CN114606265B - 一种能够实现单个aav病毒包被的迷你碱基编辑器 - Google Patents
一种能够实现单个aav病毒包被的迷你碱基编辑器 Download PDFInfo
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Abstract
本发明公开了一种能够实现单个AAV病毒包被的迷你碱基编辑器，提供了一种可单个AAV包装的miniABE与miniABE*，miniABE具较大的编辑窗口A3‑A15，miniABE*具有较窄的编辑窗口主要集中于A5，A6；解决了基于SpCas9的ABE系统无法实现单一AAV包被的技术缺点；二者是互补的关系，可根据编辑位置进行合理的版本选择，当致病基因位于距离PAM较近的时候可选择miniABE，当需要较为精确时可以选miniABE*；两种碱基编辑系统为基因治疗奠定了基础，可以用于制备基因治疗药物。
Description
技术领域
本发明公开了一种能够实现单个AAV病毒包被的迷你碱基编辑器，包括TadA-8e片段和SpCas9缺失变体片段，开发出编辑窗口大的miniABE以及编辑窗口小的miniABE*，能够实现单个AAV病毒包装，更利于基因治疗，具有良好的应用前景，属于生物技术领域。
背景技术
CRISPR/Cas9技术在目标位点引入DNA双链断裂（DSB）对生物医药领域起到革命性作用，通过修改或纠正有害基因来促进遗传病的治疗。在sgRNA引导下，Cas9蛋白能够到达指定区域并发挥核酸酶活性进行DNA双链切割。但直接使用Cas9系统往往难以实现精确编辑。基于CRISPR技术上开发出更为精确的单碱基编辑系统（ABE和CBE），可以实现四种碱基类型的转换（C-T，G-A，A-G和T-C）。已知的疾病相关的基因突变中有一半是点突变，原则上近一半的致病位点突变可以通过将A·T碱基对转化成G·C碱基对来纠正，因此ABEs对于致病等位基因的纠正特别有用。但最常用的SpCas9有1368个氨基酸组成，4104 bp。基于SpCas9的ABEs及其sgRNA远远超过了AAV载体的限制。腺相关病毒（AAV）是体内给药的最佳载体，但AAV的有效载荷能力有限（一般小于5kb）。
为了绕过AAV的有效荷载限制，将SpCas9拆成两部分并与sgRNA表达单元一起包装成两个独立的AAV，通过两个AAV中的反向末端重复（ITR）序列的重组反应或内含子介导的蛋白质反式剪接从而允许全长的SpCas9在体内重新组装。然而，与单一载体系统相比，双载体系统通常需要更高的病毒剂量，生产成本更高，难以按化学计量学进行滴定，而且操作起来也很麻烦。另一种途径是开发尺寸更小的Cas9，如SaCas9（3.2 kb）、CasX（3kb）以及CjCas9等。但结构紧凑的Cas9效率不如SpCas9，且具有更加严格的PAM。因此开发基于SpCas9的ABE载体大小缩小到AAV载体限制，有助于我们对碱基编辑器的应用，特别是在医疗领域对相关疾病进行精准基因治疗方面。
发明内容
本发明目的在于提供一种碱基编辑工具及其用途，尤其是公开一种能够实现单个AAV病毒包被的迷你碱基编辑器，解决了SpCas9的ABE系统无法实现单一AAV包被的技术缺点。
本发明公开了一种能够实现单个AAV病毒包被的迷你碱基编辑器，包括TadA-8e和SpCas9缺失变体的两种迷你碱基编辑器；其中，
TadA-8e与SpCas9缺失REC2结构域第174-296位氨基酸以及HNH结构第786-885位氨基酸构建的载体命名为miniABE，核苷酸序列如SEQIDNO.1
TadA-8e与SpCas9缺失REC2结构域第174-296位氨基酸，HNH结构第781-905位氨基酸以及TadA-8e与SpCas9的连接linker构建的载体命名为miniABE*，核苷酸序列如SEQ IDNO.2。
本发明所述的一种能够实现单个AAV病毒包被的迷你碱基编辑器，其特征在于：
包括两种AAV载体可用于AAV包被；其中，
AAV-miniABE载体核苷酸序列如SEQIDNO.3；
AAV-miniABE*载体核苷酸序列如SEQIDNO.4；
本发明所说的一种能够实现单个AAV病毒包被的迷你碱基编辑器在制备基因治疗药物中的医用用途。
本发明所说的一种能够实现单个AAV病毒包被的迷你碱基编辑器在可用于破坏小鼠PCSK9基因的用途。
本发明所说的一种能够实现单个AAV病毒包被的迷你碱基编辑器，包括以下步骤：
miniABE的构建与HEK293FT细胞验证编辑效率，将ABE8e中SpCas9缺失REC2结构域第174-296位氨基酸以及HNH结构域第786-885位氨基酸得到miniABE，在HEK293FT细胞中六个位点验证了miniABE系统的编辑效率，编辑效率在10-43%，其中SiteV位点在A15仍然保持较高的编辑效率13%。
miniABE*的构建与HEK239FT细胞验证编辑效率将ABE8e中SpCas9缺失REC2结构域第174-296位氨基酸，HNH结构域第781-905位氨基酸以及TadA-8e与SpCas9的连接linker得到miniABE*，在HEK293FT细胞中六个位点验证了miniABE系统的编辑效率，编辑效率在5-42%，且miniABE*编辑窗口明显缩小，A5-A6效率较高。
miniABE与miniABE*编辑窗口对比，miniABE与miniABE*编辑窗口对比，miniABE编辑窗口较大，A3-A15都有编辑，miniABE*窗口较小，集中于A5-A6；
miniABE与miniABE*AAV载体构建与N2a细胞转染验证，在小鼠PCSK9基因的4个位点进行验证，AAV-miniABE效率为9-21.5%，AAV-miniABE*效率为6.5-19.5%；
AAV-miniABE-P5与AAV-miniABE*-P5 AAV包装。
AAV-miniABE-P5小鼠体内验证，4周龄小鼠注射病毒1个月后肝脏编辑效率最高达1.2%。
本发明的积极效果在于：公开了一种可单个AAV包装的miniABE与miniABE*，miniABE具较大的编辑窗口A3-A15，miniABE*具有较窄的编辑窗口主要集中于A5，A6；解决了基于SpCas9的ABE系统无法实现单一AAV包被的技术缺点；二者是互补的关系，可根据编辑位置进行合理的版本选择，当致病基因位于距离PAM较近的时候可选择miniABE，当需要较为精确时可以选miniABE*；两种碱基编辑系统为基因治疗奠定了基础，可以用于制备基因治疗药物。
附图说明
图1为本发明迷你碱基编辑器示意图及细胞编辑效果图（a miniABE载体示意图，b miniABE*载体示意图，c miniABE和miniABE*HEK293FT细胞转染效率）；
图2为本发明miniABE和miniABE*编辑窗口对比图；
图3为本发明AAV载体示意图及编辑效率图（a AAV-miniABE和AAV-miniABE*载体图，b N2a细胞编辑效率图）；
图4为本发明AAV PAGE图（a AAV-miniABE-P5 AAV包装PAGE图，b AAV-miniABE*-P5 AAV包装PAGE图）；
图5为本发明AAV-miniABE-P5小鼠体内验证图（a 体内注射示意图，b AAV-miniABE-P5小鼠肝脏编辑效率图）。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。若未特别指明，实施例中所用的技术手段为本领域人员所熟知的常规手段。下述实施例中所使用的材料、试剂等如无特殊说明，均可从商业途径得到。
实施例1
1.1 miniABE载体构建
PCR将addgene来源的ABE8e（#138489）使用NEB Q5高保真聚合酶PCR成两个片段，引物为：
Fragment1F：aacatcgtgatcgaacggcagatcacaaagcacg；
Fragment1R：aaattttgctcgcgcactactcagcga；
Vector1F：gcgcgagcaaaatttaagctacaaca；
Vector1R：tcgatcacgatgttctcgggc；
之后将PCR产物进行凝胶电泳，切割Fragment1 4050，Vector1 3708条带，使用天根凝胶回收试剂盒进行胶回收；将胶回收片段1:1使用诺唯赞无缝克隆试剂盒进行重组，重组程序为50℃ 30min。随后将重组产物使用天根DH5α感受态细胞进行转化，过程为（冰浴30min，热激90s，冰浴3min，涂板）。得到单克隆以后挑菌测序，得到克隆1，随后将克隆1继续用
Fragment2F：atcaagttccggggcctgagcgcctctatgatcaag；
Fragment2R：Gcgccacgcttcccgaaggagaaagg；
Vector2F: cgggaagcgtggcgc；
Vector2R:gccccggaacttgatcatgtg；
进行PCR，胶回收，重组得到miniABE（图1a），测序正确后采用天根大提试剂盒进行质粒提取，miniABE序列如SEQIDNO.1。
1.2 sgRNA载体构建
选择6个内源基因位点，根据目标序列设计sgRNA并合成oligos，所用到的sgRNA序列如表1所示。在每个sgRNA的上游序列5’端加cacc序列接头，下游序列的5’加aaac序列接头。经合成以后，上下游通过程序（95℃ 5min室温冷却30min）退火，将退火产物连接到进BbsI酶切的74707 sgRNA骨架载体，随后经过转化，挑单克隆以及测序得到正确的sgRNA，并通过大提得到高纯度质粒。
表1. HEK293FT细胞sgRNA序列
1.3细胞培养与转染
将HEK293FT细胞接种于10% FBS的DMEM培养基中，其中含有1%双抗，在含有5% CO2的37℃细胞培养箱中进行培养。用于转染的细胞，前一天接种于6孔板中进行培养，第二天观察细胞，当细胞密度为75%左右时，用含10% FBS，不含双抗的培养基换液，使用HieffTransTM Liposomal Transfection Reagent进行转染，转染时6孔板每孔的量分别是miniABE 2μg，sgRNA 1μg。将质粒混合后用250ulOpti-MEM稀释并混匀作为试剂A。同时，将9ul的Hieff转染试剂与250μl的Opti-MEM稀释并混匀作为试剂B，静止5min。将上述试剂A与B混合，吹打混匀，静止20min后逐滴加入待转染的6孔板细胞中，放回37℃培养箱培养，12h后换一次新鲜培养基；48h后收集细胞，然后用细胞裂解液裂解（诺唯赞One Step MouseGenotyping Kit）并鉴定基因型。
1.4 miniABE在内源基因位点编辑效率检测
根据实验需求设计引物，将上述裂解产物为模板，对靶位点附近序列进行PCR扩增，将扩增产物凝胶电泳检测目的条带后，送上海生工公司进行一代测序。用于目标位点PCR扩增体系如下：2×taq（天根）12.5μl，F（10pmol/μl）1μl，R（10pmol/μl）1μl，模板1μl，ddH2O补齐到25μl。测序结束后将测序结果用EditR分析网站进行效率分析，检测结果如图1c所示。
实施例2
2.1 miniABE*载体构建
与方法1.1所述，将miniABE使用；
Fragment3F：cagagctccatcaacgacaagaagtacagcatcggcc；
Franment3R:cttatcagctccctcgtcacctcccag；
Vector3F:gagggagctgataagcgcac；
Vector3R:gttgatggagctctgggcct；
进行PCR扩增，胶回收重组，转化，测序得到克隆2，再将克隆2使用：
Fragment4F：aacagccgcgagagaggcggcctgagc；
Fragment4R：gagctgaatgaagccataccaaacgac；
Vector4F：ggcttcattcagctccggttc；
Vector4R：tctctcgcggctgttcttctg；
进行PCR扩增，胶回收，重组，转化，测序，大提质粒得到miniABE*（图1b），序列如SEQIDNO.2所示。
2.2 细胞培养与转染
与1.3所述一致。
2.3 miniABE*在内源性基因位点编辑效率检测
与1.4所述一致，结果如图1c所示。
2.4 miniABE与miniABE*编辑窗口对比
按照1.2所述，构建含有不同A的sgRNA，序列如表2所示，按1.3,1.4转染细胞，并评估编辑效率，统计A3-A15的编辑效率，得到miniABE和miniABE*的大致窗口范围，如图2所示。
表2.窗口评估细胞sgRNA序列
实施例3
3.1 AAV-miniABE载体构建
由金斯瑞生物公司合成miniCMV以及短Poly（A）替换miniABE中的CMV启动子及BGHPA序列得到克隆3，并将U6启动子驱动的sgRNA序列重组在克隆3上得到克隆4.之后使用：
Fragment5F：ggtacaattcacgcggactcacggggatttccaag；
Fragment5R：ggggttcctgcggccaaaaaaagcaccgactcggt；
PCR扩增克隆4并连接到经MluI和NotI酶切的AAV骨架载体上，转化，测序，提取DNA后得到AAV-miniABE如图3a上所示，序列见SEQIDNO.3。
3.2 AAV-miniABE*载体构建
由金斯瑞生物公司合成miniCMV以及短Poly（A）序列替换miniABE*中的CMV启动子及BGH PA序列得到克隆5，并将U6启动子驱动的sgRNA序列重组在克隆3上得到克隆6.之后使用：
Fragment5F：ggtacaattcacgcggactcacggggatttccaag；
Fragment5R：ggggttcctgcggccaaaaaaagcaccgactcggt；
PCR扩增克隆6并连接到经MluI和NotI酶切的AAV骨架载体上，转化，测序，提取DNA后得到AAV-miniABE*如图3a下所示，序列见SEQIDNO.4。
3.3 Pcsk9sgRNA构建
选择5个小鼠PCSK9基因位点，根据目标序列设计sgRNA并合成oligos，所用到的sgRNA序列如表3所示。按照1.2所述将sgRNA连接到BbsI酶切后的AAV-miniABE和AAV-miniABE*载体中，随后通过DH5α转化，挑菌，测序得到正确连接的sgRNA，并通过大提质粒得到高纯度的质粒。
表3. N2a细胞sgRNA序列
3.4 细胞培养与转染
将N2a细胞接种于10% FBS的DMEM培养基中，其中含有1%双抗，在含有5% CO2的37℃细胞培养箱中进行培养。用于转染的细胞，前一天接种于6孔板中进行培养，第二天观察细胞，当细胞密度为75%左右时，用含10% FBS，不含双抗的培养基换液，使用HieffTransTMLiposomal Transfection Reagent进行转染，转染时6孔板每孔的量分别是AAV载体3μg。将质粒混合后用250ul Opti-MEM稀释并混匀作为试剂A。同时，将9ul的Hieff转染试剂与250μl的Opti-MEM稀释并混匀作为试剂B，静止5min。将上述试剂A与B混合，吹打混匀，静止20min后逐滴加入待转染的6孔板细胞中，放回37℃培养箱培养，12h后换一次新鲜培养基。48h后收集细胞，然后用细胞裂解液裂解（诺唯赞One Step Mouse Genotyping Kit）并鉴定基因型。
3.4 AAV载体在内源基因位点编辑效率检测
方法通1.4所述，经过比较发现P5位点效率最高如图3b所示，因此将上述AAV-miniABE-P5与AAV-miniABE*-P5的两种载体进行AAV包装。
实施例4
将AAV-miniABE-P5送至派真生物公司进行包装，使用qPCR检测滴度，使用SDS-PAGE检测纯度图4a。将AAV-miniABE*-P5送至吉满生物公司进行AAV包被，使用SDS-PAGE检测纯度图4b，同样检测滴度。
包装滴度分别如表4：
表4. AAV生产滴度
实施例5
5.1 AAV病毒注射
将AAV包被的miniABE病毒颗粒置于冰上解冻，用胰岛素注射针吸取病毒，尾静脉注射8周龄的雌鼠三只，每只的注射量大约为4E+12GCs的病毒，同样给与对照组小鼠注射相同的量的PBS，如图5a。
5.2 编辑效率检测
1个月后，将小鼠处死，取肝脏组织进行基因组提取，随后将目的基因PCR扩增，PCR引物加深度测序接头，将产物送杭州水稻所进行深度测序，计算突变比例如图5b所示。
综上所述，本发明有效克服了现有技术无法满足AAV包装的缺点，制备了两个窗口互补的迷你碱基编辑器，并成功用于AAV的包装，并将AAV注射到小鼠体内后产生一定编辑效果，上述迷你碱基编辑器具有高度产业利用价值。
上述实施例仅例示性说明本发明的原理及其功效，而非用于限制本发明。任何熟悉此技术的人士皆可在不违背本发明的精神及范畴下，对上述实施例进行修饰或改变。因此，举凡所属技术领域中具有通常知识者在未脱离本发明所揭示的精神与技术思想下所完成的一切等效修饰或改变，仍应由本发明的权利要求所涵盖。
序列表
<110> 吉林大学
<120> 一种能够实现单个AAV病毒包被的迷你碱基编辑器
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 4178
<212> DNA
<213> miniABE(人工合成)
<400> 1
atgaaacgga cagccgacgg aagcgagttc gagtcaccaa agaagaagcg gaaagtctct 60
gaggtggagt tttcccacga gtactggatg agacatgccc tgaccctggc caagagggca 120
cgggatgaga gggaggtgcc tgtgggagcc gtgctggtgc tgaacaatag agtgatcggc 180
gagggctgga acagagccat cggcctgcac gacccaacag cccatgccga aattatggcc 240
ctgagacagg gcggcctggt catgcagaac tacagactga ttgacgccac cctgtacgtg 300
acattcgagc cttgcgtgat gtgcgccggc gccatgatcc actctaggat cggccgcgtg 360
gtgtttggcg tgaggaactc aaaaagaggc gccgcaggct ccctgatgaa cgtgctgaac 420
taccccggca tgaatcaccg cgtcgaaatt accgagggaa tcctggcaga tgaatgtgcc 480
gccctgctgt gcgatttcta tcggatgcct agacaggtgt tcaatgctca gaagaaggcc 540
cagagctcca tcaactccgg aggatctagc ggaggctcct ctggctctga gacacctggc 600
acaagcgaga gcgcaacacc tgaaagcagc gggggcagca gcggggggtc agacaagaag 660
tacagcatcg gcctggccat cggcaccaac tctgtgggct gggccgtgat caccgacgag 720
tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca cagcatcaag 780
aagaacctga tcggagccct gctgttcgac agcggcgaaa cagccgaggc cacccggctg 840
aagagaaccg ccagaagaag atacaccaga cggaagaacc ggatctgcta tctgcaagag 900
atcttcagca acgagatggc caaggtggac gacagcttct tccacagact ggaagagtcc 960
ttcctggtgg aagaggataa gaagcacgag cggcacccca tcttcggcaa catcgtggac 1020
gaggtggcct accacgagaa gtaccccacc atctaccacc tgagaaagaa actggtggac 1080
agcaccgaca aggccgacct gcggctgatc tatctggccc tggcccacat gatcaagttc 1140
cggggccact tcctgatcga gggcgactcc gacgccatcc tgctgagcga catcctgaga 1200
gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag atacgacgag 1260
caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac 1320
aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacattga cggcggagcc 1380
agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga cggcaccgag 1440
gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac cttcgacaac 1500
ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg gcggcaggaa 1560
gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct gaccttccgc 1620
atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg gatgaccaga 1680
aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa gggcgcttcc 1740
gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa cgagaaggtg 1800
ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt ataacgagct gaccaaagtg 1860
aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca gaaaaaggcc 1920
atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac 1980
tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga agatcggttc 2040
aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg 2100
gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac actgtttgag 2160
gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga cgacaaagtg 2220
atgaagcagc tgaagcggcg gagatacacc ggctggggca ggctgagccg gaagctgatc 2280
aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa gtccgacggc 2340
ttcgccaaca gaaacttcat gcagctgatc cacgacgaca gcctgacctt taaagaggac 2400
atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat tgccaatctg 2460
gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt ggacgagctc 2520
gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc cagagagaac 2580
cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat cgaactgctg 2640
aacgccaagc tgattaccca gagaaagttc gacaatctga ccaaggccga gagaggcggc 2700
ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac ccggcagatc 2760
acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga cgagaatgac 2820
aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc cgatttccgg 2880
aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc ccacgacgcc 2940
tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctaagct ggaaagcgag 3000
ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa gagcgagcag 3060
gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa ctttttcaag 3120
accgagatta ccctggccaa cggcgagatc cggaagcggc ctctgatcga gacaaacggc 3180
gaaaccgggg agatcgtgtg ggataagggc cgggattttg ccaccgtgcg gaaagtgctg 3240
agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa 3300
gagtctatcc tgcccaagag gaacagcgat aagctgatcg ccagaaagaa ggactgggac 3360
cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct ggtggtggcc 3420
aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct ggggatcacc 3480
atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc caagggctac 3540
aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt cgagctggaa 3600
aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa cgaactggcc 3660
ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa gctgaagggc 3720
tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaagca ctacctggac 3780
gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga cgctaatctg 3840
gacaaagtgc tgtccgccta caacaagcac cgggataagc ccatcagaga gcaggccgag 3900
aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt caagtacttt 3960
gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga cgccaccctg 4020
atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca gctgggaggt 4080
gacgagggag ctgataagcg caccgccgat ggttccgagt tcgaaagccc caagaagaag 4140
aggaaagtct aaccggtcat catcaccatc accattga 4178
<210> 2
<211> 4007
<212> DNA
<213> miniABE*(人工合成)
<400> 2
atgaaacgga cagccgacgg aagcgagttc gagtcaccaa agaagaagcg gaaagtctct 60
gaggtggagt tttcccacga gtactggatg agacatgccc tgaccctggc caagagggca 120
cgggatgaga gggaggtgcc tgtgggagcc gtgctggtgc tgaacaatag agtgatcggc 180
gagggctgga acagagccat cggcctgcac gacccaacag cccatgccga aattatggcc 240
ctgagacagg gcggcctggt catgcagaac tacagactga ttgacgccac cctgtacgtg 300
acattcgagc cttgcgtgat gtgcgccggc gccatgatcc actctaggat cggccgcgtg 360
gtgtttggcg tgaggaactc aaaaagaggc gccgcaggct ccctgatgaa cgtgctgaac 420
taccccggca tgaatcaccg cgtcgaaatt accgagggaa tcctggcaga tgaatgtgcc 480
gccctgctgt gcgatttcta tcggatgcct agacaggtgt tcaatgctca gaagaaggcc 540
cagagctcca tcaacgacaa gaagtacagc atcggcctgg ccatcggcac caactctgtg 600
ggctgggccg tgatcaccga cgagtacaag gtgcccagca agaaattcaa ggtgctgggc 660
aacaccgacc ggcacagcat caagaagaac ctgatcggag ccctgctgtt cgacagcggc 720
gaaacagccg aggccacccg gctgaagaga accgccagaa gaagatacac cagacggaag 780
aaccggatct gctatctgca agagatcttc agcaacgaga tggccaaggt ggacgacagc 840
ttcttccaca gactggaaga gtccttcctg gtggaagagg ataagaagca cgagcggcac 900
cccatcttcg gcaacatcgt ggacgaggtg gcctaccacg agaagtaccc caccatctac 960
cacctgagaa agaaactggt ggacagcacc gacaaggccg acctgcggct gatctatctg 1020
gccctggccc acatgatcaa gttccggggc cacttcctga tcgagggcga ctccgacgcc 1080
atcctgctga gcgacatcct gagagtgaac accgagatca ccaaggcccc cctgagcgcc 1140
tctatgatca agagatacga cgagcaccac caggacctga ccctgctgaa agctctcgtg 1200
cggcagcagc tgcctgagaa gtacaaagag attttcttcg accagagcaa gaacggctac 1260
gccggctaca ttgacggcgg agccagccag gaagagttct acaagttcat caagcccatc 1320
ctggaaaaga tggacggcac cgaggaactg ctcgtgaagc tgaacagaga ggacctgctg 1380
cggaagcagc ggaccttcga caacggcagc atcccccacc agatccacct gggagagctg 1440
cacgccattc tgcggcggca ggaagatttt tacccattcc tgaaggacaa ccgggaaaag 1500
atcgagaaga tcctgacctt ccgcatcccc tactacgtgg gccctctggc caggggaaac 1560
agcagattcg cctggatgac cagaaagagc gaggaaacca tcaccccctg gaacttcgag 1620
gaagtggtgg acaagggcgc ttccgcccag agcttcatcg agcggatgac caacttcgat 1680
aagaacctgc ccaacgagaa ggtgctgccc aagcacagcc tgctgtacga gtacttcacc 1740
gtgtataacg agctgaccaa agtgaaatac gtgaccgagg gaatgagaaa gcccgccttc 1800
ctgagcggcg agcagaaaaa ggccatcgtg gacctgctgt tcaagaccaa ccggaaagtg 1860
accgtgaagc agctgaaaga ggactacttc aagaaaatcg agtgcttcga ctccgtggaa 1920
atctccggcg tggaagatcg gttcaacgcc tccctgggca cataccacga tctgctgaaa 1980
attatcaagg acaaggactt cctggacaat gaggaaaacg aggacattct ggaagatatc 2040
gtgctgaccc tgacactgtt tgaggacaga gagatgatcg aggaacggct gaaaacctat 2100
gcccacctgt tcgacgacaa agtgatgaag cagctgaagc ggcggagata caccggctgg 2160
ggcaggctga gccggaagct gatcaacggc atccgggaca agcagtccgg caagacaatc 2220
ctggatttcc tgaagtccga cggcttcgcc aacagaaact tcatgcagct gatccacgac 2280
gacagcctga cctttaaaga ggacatccag aaagcccagg tgtccggcca gggcgatagc 2340
ctgcacgagc acattgccaa tctggccggc agccccgcca ttaagaaggg catcctgcag 2400
acagtgaagg tggtggacga gctcgtgaaa gtgatgggcc ggcacaagcc cgagaacatc 2460
gtgatcgaaa tggccagaga gaaccagacc acccagaagg gacagaagaa cagccgcgag 2520
agaggcggcc tgagcgaact ggataaggcc ggcttcatca agagacagct ggtggaaacc 2580
cggcagatca caaagcacgt ggcacagatc ctggactccc ggatgaacac taagtacgac 2640
gagaatgaca agctgatccg ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc 2700
gatttccgga aggatttcca gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc 2760
cacgacgcct acctgaacgc cgtcgtggga accgccctga tcaaaaagta ccctaagctg 2820
gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat gatcgccaag 2880
agcgagcagg aaatcggcaa ggctaccgcc aagtacttct tctacagcaa catcatgaac 2940
tttttcaaga ccgagattac cctggccaac ggcgagatcc ggaagcggcc tctgatcgag 3000
acaaacggcg aaaccgggga gatcgtgtgg gataagggcc gggattttgc caccgtgcgg 3060
aaagtgctga gcatgcccca agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc 3120
ttcagcaaag agtctatcct gcccaagagg aacagcgata agctgatcgc cagaaagaag 3180
gactgggacc ctaagaagta cggcggcttc gacagcccca ccgtggccta ttctgtgctg 3240
gtggtggcca aagtggaaaa gggcaagtcc aagaaactga agagtgtgaa agagctgctg 3300
gggatcacca tcatggaaag aagcagcttc gagaagaatc ccatcgactt tctggaagcc 3360
aagggctaca aagaagtgaa aaaggacctg atcatcaagc tgcctaagta ctccctgttc 3420
gagctggaaa acggccggaa gagaatgctg gcctctgccg gcgaactgca gaagggaaac 3480
gaactggccc tgccctccaa atatgtgaac ttcctgtacc tggccagcca ctatgagaag 3540
ctgaagggct cccccgagga taatgagcag aaacagctgt ttgtggaaca gcacaagcac 3600
tacctggacg agatcatcga gcagatcagc gagttctcca agagagtgat cctggccgac 3660
gctaatctgg acaaagtgct gtccgcctac aacaagcacc gggataagcc catcagagag 3720
caggccgaga atatcatcca cctgtttacc ctgaccaatc tgggagcccc tgccgccttc 3780
aagtactttg acaccaccat cgaccggaag aggtacacca gcaccaaaga ggtgctggac 3840
gccaccctga tccaccagag catcaccggc ctgtacgaga cacggatcga cctgtctcag 3900
ctgggaggtg acgagggagc tgataagcgc accgccgatg gttccgagtt cgaaagcccc 3960
aagaagaaga ggaaagtcta accggtcatc atcaccatca ccattga 4007
<210> 3
<211> 7272
<212> DNA
<213> AAV-miniABE(人工合成)
<400> 3
gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac 60
caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc 120
ggtaggcgtg tacggtggga ggtctatata agcagagctc gtttagtgaa ccgtcagatc 180
gaccggttct agagcgctgc caccatgaaa cggacagccg acggaagcga gttcgagtca 240
ccaaagaaga agcggaaagt ctctgaggtg gagttttccc acgagtactg gatgagacat 300
gccctgaccc tggccaagag ggcacgggat gagagggagg tgcctgtggg agccgtgctg 360
gtgctgaaca atagagtgat cggcgagggc tggaacagag ccatcggcct gcacgaccca 420
acagcccatg ccgaaattat ggccctgaga cagggcggcc tggtcatgca gaactacaga 480
ctgattgacg ccaccctgta cgtgacattc gagccttgcg tgatgtgcgc cggcgccatg 540
atccactcta ggatcggccg cgtggtgttt ggcgtgagga actcaaaaag aggcgccgca 600
ggctccctga tgaacgtgct gaactacccc ggcatgaatc accgcgtcga aattaccgag 660
ggaatcctgg cagatgaatg tgccgccctg ctgtgcgatt tctatcggat gcctagacag 720
gtgttcaatg ctcagaagaa ggcccagagc tccatcaact ccggaggatc tagcggaggc 780
tcctctggct ctgagacacc tggcacaagc gagagcgcaa cacctgaaag cagcgggggc 840
agcagcgggg ggtcagacaa gaagtacagc atcggcctgg ccatcggcac caactctgtg 900
ggctgggccg tgatcaccga cgagtacaag gtgcccagca agaaattcaa ggtgctgggc 960
aacaccgacc ggcacagcat caagaagaac ctgatcggag ccctgctgtt cgacagcggc 1020
gaaacagccg aggccacccg gctgaagaga accgccagaa gaagatacac cagacggaag 1080
aaccggatct gctatctgca agagatcttc agcaacgaga tggccaaggt ggacgacagc 1140
ttcttccaca gactggaaga gtccttcctg gtggaagagg ataagaagca cgagcggcac 1200
cccatcttcg gcaacatcgt ggacgaggtg gcctaccacg agaagtaccc caccatctac 1260
cacctgagaa agaaactggt ggacagcacc gacaaggccg acctgcggct gatctatctg 1320
gccctggccc acatgatcaa gttccggggc cacttcctga tcgagggcga ctccgacgcc 1380
atcctgctga gcgacatcct gagagtgaac accgagatca ccaaggcccc cctgagcgcc 1440
tctatgatca agagatacga cgagcaccac caggacctga ccctgctgaa agctctcgtg 1500
cggcagcagc tgcctgagaa gtacaaagag attttcttcg accagagcaa gaacggctac 1560
gccggctaca ttgacggcgg agccagccag gaagagttct acaagttcat caagcccatc 1620
ctggaaaaga tggacggcac cgaggaactg ctcgtgaagc tgaacagaga ggacctgctg 1680
cggaagcagc ggaccttcga caacggcagc atcccccacc agatccacct gggagagctg 1740
cacgccattc tgcggcggca ggaagatttt tacccattcc tgaaggacaa ccgggaaaag 1800
atcgagaaga tcctgacctt ccgcatcccc tactacgtgg gccctctggc caggggaaac 1860
agcagattcg cctggatgac cagaaagagc gaggaaacca tcaccccctg gaacttcgag 1920
gaagtggtgg acaagggcgc ttccgcccag agcttcatcg agcggatgac caacttcgat 1980
aagaacctgc ccaacgagaa ggtgctgccc aagcacagcc tgctgtacga gtacttcacc 2040
gtgtataacg agctgaccaa agtgaaatac gtgaccgagg gaatgagaaa gcccgccttc 2100
ctgagcggcg agcagaaaaa ggccatcgtg gacctgctgt tcaagaccaa ccggaaagtg 2160
accgtgaagc agctgaaaga ggactacttc aagaaaatcg agtgcttcga ctccgtggaa 2220
atctccggcg tggaagatcg gttcaacgcc tccctgggca cataccacga tctgctgaaa 2280
attatcaagg acaaggactt cctggacaat gaggaaaacg aggacattct ggaagatatc 2340
gtgctgaccc tgacactgtt tgaggacaga gagatgatcg aggaacggct gaaaacctat 2400
gcccacctgt tcgacgacaa agtgatgaag cagctgaagc ggcggagata caccggctgg 2460
ggcaggctga gccggaagct gatcaacggc atccgggaca agcagtccgg caagacaatc 2520
ctggatttcc tgaagtccga cggcttcgcc aacagaaact tcatgcagct gatccacgac 2580
gacagcctga cctttaaaga ggacatccag aaagcccagg tgtccggcca gggcgatagc 2640
ctgcacgagc acattgccaa tctggccggc agccccgcca ttaagaaggg catcctgcag 2700
acagtgaagg tggtggacga gctcgtgaaa gtgatgggcc ggcacaagcc cgagaacatc 2760
gtgatcgaaa tggccagaga gaaccagacc acccagaagg gacagaagaa cagccgcgag 2820
agaatgaagc ggatcgaact gctgaacgcc aagctgatta cccagagaaa gttcgacaat 2880
ctgaccaagg ccgagagagg cggcctgagc gaactggata aggccggctt catcaagaga 2940
cagctggtgg aaacccggca gatcacaaag cacgtggcac agatcctgga ctcccggatg 3000
aacactaagt acgacgagaa tgacaagctg atccgggaag tgaaagtgat caccctgaag 3060
tccaagctgg tgtccgattt ccggaaggat ttccagtttt acaaagtgcg cgagatcaac 3120
aactaccacc acgcccacga cgcctacctg aacgccgtcg tgggaaccgc cctgatcaaa 3180
aagtacccta agctggaaag cgagttcgtg tacggcgact acaaggtgta cgacgtgcgg 3240
aagatgatcg ccaagagcga gcaggaaatc ggcaaggcta ccgccaagta cttcttctac 3300
agcaacatca tgaacttttt caagaccgag attaccctgg ccaacggcga gatccggaag 3360
cggcctctga tcgagacaaa cggcgaaacc ggggagatcg tgtgggataa gggccgggat 3420
tttgccaccg tgcggaaagt gctgagcatg ccccaagtga atatcgtgaa aaagaccgag 3480
gtgcagacag gcggcttcag caaagagtct atcctgccca agaggaacag cgataagctg 3540
atcgccagaa agaaggactg ggaccctaag aagtacggcg gcttcgacag ccccaccgtg 3600
gcctattctg tgctggtggt ggccaaagtg gaaaagggca agtccaagaa actgaagagt 3660
gtgaaagagc tgctggggat caccatcatg gaaagaagca gcttcgagaa gaatcccatc 3720
gactttctgg aagccaaggg ctacaaagaa gtgaaaaagg acctgatcat caagctgcct 3780
aagtactccc tgttcgagct ggaaaacggc cggaagagaa tgctggcctc tgccggcgaa 3840
ctgcagaagg gaaacgaact ggccctgccc tccaaatatg tgaacttcct gtacctggcc 3900
agccactatg agaagctgaa gggctccccc gaggataatg agcagaaaca gctgtttgtg 3960
gaacagcaca agcactacct ggacgagatc atcgagcaga tcagcgagtt ctccaagaga 4020
gtgatcctgg ccgacgctaa tctggacaaa gtgctgtccg cctacaacaa gcaccgggat 4080
aagcccatca gagagcaggc cgagaatatc atccacctgt ttaccctgac caatctggga 4140
gcccctgccg ccttcaagta ctttgacacc accatcgacc ggaagaggta caccagcacc 4200
aaagaggtgc tggacgccac cctgatccac cagagcatca ccggcctgta cgagacacgg 4260
atcgacctgt ctcagctggg aggtgacgag ggagctgata agcgcaccgc cgatggttcc 4320
gagttcgaaa gccccaagaa gaagaggaaa gtctaaccgg tcatcatcac catcaccatt 4380
gagaattcaa taaaagatct ttattttcat tagatctgtg tgttggtttt ttgtgtgggt 4440
accgagggcc tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga 4500
gagataatta gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt 4560
agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat 4620
catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa 4680
ggacgaaaca ccgttgcagg cctggagttt attgtttaag agctatgctg gaaacagcat 4740
agcaagttta aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct 4800
ttttttcttc tgaggcggaa agaaccagct ggggctcgat accgtcgacc tctagctaga 4860
gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc 4920
cacacaacat acgagccgga agcataaagt gtaaagccta gggtgcctaa tgagtgagct 4980
aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc 5040
agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt 5100
ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag 5160
ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca 5220
tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt 5280
tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc 5340
gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct 5400
ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg 5460
tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca 5520
agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact 5580
atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta 5640
acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta 5700
actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct 5760
tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt 5820
tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga 5880
tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca 5940
tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat 6000
caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg 6060
cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt 6120
agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag 6180
acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc 6240
gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag 6300
ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca 6360
tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa 6420
ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga 6480
tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata 6540
attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca 6600
agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg 6660
ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg 6720
ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg 6780
cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag 6840
gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac 6900
tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca 6960
tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag 7020
tgccacctga cgtcgacgga tcgggagatc gatctcccga tcccctaggg tcgactctca 7080
gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg 7140
aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca 7200
attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca 7260
gatatacgcg tt 7272
<210> 4
<211> 7101
<212> DNA
<213> AAV-miniABE*(人工合成)
<400> 4
gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac 60
caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc 120
ggtaggcgtg tacggtggga ggtctatata agcagagctc gtttagtgaa ccgtcagatc 180
gaccggttct agagcgctgc caccatgaaa cggacagccg acggaagcga gttcgagtca 240
ccaaagaaga agcggaaagt ctctgaggtg gagttttccc acgagtactg gatgagacat 300
gccctgaccc tggccaagag ggcacgggat gagagggagg tgcctgtggg agccgtgctg 360
gtgctgaaca atagagtgat cggcgagggc tggaacagag ccatcggcct gcacgaccca 420
acagcccatg ccgaaattat ggccctgaga cagggcggcc tggtcatgca gaactacaga 480
ctgattgacg ccaccctgta cgtgacattc gagccttgcg tgatgtgcgc cggcgccatg 540
atccactcta ggatcggccg cgtggtgttt ggcgtgagga actcaaaaag aggcgccgca 600
ggctccctga tgaacgtgct gaactacccc ggcatgaatc accgcgtcga aattaccgag 660
ggaatcctgg cagatgaatg tgccgccctg ctgtgcgatt tctatcggat gcctagacag 720
gtgttcaatg ctcagaagaa ggcccagagc tccatcaacg acaagaagta cagcatcggc 780
ctggccatcg gcaccaactc tgtgggctgg gccgtgatca ccgacgagta caaggtgccc 840
agcaagaaat tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa gaacctgatc 900
ggagccctgc tgttcgacag cggcgaaaca gccgaggcca cccggctgaa gagaaccgcc 960
agaagaagat acaccagacg gaagaaccgg atctgctatc tgcaagagat cttcagcaac 1020
gagatggcca aggtggacga cagcttcttc cacagactgg aagagtcctt cctggtggaa 1080
gaggataaga agcacgagcg gcaccccatc ttcggcaaca tcgtggacga ggtggcctac 1140
cacgagaagt accccaccat ctaccacctg agaaagaaac tggtggacag caccgacaag 1200
gccgacctgc ggctgatcta tctggccctg gcccacatga tcaagttccg gggccacttc 1260
ctgatcgagg gcgactccga cgccatcctg ctgagcgaca tcctgagagt gaacaccgag 1320
atcaccaagg cccccctgag cgcctctatg atcaagagat acgacgagca ccaccaggac 1380
ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg agaagtacaa agagattttc 1440
ttcgaccaga gcaagaacgg ctacgccggc tacattgacg gcggagccag ccaggaagag 1500
ttctacaagt tcatcaagcc catcctggaa aagatggacg gcaccgagga actgctcgtg 1560
aagctgaaca gagaggacct gctgcggaag cagcggacct tcgacaacgg cagcatcccc 1620
caccagatcc acctgggaga gctgcacgcc attctgcggc ggcaggaaga tttttaccca 1680
ttcctgaagg acaaccggga aaagatcgag aagatcctga ccttccgcat cccctactac 1740
gtgggccctc tggccagggg aaacagcaga ttcgcctgga tgaccagaaa gagcgaggaa 1800
accatcaccc cctggaactt cgaggaagtg gtggacaagg gcgcttccgc ccagagcttc 1860
atcgagcgga tgaccaactt cgataagaac ctgcccaacg agaaggtgct gcccaagcac 1920
agcctgctgt acgagtactt caccgtgtat aacgagctga ccaaagtgaa atacgtgacc 1980
gagggaatga gaaagcccgc cttcctgagc ggcgagcaga aaaaggccat cgtggacctg 2040
ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga aagaggacta cttcaagaaa 2100
atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag atcggttcaa cgcctccctg 2160
ggcacatacc acgatctgct gaaaattatc aaggacaagg acttcctgga caatgaggaa 2220
aacgaggaca ttctggaaga tatcgtgctg accctgacac tgtttgagga cagagagatg 2280
atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg acaaagtgat gaagcagctg 2340
aagcggcgga gatacaccgg ctggggcagg ctgagccgga agctgatcaa cggcatccgg 2400
gacaagcagt ccggcaagac aatcctggat ttcctgaagt ccgacggctt cgccaacaga 2460
aacttcatgc agctgatcca cgacgacagc ctgaccttta aagaggacat ccagaaagcc 2520
caggtgtccg gccagggcga tagcctgcac gagcacattg ccaatctggc cggcagcccc 2580
gccattaaga agggcatcct gcagacagtg aaggtggtgg acgagctcgt gaaagtgatg 2640
ggccggcaca agcccgagaa catcgtgatc gaaatggcca gagagaacca gaccacccag 2700
aagggacaga agaacagccg cgagagaggc ggcctgagcg aactggataa ggccggcttc 2760
atcaagagac agctggtgga aacccggcag atcacaaagc acgtggcaca gatcctggac 2820
tcccggatga acactaagta cgacgagaat gacaagctga tccgggaagt gaaagtgatc 2880
accctgaagt ccaagctggt gtccgatttc cggaaggatt tccagtttta caaagtgcgc 2940
gagatcaaca actaccacca cgcccacgac gcctacctga acgccgtcgt gggaaccgcc 3000
ctgatcaaaa agtaccctaa gctggaaagc gagttcgtgt acggcgacta caaggtgtac 3060
gacgtgcgga agatgatcgc caagagcgag caggaaatcg gcaaggctac cgccaagtac 3120
ttcttctaca gcaacatcat gaactttttc aagaccgaga ttaccctggc caacggcgag 3180
atccggaagc ggcctctgat cgagacaaac ggcgaaaccg gggagatcgt gtgggataag 3240
ggccgggatt ttgccaccgt gcggaaagtg ctgagcatgc cccaagtgaa tatcgtgaaa 3300
aagaccgagg tgcagacagg cggcttcagc aaagagtcta tcctgcccaa gaggaacagc 3360
gataagctga tcgccagaaa gaaggactgg gaccctaaga agtacggcgg cttcgacagc 3420
cccaccgtgg cctattctgt gctggtggtg gccaaagtgg aaaagggcaa gtccaagaaa 3480
ctgaagagtg tgaaagagct gctggggatc accatcatgg aaagaagcag cttcgagaag 3540
aatcccatcg actttctgga agccaagggc tacaaagaag tgaaaaagga cctgatcatc 3600
aagctgccta agtactccct gttcgagctg gaaaacggcc ggaagagaat gctggcctct 3660
gccggcgaac tgcagaaggg aaacgaactg gccctgccct ccaaatatgt gaacttcctg 3720
tacctggcca gccactatga gaagctgaag ggctcccccg aggataatga gcagaaacag 3780
ctgtttgtgg aacagcacaa gcactacctg gacgagatca tcgagcagat cagcgagttc 3840
tccaagagag tgatcctggc cgacgctaat ctggacaaag tgctgtccgc ctacaacaag 3900
caccgggata agcccatcag agagcaggcc gagaatatca tccacctgtt taccctgacc 3960
aatctgggag cccctgccgc cttcaagtac tttgacacca ccatcgaccg gaagaggtac 4020
accagcacca aagaggtgct ggacgccacc ctgatccacc agagcatcac cggcctgtac 4080
gagacacgga tcgacctgtc tcagctggga ggtgacgagg gagctgataa gcgcaccgcc 4140
gatggttccg agttcgaaag ccccaagaag aagaggaaag tctaaccggt catcatcacc 4200
atcaccattg agaattcaat aaaagatctt tattttcatt agatctgtgt gttggttttt 4260
tgtgtgggta ccgagggcct atttcccatg attccttcat atttgcatat acgatacaag 4320
gctgttagag agataattag aattaatttg actgtaaaca caaagatatt agtacaaaat 4380
acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa 4440
atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc 4500
ttgtggaaag gacgaaacac cgttgcaggc ctggagttta ttgtttaaga gctatgctgg 4560
aaacagcata gcaagtttaa ataaggctag tccgttatca acttgaaaaa gtggcaccga 4620
gtcggtgctt tttttcttct gaggcggaaa gaaccagctg gggctcgata ccgtcgacct 4680
ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc 4740
tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctag ggtgcctaat 4800
gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc 4860
tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg 4920
ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag 4980
cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag 5040
gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 5100
tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc 5160
agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 5220
tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 5280
cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg 5340
ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 5400
ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 5460
ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 5520
ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc 5580
cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 5640
gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 5700
atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga 5760
ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa 5820
gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa 5880
tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc 5940
ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga 6000
taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa 6060
gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt 6120
gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg 6180
ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc 6240
aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg 6300
gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag 6360
cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt 6420
actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt 6480
caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac 6540
gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac 6600
ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag 6660
caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa 6720
tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga 6780
gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc 6840
cccgaaaagt gccacctgac gtcgacggat cgggagatcg atctcccgat cccctagggt 6900
cgactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt 6960
gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct 7020
tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg 7080
tacgggccag atatacgcgt t 7101
Claims (4)
1.一种能够实现单个AAV病毒包被的迷你碱基编辑器，其特征在于：包括TadA-8e和SpCas9缺失变体的两种迷你碱基编辑器；其中，
TadA-8e与SpCas9缺失REC2结构域第174-296位氨基酸以及HNH结构第786-885位氨基酸构建的载体命名为miniABE，核苷酸序列如SEQ ID NO.1
TadA-8e与SpCas9缺失REC2结构域第174-296位氨基酸，HNH结构第781-905位氨基酸以及TadA-8e与SpCas9的连接linker构建的载体命名为miniABE*，核苷酸序列如SEQ IDNO.2。
2.一种AAV载体，其特征在于：
AAV-miniABE载体核苷酸序列如SEQIDNO.3；
AAV-miniABE*载体核苷酸序列如SEQIDNO.4。
3.如权利要求1所述的一种能够实现单个AAV病毒包被的迷你碱基编辑器在制备基因治疗药物中的医用用途。
4.如权利要求1所述的一种能够实现单个AAV病毒包被的迷你碱基编辑器在破坏小鼠PCSK9基因中的的用途。
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| CN110467679A (zh) * | 2019-08-06 | 2019-11-19 | 广州大学 | 一种融合蛋白、碱基编辑工具和方法及其应用 |
| CN110734900A (zh) * | 2019-11-06 | 2020-01-31 | 上海科技大学 | 一种胞嘧啶碱基编辑工具及其用途 |
| CN113717266A (zh) * | 2021-09-14 | 2021-11-30 | 中国农业科学院生物技术研究所 | Osnop2蛋白及其编码基因关于耐逆性和产量相关的应用 |
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| CN110734900A (zh) * | 2019-11-06 | 2020-01-31 | 上海科技大学 | 一种胞嘧啶碱基编辑工具及其用途 |
| CN113717266A (zh) * | 2021-09-14 | 2021-11-30 | 中国农业科学院生物技术研究所 | Osnop2蛋白及其编码基因关于耐逆性和产量相关的应用 |
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