctggttagaccagatctgagcctgggagctctctcgctaactagg3060gaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccg3120tctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaat3180ctctagcagtagtagttcatgtcatcttattattcagtatttataacttgcaaagaaatg3240aatatcagagagtgagaggaacttgtttattgcagcttataatggttacaaataaagcaa3300tagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtc3360caaactcatcaatgtatcttatcatgtctggctctagctatcccgcccctaactccgccc3420atcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttt3480tttatttatgcagagcaaaccgcccagagtagaagatggattggggcacgctgcagacga3540tcctggggggtgtgaacaaacactccaccagcattggaaagatctggctcaccgtcctct3600tcatttttcgcattatgatcctcgttgtggctgcaaaggaggtgtggggagatgagcagg3660ccgactttgtctgcaacaccctgcagccaggctgcaagaacgtgtgctacgatcactact3720tccccatctcccacatccggctatgggccctgcagctgatcttcgtgtccacgccagcgc3780tcctagtggccatgcacgtggcctaccggagacat381573814DNA人工序列GJB2-EX35-AFprim_转录1..38147ggggtgcggttaaaaggcgccacggcgggagacaggtgttgcggccccgcagcgcccgcg60cgctcctctccccgactcggagcccctcggcggcgcccggcccaggacccgcctaggagc120gcaggagccccagcgcagagaccccaacgccgagacccccgccccggccccgccgcgctt180cctcccgacgcaggtaggtaagttaggcagggatattcaccattatcgtttcagacccac240ctcccaaccccgaggggacccgacaggcccgaaggaatagaagaagaaggtggagagaga300gacagagacagatccattcgattagtgaacggatctcgacggtatcgatactagtattat360gcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatc420gctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgac480tcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaa540aatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggt600aggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccgtcagatcgcc660tggagacgccatccacgctgttttgacctccatagaagattctagaaccatggctagcgt720gagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcga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权利要求：1.一种用于编辑目标核酸序列的组合物，包括：一或多个核酸分子，所述核酸分子各自包含两端与封端序列相接的人工核酸序列，其中，所述封端序列各自与位于所述目标核酸序列中的区域为同源，以及核酸外切酶λ-核酸重组酶β蛋白质，或包含编码所述核酸外切酶λ-核酸重组酶β蛋白质的核酸序列的线性或环状载体。2.根据权利要求1所述的组合物，其中，所述核酸分子是双链DNA。3.根据权利要求1所述的组合物，其中，所述人工核酸序列是内含子序列。4.根据权利要求3所述的组合物，其中，所述人工核酸序列包括剪接供体位点、剪接受体位点、分支位点、选择标记及其组合中的至少一者。5.根据权利要求4所述的组合物，其中，所述选择标记包括可操作地连接至报道基因的启动子。6.根据权利要求5所述的组合物，其中，所述启动子是组成型启动子、可诱导型启动子、或细胞或组织专一性启动子，以及所述报道基因是荧光报道基因、酶报道基因或抗生素选择基因。7.根据权利要求1所述的组合物，其中，位于所述目标核酸序列中的所述区域是外显子或内含子。8.根据权利要求1所述的组合物，进一步包括核酸外切酶及抗RecBCD蛋白质中的至少一者。9.根据权利要求1所述的组合物，其中，所述载体进一步包括可操作地连接至所述编码核酸外切酶λ-核酸重组酶β蛋白质的核酸序列的启动子。10.根据权利要求9所述的组合物，其中，所述载体包括选自编码核酸外切酶的核酸序列、编码抗RecBCD蛋白质的核酸序列及报道基因所组成的组中的至少一者。11.根据权利要求10所述的组合物，其中，所述启动子是组成型启动子、可诱导型启动子、或细胞或组织专一性启动子，以及所述报道基因是荧光报道基因、酶报道基因或抗生素选择基因。12.根据权利要求10所述的组合物，其中，所述载体沿5’至3’的下游方向包含：所述启动子、所述编码核酸外切酶λ-核酸重组酶β蛋白质的核酸序列、所述编码核酸外切酶的核酸序列及所述报道基因。13.根据权利要求1所述的组合物，其中，所述核酸分子以0.05μg至5μg的量存在。14.根据权利要求1所述的组合物，与选自锌指核酸酶、类转录活化因子效应物核酸酶及成簇规律间隔短回文重复序列Cas系统中的至少一者组合使用。15.一种编辑目标核酸序列的方法，包括：将根据权利要求1所述的组合物引入细胞中，以诱导所述目标核酸序列中发生遗传改变。16.根据权利要求15所述的方法，进一步包括检测具有所述遗传改变的所述细胞。17.根据权利要求15所述的方法，其中，所述目标核酸序列的编辑选自重组工程、基因组修饰、基因敲入及基因敲除所组成的组中的至少一者。18.根据权利要求15所述的方法，其中，所述细胞是真核细胞。19.根据权利要求18所述的方法，其中，所述真核细胞是哺乳动物细胞。20.根据权利要求19所述的方法，其中，所述哺乳动物细胞是人类细胞。

百度查询： 许景焜;林隆志;许益华 编辑核酸序列的组合物及方法

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