CN112538500A - 一种碱基编辑器及其制备方法和应用 - Google Patents
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Abstract
本发明公开了一种碱基编辑器，所述碱基编辑器为PX459重组质粒，所述PX459重组质粒包括PX459‑U6‑sgRNA‑CBh‑T2A‑PURO载体骨架和rAPOBEC1(W90Y+R126E；YE1)‑SpCas9n(D10A)‑UGI核苷酸序列。所述碱基编辑器制备方法简便，且在应用中采用单质粒进行细胞转染操作，这样不仅可以提高基因编辑效率，能够特异的将将靶位点的胞嘧啶C突变成胸腺嘧啶T，而且对非靶位点的碱基没有任何影响，所述碱基编辑器应用领域广泛，不仅能够应用于猪体细胞基因编辑，还可应用到牛、羊等经济动物细胞基因编辑上。
Description
技术领域
本公开属于基因编辑技术领域，具体涉及一种碱基编辑器及其制备方法和应用。
背景技术
基因编辑(gene editing)，又称基因组编辑(genome edting)或基因组工程(genome engineering)，是一种新兴的比较精确的能对生物体基因组特定目标基因进行修饰的一种基因工程技术。基因编辑技术指能够让人类对目标基因进行定点“编辑”，实现对特定DNA片段的修饰。当前CRISPR/Cas9是最常用的基因编辑工具，其原理是核酸酶导致DNA双链断裂(Double strand break,DSB)，诱导细胞激活同源重组(Homology-directedrepair,HDR)或非同源末端连接(Non-homologous end joining,NHEJ)来修复DSB，修复的过程就能引入基因突变，实现基因敲除或敲入【1】。
体细胞核移植(Somatic cell nuclear transfer)，简称“核移植”，是指将一个物种的体细胞移植到去核卵母细胞中，移入的供体细胞核在受体卵胞质中完成重编程，然后移植到代孕母体以生产新动物个体的技术【2】。由于细胞遗传物质包含在核内，得到的后代具有与核供体完全相同的遗传信息，因此体细胞核移植也被称为“体细胞克隆”(Somaticcell cloning)。由于体细胞克隆猪遗传信息全部来自供体细胞，因此对供体细胞进行基因编辑，再用基因编辑细胞进行克隆猪生产，就能得到表达预定表型的基因编辑动物。目前以CRISPR/Cas9为代表的基因编辑技术结合体细胞克隆已经在包括猪、牛、羊在内的多种动物基因修饰上得到了广泛应用，短短几年内公开报道的以改良经济性状和疾病模型为目的的基因编辑猪就多达100多种，为农业生产和生物医学研究带来了强大助力【3】。
胞嘧啶碱基编辑器(Cytosine base editor,CBE)是在传统CRISPR-Cas9系统中只具有单链切割能力Cas9n(D10A)蛋白上融合大鼠胞嘧啶脱氨酶rAPOBEC1和尿嘧啶DNA糖基化酶抑制剂UGI而成，该融合蛋白能够在单链向导RNA(sgRNA)的引导下特异性地针对靶序列C·G碱基对高效转化为T·A碱基对，实现单个碱基突变以改变基因功能【4】。相比CRISPR/Cas9而言，CBE由于仅针对目标区域的C碱基定向突变，不导致DSB，因而编辑结果能够实现预测，而应具有更高的精准度和安全性，势必在猪的遗传改良和疾病模型构建中得到更广泛的应用。研究表明CBE也存在广泛性基因脱靶这一重要不足。
参考文献：
[1]Hsu PD,Lander ES,Zhang F.Development and applications of CRISPR-Cas9 for genome engineering.Cell,2014,157:1262-1278.
[2]Keefer CL.Artificial cloning of domestic animals.Proc.Natl.Acad.Sci.U.S.A.,2015,112:8874-8878.
[3]Zhao JG,Lai L,Ji W,Zhou Q.Genome editing in large animals:Currentstatus and future prospects.Natl.Sci.Rev.,2019,6:402-420.
[4]Komor AC,Kim YB,Packer MS,Zuris JA,Liu DR.Programmableediting of atarget base in genomic DNA without double-stranded DNA cleavage.Nature,2016,533:420-424.
发明内容
本发明公开了一种碱基编辑器，以解决现有技术中所存在的一个或多个技术问题，至少提供一种有益的选择或创造条件。
一种碱基编辑器，其特征在于，所述碱基编辑器为PX459重组质粒，所述PX459重组质粒含有如SEQ ID NO.2所示的rAPOBEC1(W90Y+R126E；YE1)-SpCas9n(D10A)-UGI核苷酸序列。
经过系统性研究，CBE存在广泛性基因脱靶的不足可以通过序列突变来降低，例如携带W90Y+R126E突变的APOBEC1(YE1)就能最大限度降低其基因组脱靶活性。但实践表明，经过改造的CBE，虽然基因组脱靶毒性显著下降，但同时其基因编辑活性也受损害，因此在实际应用CBE时还需改进操作方法以提高基因编辑效率。利用CBE制备基因编辑细胞，是利用体细胞克隆生产单碱基编辑猪的第一步。最常用的方式是采用双质粒(一个携带腺嘌呤碱基编辑器，一个携带sgRNA)进行细胞共转染。该方法在实践应用中，需要制备2种质粒并同时转染进细胞，程序复杂，且共转染效率低，同时由于两种质粒不匹配表达也将影响碱基编辑效率，因此在细胞上进行单碱基编辑效果不佳。
进一步，所述PX459重组质粒包括PX459-U6-sgRNA-CBh-T2A-PURO载体骨架和所述rAPOBEC1(W90Y+R126E；YE1)-SpCas9n(D10A)-UGI核苷酸序列。
进一步，所述PX459重组质粒的核苷酸序列如SEQ ID NO.1所示。
另一方面，本发明还公开了上述碱基编辑器的制备方法，具体步骤为：
(I)用带有双酶切位点的引物对PX459质粒进行PCR扩增，回收PCR产物经双酶切获得的PX459-U6-sgRNA-CBh-T2A-PURO载体骨架；
(II)用带有双酶切位点的引物对YE1-BE4max-NG质粒进行PCR扩增，回收PCR产物经双酶切获得的rAPOBEC1(W90Y+R126E；YE1)-SpCas9n(D10A)-UGI核苷酸序列；
(III)利用DNA连接酶将PX459-U6-sgRNA-CBh-T2A-PURO载体骨架和rAPOBEC1(W90Y+R126E；YE1)-SpCas9n(D10A)-UGI核苷酸序列连接，获得所述PX459重组质粒。
进一步，进行双酶切所使用的酶为AgeI限制性内切酶和SalI限制性内切酶。
前述的碱基编辑器可应用于基因编辑中。具体应用的方法为：
(1)确定待编辑基因的靶位点；
(2)根据基因的靶位点合成gRNA引物，然后将gRNA引物进行高温退火形成引物二聚体；
(3)利用限制性内切酶将所述PX459重组质粒进行线性化，然后与引物二聚体混合后进行连接，获得连接产物；
(4)将连接产物进行转化感受态细胞，扩增培养后回收纯化质粒；
(5)将回收的纯化质粒进行细胞转染，进行传代培养；
(6)加入抗生素进行细胞的抗性筛选；
(7)撤除抗生素继续进行细胞培养，直至长出单细胞克隆。
进一步，步骤(3)中所述限制性内切酶为BbsI限制性内切酶。
进一步，步骤(5)中细胞转染是采用Lipofectamine 3000试剂进行细胞转染，优选的所述细胞转染的时间为6h。
进一步，所述步骤(5)中细胞传代培养的密度为1万/35mm直径培养皿，优选的所述细胞传代培养的时间为2天。
进一步，所述步骤(6)中抗生素为嘌呤霉素；优选的所述嘌呤霉素的使用量为1μg/mL；优选的所述细胞抗性筛选的时间为72h。
本发明提供的碱基编辑器为单种重组质粒，包含了胞嘧啶碱基编辑器、gRNA表达以及其它进行细胞基因编辑所需要的筛选标记等基因元件，在应用中利用单种质粒进行细胞转染操作，这样不仅可以提高碱基编辑效率，能够特异的将将靶位点的胞嘧啶C突变成胸腺嘧啶T，而且对靶点的非C碱基以及非靶位点的C碱基没有任何影响。本发明提供的碱基编辑器制备方法简便，同时其应用领域广泛，不仅能够应用于猪体细胞基因编辑，还可应用到牛、羊等动物细胞基因编辑上。
附图说明
图1碱基编辑器结构示意图；
图2是实施例3所述Sanger测序结果图；
图3是实施例3所述二代测序结果图。
具体实施方式
以下各步骤仅用以说明本公开的技术方案，而非对其限制；尽管参照前述各步骤对本公开进行了详细的说明，但本领域的普通技术人员应当理解：其依然可以对前述各步骤所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本公开各步骤技术方案的范围。
实施例1
一种碱基编辑器的制备方法，所述制备方法的具体步骤为：
(1)以PX459质粒(Addgene Plasmid#62988)制备载体基础结构，其包括sgRNA表达元件以及由T2A连接的PURO筛选基因表达元件，可用于真核细胞筛选表达。用带有双酶切位点的引物(上游引物SEQ ID NO.3：ATCATCGTCGACAAAAGGCCGGCGGCCA；下游引物SEQ IDNO.4：CATGGTGGCACCGGTCCAAC)对PX459质粒进行PCR扩增，扩增产物4968bp，回收PCR产物经SalI/AgeI双酶切获得PX459-U6-sgRNA-CBh-T2A-PURO载体骨架；
(2)用带有双酶切位点的引物(上游引物SEQ ID NO.5：ATCATCACCGGTGCCACCATGAAACGGACA；下游引物SEQ ID NO.6：CCTTTTGTCGACTGAGCCGCCAGACAGCAT)对YE1-BE4max-NG质粒进行PCR扩增，扩增产物5538bp，回收PCR产物经SalI/AgeI双酶切获得rAPOBEC1(W90Y+R126E；YE1)-SpCas9n(D10A)-UGI核苷酸序列；
(3)用T4 DNA连接酶将PX459-U6-sgRNA-CBh-T2A-PURO载体骨架和rAPOBEC1(W90Y+R126E；YE1)-SpCas9n(D10A)-UGI核苷酸序列连接，其结构图如图1所示。
将步骤(3)所得PX459重组质粒转化感受态细胞，涂平板培养，提取纯化质粒后经测序鉴定，其核苷酸序列如SEQ ID NO.1所示。
实施例2
一种碱基编辑器在猪体细胞(选择巴马香猪低密度脂蛋白受体基因LDLR)基因编辑中的应用其具体操作步骤为：
(1)利用巴马香猪LDLR基因特异性引物(上游引物SEQ ID NO.7：CTCACTGGGCTTGGCCTG；下游引物SEQ ID NO.8：AGTTCTCCTCGTCAGACTTG)对巴马香猪基因组DNA进行PCR扩增，经基因测序获得其核苷酸序列如SEQ ID NO.9(片段大小为489bp)；
(2)利用CRISPR/Cas9基因编辑在线设计工具CRISPOR(http://crispor.tefor.net/)设计位于低密度脂蛋白受体基因第1外显子的靶点，发现序列SEQ IDNO.10：CCTGCATCCCTGAGCTGTGGGCC评分较高，选作编辑靶点(互补链)，序列中大写G即为胞嘧啶碱基编辑器可能靶向的碱基靶标(互补碱基)，序列中CCT即为PAM序列。
(3)根据上述序列合成gRNA引物(上游引物SEQ ID NO.11：CACCGGCCCACAGCTCAGGGATGC；下游引物SEQ ID NO.12：AAACGCATCCCTGAGCTGTGGGCC)，高温退火形成引物二聚体；
(4)用BbsI限制性内切酶将碱基编辑器质粒线性化，琼脂糖凝胶电泳分裂基因片段，切胶回收，与gRNA引物退火二聚体混合后用DNA连接酶于4℃反应过夜，得到连接产物；
(5)将连接产物转化感受态细胞，涂板筛选培养，挑阳性菌扩大培养，对阳性菌液抽提质粒，经基因测序确定gRNA引物连接无误，将质粒于-20℃冰箱保存，将用于细胞转染；
(6)培养低代次巴马香猪成纤维细胞，采用Lipofectamine 3000试剂进行细胞转染；
(7)转染24小时后，将细胞以1万/35mm培养皿的密度传代；
(8)细胞传代培养培养2天后，加入11μg/mL嘌呤霉素进行抗性筛选72小时；
(9)进行抗性筛选后，撤除抗生素继续培养6-8天，消化收集细胞，提取细胞基因组DNA，然后用巴马香猪LDLR基因特异性引物(上游引物为：SEQ ID NO.7；下游引物为：SEQ IDNO.8)对巴马香猪基因组DNA进行PCR扩增，将PCR产物回收后送基因测序公司分别经Sanger和二代测序，分析序列组成和碱基编辑效率。
实施例3
实验组：利用上述实施例2中的碱基编辑器在猪体细胞基因编辑中的应用的方法，在进行细胞转染的步骤中设置3次平行实验。
对照组：采用“线性化PX459+YE1-BE4max-NG”双质粒同时转染作为对照，操作方法与实施例2相同；线性化PX459质粒操作方法也与实施例2相同，同时在步骤(4)得到携带与实验组碱基编辑器携带相同gRNA之后，用BglII限制性内切酶进行线性化以破坏Cas9表达元件并作为gRNA表达质粒，回收线性化基因产物，与YE1-BE4max-NG质粒同时转染细胞，作为双质粒基因编辑对照试验。
完成细胞转染和抗性筛选后，撤除抗生素继续培养6-8天，消化收集细胞，提取细胞基因组DNA，用编辑位点特异性引物(上游引物为：SEQ ID NO.7；下游引物为：SEQ IDNO.8)进行PCR扩增，回收PCR产物送基因测序公司分别进行Sanger和二代测序，分析目标靶点胞嘧啶C(或互补链G)突变成胸腺嘧啶T(或互补链A)的比例。经统计分析两组试验效率，统计方法为Student’s t-test，编辑效率用平均值±标准差表示，百分比数值经反正弦函数转换后进行统计分析，当P＜0.05时表明具有统计学差异。
基因编辑的Sanger测序结果如图2所示。实验组开展的用单个CBE碱基编辑器质粒在猪体细胞中编辑LDLR基因靶点(SEQ ID NO.8：CCTGCATCCCTGAGCTGTGGGCC，序列中大写G即为胞嘧啶碱基编辑器可能靶向的碱基靶标(互补碱基)，序列中CCT即为PAM序列)，预期的靶点G碱基均有一定程度的突变成A碱基；而在对照组，采用“线性化PX459+YE1-BE4max-NG”双质粒同时转染的方法编辑猪体细胞中编辑LDLR基因靶点，则在预期的靶点G碱基观察不到突变。
基因编辑的二代测序结果如图3所示。实验组开展的用单个CBE碱基编辑器质粒在猪体细胞中编辑LDLR基因靶点，预期的靶点G碱基突变成A碱基的效率为29.7±4.1％，显著高于对照组1.9±0.7％(P＜0.001)。
综上所述，表明本公开所述的碱基编辑器在使用中不仅简化了操作程序，且大幅度提升了靶向胞嘧啶碱基编辑效率，因而具有重要的利用价值和应用前景。
SEQUENCE LISTING
<110> 佛山科学技术学院
<120> 一种碱基编辑器及其制备方法和应用
<130> 2020
<160> 12
<170> PatentIn version 3.5
<210> 1
<211> 10467
<212> DNA
<213> 人工序列
<400> 1
gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60
ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120
aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180
atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240
cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag 300
gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct 360
agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac 420
aaatggctct agaggtaccc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 480
ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc cattgacgtc 540
aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc 600
caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tgtgcccagt 660
acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta 720
ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac 780
ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg 840
ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga 900
gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc 960
ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgcgct 1020
gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg cccgccccgg ctctgactga 1080
ccgcgttact cccacaggtg agcgggcggg acggcccttc tcctccgggc tgtaattagc 1140
tgagcaagag gtaagggttt aagggatggt tggttggtgg ggtattaatg tttaattacc 1200
tggagcacct gcctgaaatc actttttttc aggttggacc ggtgccacca tgaaacggac 1260
agccgacgga agcgagttcg agtcaccaaa gaagaagcgg aaagtctcct cagagactgg 1320
gcctgtcgcc gtcgatccaa ccctgcgccg ccggattgaa cctcacgagt ttgaagtgtt 1380
ctttgacccc cgggagctga gaaaggagac atgcctgctg tacgagatca actggggagg 1440
caggcactcc atctggaggc acacctctca gaacacaaat aagcacgtgg aggtgaactt 1500
catcgagaag tttaccacag agcggtactt ctgccccaat accagatgta gcatcacatg 1560
gtttctgagc tattcccctt gcggagagtg tagcagggcc atcaccgagt tcctgtccag 1620
atatccacac gtgacactgt ttatctacat cgccaggctg tatcaccacg cagacccaga 1680
gaataggcag ggcctgcgcg atctgatcag ctccggcgtg accatccaga tcatgacaga 1740
gcaggagtcc ggctactgct ggcggaactt cgtgaattat tctcctagca acgaggccca 1800
ctggcctagg tacccacacc tgtgggtgcg cctgtacgtg ctggagctgt attgcatcat 1860
cctgggcctg cccccttgtc tgaatatcct gcggagaaag cagccccagc tgaccttctt 1920
tacaatcgcc ctgcagtctt gtcactatca gaggctgcca ccccacatcc tgtgggccac 1980
aggcctgaag tctggaggat ctagcggagg atcctctggc agcgagacac caggaacaag 2040
cgagtcagca acaccagaga gcagtggcgg cagcagcggc ggcagcgaca agaagtacag 2100
catcggcctg gccatcggca ccaactctgt gggctgggcc gtgatcaccg acgagtacaa 2160
ggtgcccagc aagaaattca aggtgctggg caacaccgac cggcacagca tcaagaagaa 2220
cctgatcgga gccctgctgt tcgacagcgg cgaaacagcc gaggccaccc ggctgaagag 2280
aaccgccaga agaagataca ccagacggaa gaaccggatc tgctatctgc aagagatctt 2340
cagcaacgag atggccaagg tggacgacag cttcttccac agactggaag agtccttcct 2400
ggtggaagag gataagaagc acgagcggca ccccatcttc ggcaacatcg tggacgaggt 2460
ggcctaccac gagaagtacc ccaccatcta ccacctgaga aagaaactgg tggacagcac 2520
cgacaaggcc gacctgcggc tgatctatct ggccctggcc cacatgatca agttccgggg 2580
ccacttcctg atcgagggcg acctgaaccc cgacaacagc gacgtggaca agctgttcat 2640
ccagctggtg cagacctaca accagctgtt cgaggaaaac cccatcaacg ccagcggcgt 2700
ggacgccaag gccatcctgt ctgccagact gagcaagagc agacggctgg aaaatctgat 2760
cgcccagctg cccggcgaga agaagaatgg cctgttcgga aacctgattg ccctgagcct 2820
gggcctgacc cccaacttca agagcaactt cgacctggcc gaggatgcca aactgcagct 2880
gagcaaggac acctacgacg acgacctgga caacctgctg gcccagatcg gcgaccagta 2940
cgccgacctg tttctggccg ccaagaacct gtccgacgcc atcctgctga gcgacatcct 3000
gagagtgaac accgagatca ccaaggcccc cctgagcgcc tctatgatca agagatacga 3060
cgagcaccac caggacctga ccctgctgaa agctctcgtg cggcagcagc tgcctgagaa 3120
gtacaaagag attttcttcg accagagcaa gaacggctac gccggctaca ttgacggcgg 3180
agccagccag gaagagttct acaagttcat caagcccatc ctggaaaaga tggacggcac 3240
cgaggaactg ctcgtgaagc tgaacagaga ggacctgctg cggaagcagc ggaccttcga 3300
caacggcagc atcccccacc agatccacct gggagagctg cacgccattc tgcggcggca 3360
ggaagatttt tacccattcc tgaaggacaa ccgggaaaag atcgagaaga tcctgacctt 3420
ccgcatcccc tactacgtgg gccctctggc caggggaaac agcagattcg cctggatgac 3480
cagaaagagc gaggaaacca tcaccccctg gaacttcgag gaagtggtgg acaagggcgc 3540
ttccgcccag agcttcatcg agcggatgac caacttcgat aagaacctgc ccaacgagaa 3600
ggtgctgccc aagcacagcc tgctgtacga gtacttcacc gtgtataacg agctgaccaa 3660
agtgaaatac gtgaccgagg gaatgagaaa gcccgccttc ctgagcggcg agcagaaaaa 3720
ggccatcgtg gacctgctgt tcaagaccaa ccggaaagtg accgtgaagc agctgaaaga 3780
ggactacttc aagaaaatcg agtgcttcga ctccgtggaa atctccggcg tggaagatcg 3840
gttcaacgcc tccctgggca cataccacga tctgctgaaa attatcaagg acaaggactt 3900
cctggacaat gaggaaaacg aggacattct ggaagatatc gtgctgaccc tgacactgtt 3960
tgaggacaga gagatgatcg aggaacggct gaaaacctat gcccacctgt tcgacgacaa 4020
agtgatgaag cagctgaagc ggcggagata caccggctgg ggcaggctga gccggaagct 4080
gatcaacggc atccgggaca agcagtccgg caagacaatc ctggatttcc tgaagtccga 4140
cggcttcgcc aacagaaact tcatgcagct gatccacgac gacagcctga cctttaaaga 4200
ggacatccag aaagcccagg tgtccggcca gggcgatagc ctgcacgagc acattgccaa 4260
tctggccggc agccccgcca ttaagaaggg catcctgcag acagtgaagg tggtggacga 4320
gctcgtgaaa gtgatgggcc ggcacaagcc cgagaacatc gtgatcgaaa tggccagaga 4380
gaaccagacc acccagaagg gacagaagaa cagccgcgag agaatgaagc ggatcgaaga 4440
gggcatcaaa gagctgggca gccagatcct gaaagaacac cccgtggaaa acacccagct 4500
gcagaacgag aagctgtacc tgtactacct gcagaatggg cgggatatgt acgtggacca 4560
ggaactggac atcaaccggc tgtccgacta cgatgtggac catatcgtgc ctcagagctt 4620
tctgaaggac gactccatcg acaacaaggt gctgaccaga agcgacaaga accggggcaa 4680
gagcgacaac gtgccctccg aagaggtcgt gaagaagatg aagaactact ggcggcagct 4740
gctgaacgcc aagctgatta cccagagaaa gttcgacaat ctgaccaagg ccgagagagg 4800
cggcctgagc gaactggata aggccggctt catcaagaga cagctggtgg aaacccggca 4860
gatcacaaag cacgtggcac agatcctgga ctcccggatg aacactaagt acgacgagaa 4920
tgacaagctg atccgggaag tgaaagtgat caccctgaag tccaagctgg tgtccgattt 4980
ccggaaggat ttccagtttt acaaagtgcg cgagatcaac aactaccacc acgcccacga 5040
cgcctacctg aacgccgtcg tgggaaccgc cctgatcaaa aagtacccta agctggaaag 5100
cgagttcgtg tacggcgact acaaggtgta cgacgtgcgg aagatgatcg ccaagagcga 5160
gcaggaaatc ggcaaggcta ccgccaagta cttcttctac agcaacatca tgaacttttt 5220
caagaccgag attaccctgg ccaacggcga gatccggaag cggcctctga tcgagacaaa 5280
cggcgaaacc ggggagatcg tgtgggataa gggccgggat tttgccaccg tgcggaaagt 5340
gctgagcatg ccccaagtga atatcgtgaa aaagaccgag gtgcagacag gcggcttcag 5400
caaagagtct atcctgccca agaggaacag cgataagctg atcgccagaa agaaggactg 5460
ggaccctaag aagtacggcg gcttcgacag ccccaccgtg gcctattctg tgctggtggt 5520
ggccaaagtg gaaaagggca agtccaagaa actgaagagt gtgaaagagc tgctggggat 5580
caccatcatg gaaagaagca gcttcgagaa gaatcccatc gactttctgg aagccaaggg 5640
ctacaaagaa gtgaaaaagg acctgatcat caagctgcct aagtactccc tgttcgagct 5700
ggaaaacggc cggaagagaa tgctggcctc tgccggcgaa ctgcagaagg gaaacgaact 5760
ggccctgccc tccaaatatg tgaacttcct gtacctggcc agccactatg agaagctgaa 5820
gggctccccc gaggataatg agcagaaaca gctgtttgtg gaacagcaca agcactacct 5880
ggacgagatc atcgagcaga tcagcgagtt ctccaagaga gtgatcctgg ccgacgctaa 5940
tctggacaaa gtgctgtccg cctacaacaa gcaccgggat aagcccatca gagagcaggc 6000
cgagaatatc atccacctgt ttaccctgac caatctggga gcccctgccg ccttcaagta 6060
ctttgacacc accatcgacc ggaagaggta caccagcacc aaagaggtgc tggacgccac 6120
cctgatccac cagagcatca ccggcctgta cgagacacgg atcgacctgt ctcagctggg 6180
aggtgacagc ggcgggagcg gcgggagcgg ggggagcact aatctgagcg acatcattga 6240
gaaggagact gggaaacagc tggtcattca ggagtccatc ctgatgctgc ctgaggaggt 6300
ggaggaagtg atcggcaaca agccagagtc tgacatcctg gtgcacaccg cctacgacga 6360
gtccacagat gagaatgtga tgctgctgac ctctgacgcc cccgagtata agccttgggc 6420
cctggtcatc caggattcta acggcgagaa taagatcaag atgctgagcg gaggatccgg 6480
aggatctgga ggcagcacca acctgtctga catcatcgag aaggagacag gcaagcagct 6540
ggtcatccag gagagcatcc tgatgctgcc cgaagaagtc gaagaagtga tcggaaacaa 6600
gcctgagagc gatatcctgg tccataccgc ctacgacgag agtaccgacg aaaatgtgat 6660
gctgctgaca tccgacgccc cagagtataa gccctgggct ctggtcatcc aggattccaa 6720
cggagagaac aaaatcaaaa tgctgtctgg cggctcagtc gacaaaaggc cggcggccac 6780
gaaaaaggcc ggccaggcaa aaaagaaaaa ggaattcggc agtggagagg gcagaggaag 6840
tctgctaaca tgcggtgacg tcgaggagaa tcctggccca atgaccgagt acaagcccac 6900
ggtgcgcctc gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt 6960
cgccgactac cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac 7020
cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc 7080
ggacgacggc gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt 7140
gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca 7200
acagatggaa ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac 7260
cgtcggagtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg 7320
agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa 7380
cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg 7440
accgcgcacc tggtgcatga cccgcaagcc cggtgcctga gaattctaac tagagctcgc 7500
tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg 7560
ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt 7620
gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc 7680
aagggggagg attgggaaga gaatagcagg catgctgggg agcggccgca ggaaccccta 7740
gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca 7800
aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc 7860
tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac 7920
cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg 7980
tggtggttac gcgcagcgtg accgctacac ttgccagcgc cttagcgccc gctcctttcg 8040
ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg 8100
ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt 8160
tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt 8220
tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaactcta 8280
tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggtctat tggttaaaaa 8340
atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt 8400
tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc 8460
cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac 8520
aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac 8580
gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa 8640
tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt 8700
tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc 8760
ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc 8820
ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa 8880
aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg 8940
gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag 9000
ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc 9060
gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta 9120
cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg 9180
cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca 9240
acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac 9300
caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat 9360
taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg 9420
ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata 9480
aatctggagc cggtgagcgt ggaagccgcg gtatcattgc agcactgggg ccagatggta 9540
agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa 9600
atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag 9660
tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg 9720
tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact 9780
gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg 9840
taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc 9900
aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata 9960
ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta 10020
catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc 10080
ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg 10140
ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac 10200
agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg 10260
taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt 10320
atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct 10380
cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg 10440
ccttttgctg gccttttgct cacatgt 10467
<210> 2
<211> 5527
<212> DNA
<213> 人工序列
<400> 2
gaccggtgcc accatgaaac ggacagccga cggaagcgag ttcgagtcac caaagaagaa 60
gcggaaagtc tcctcagaga ctgggcctgt cgccgtcgat ccaaccctgc gccgccggat 120
tgaacctcac gagtttgaag tgttctttga cccccgggag ctgagaaagg agacatgcct 180
gctgtacgag atcaactggg gaggcaggca ctccatctgg aggcacacct ctcagaacac 240
aaataagcac gtggaggtga acttcatcga gaagtttacc acagagcggt acttctgccc 300
caataccaga tgtagcatca catggtttct gagctattcc ccttgcggag agtgtagcag 360
ggccatcacc gagttcctgt ccagatatcc acacgtgaca ctgtttatct acatcgccag 420
gctgtatcac cacgcagacc cagagaatag gcagggcctg cgcgatctga tcagctccgg 480
cgtgaccatc cagatcatga cagagcagga gtccggctac tgctggcgga acttcgtgaa 540
ttattctcct agcaacgagg cccactggcc taggtaccca cacctgtggg tgcgcctgta 600
cgtgctggag ctgtattgca tcatcctggg cctgccccct tgtctgaata tcctgcggag 660
aaagcagccc cagctgacct tctttacaat cgccctgcag tcttgtcact atcagaggct 720
gccaccccac atcctgtggg ccacaggcct gaagtctgga ggatctagcg gaggatcctc 780
tggcagcgag acaccaggaa caagcgagtc agcaacacca gagagcagtg gcggcagcag 840
cggcggcagc gacaagaagt acagcatcgg cctggccatc ggcaccaact ctgtgggctg 900
ggccgtgatc accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac 960
cgaccggcac agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac 1020
agccgaggcc acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg 1080
gatctgctat ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt 1140
ccacagactg gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat 1200
cttcggcaac atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct 1260
gagaaagaaa ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct 1320
ggcccacatg atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa 1380
cagcgacgtg gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga 1440
aaaccccatc aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa 1500
gagcagacgg ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt 1560
cggaaacctg attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct 1620
ggccgaggat gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct 1680
gctggcccag atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga 1740
cgccatcctg ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag 1800
cgcctctatg atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct 1860
cgtgcggcag cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg 1920
ctacgccggc tacattgacg gcggagccag ccaggaagag ttctacaagt tcatcaagcc 1980
catcctggaa aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct 2040
gctgcggaag cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga 2100
gctgcacgcc attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga 2160
aaagatcgag aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg 2220
aaacagcaga ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt 2280
cgaggaagtg gtggacaagg gcgcttccgc ccagagcttc atcgagcgga tgaccaactt 2340
cgataagaac ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt 2400
caccgtgtat aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc 2460
cttcctgagc ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa 2520
agtgaccgtg aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt 2580
ggaaatctcc ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct 2640
gaaaattatc aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga 2700
tatcgtgctg accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac 2760
ctatgcccac ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg 2820
ctggggcagg ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac 2880
aatcctggat ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca 2940
cgacgacagc ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga 3000
tagcctgcac gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct 3060
gcagacagtg aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa 3120
catcgtgatc gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg 3180
cgagagaatg aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga 3240
acaccccgtg gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa 3300
tgggcgggat atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt 3360
ggaccatatc gtgcctcaga gctttctgaa ggacgactcc atcgacaaca aggtgctgac 3420
cagaagcgac aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa 3480
gatgaagaac tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga 3540
caatctgacc aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa 3600
gagacagctg gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg 3660
gatgaacact aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct 3720
gaagtccaag ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat 3780
caacaactac caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat 3840
caaaaagtac cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt 3900
gcggaagatg atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt 3960
ctacagcaac atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg 4020
gaagcggcct ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg 4080
ggattttgcc accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac 4140
cgaggtgcag acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgataa 4200
gctgatcgcc agaaagaagg actgggaccc taagaagtac ggcggcttcg acagccccac 4260
cgtggcctat tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa 4320
gagtgtgaaa gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc 4380
catcgacttt ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct 4440
gcctaagtac tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg 4500
cgaactgcag aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct 4560
ggccagccac tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt 4620
tgtggaacag cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa 4680
gagagtgatc ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg 4740
ggataagccc atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct 4800
gggagcccct gccgccttca agtactttga caccaccatc gaccggaaga ggtacaccag 4860
caccaaagag gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac 4920
acggatcgac ctgtctcagc tgggaggtga cagcggcggg agcggcggga gcggggggag 4980
cactaatctg agcgacatca ttgagaagga gactgggaaa cagctggtca ttcaggagtc 5040
catcctgatg ctgcctgagg aggtggagga agtgatcggc aacaagccag agtctgacat 5100
cctggtgcac accgcctacg acgagtccac agatgagaat gtgatgctgc tgacctctga 5160
cgcccccgag tataagcctt gggccctggt catccaggat tctaacggcg agaataagat 5220
caagatgctg agcggaggat ccggaggatc tggaggcagc accaacctgt ctgacatcat 5280
cgagaaggag acaggcaagc agctggtcat ccaggagagc atcctgatgc tgcccgaaga 5340
agtcgaagaa gtgatcggaa acaagcctga gagcgatatc ctggtccata ccgcctacga 5400
cgagagtacc gacgaaaatg tgatgctgct gacatccgac gccccagagt ataagccctg 5460
ggctctggtc atccaggatt ccaacggaga gaacaaaatc aaaatgctgt ctggcggctc 5520
agtcgac 5527
<210> 3
<211> 28
<212> DNA
<213> 人工序列
<400> 3
atcatcgtcg acaaaaggcc ggcggcca 28
<210> 4
<211> 20
<212> DNA
<213> 人工序列
<400> 4
catggtggca ccggtccaac 20
<210> 5
<211> 30
<212> DNA
<213> 人工序列
<400> 5
atcatcaccg gtgccaccat gaaacggaca 30
<210> 6
<211> 30
<212> DNA
<213> 人工序列
<400> 6
ccttttgtcg actgagccgc cagacagcat 30
<210> 7
<211> 18
<212> DNA
<213> 人工序列
<400> 7
ctcactgggc ttggcctg 18
<210> 8
<211> 20
<212> DNA
<213> 人工序列
<400> 8
agttctcctc gtcagacttg 20
<210> 9
<211> 489
<212> DNA
<213> 人工序列
<400> 9
ctcactgggc ttggcctgtc ctgggctcag tgtccccatc tatgcagtgg gctggtgtag 60
ggcctccccg gtaacctggc tgtgatcttc tgtctatttc tgaagccccc aagacgtgct 120
cccaagatga gttccgctgc caggacggca agtgcatcgc cccaaagttt gtctgtgact 180
cggaccggga ctgcctggac ggctcggatg aagcatcctg ccccacaccc acctgtggcc 240
ccgccagctt ccagtgcaac agctccacct gcatccctga gctgtgggcc tgtgatggtg 300
atcctgactg cgaggacggc tcagacgagt ggccacagca ctgcaggagc cacagctcat 360
cactccccga gaggagcaac aacccctgct cagccctcga gttccactgc cacagtggcg 420
agtgcatcca ctccagctgg cgctgcgacg gagacactga ctgcaaggac aagtctgacg 480
aggagaact 489
<210> 10
<211> 23
<212> DNA
<213> 人工序列
<400> 10
cctgcatccc tgagctgtgg gcc 23
<210> 11
<211> 24
<212> DNA
<213> 人工序列
<400> 11
caccggccca cagctcaggg atgc 24
<210> 12
<211> 24
<212> DNA
<213> 人工序列
<400> 12
aaacgcatcc ctgagctgtg ggcc 24
Claims (10)
1.一种碱基编辑器，其特征在于，所述碱基编辑器为PX459重组质粒，所述PX459重组质粒含有如SEQ ID NO.2所示的rAPOBEC1(W90Y+R126E；YE1)-SpCas9n(D10A)-UGI核苷酸序列。
2.根据权利要求1中所述的碱基编辑器，其特征在于，所述PX459重组质粒包括PX459-U6-sgRNA-CBh-T2A-PURO载体骨架和所述rAPOBEC1(W90Y+R126E；YE1)-SpCas9n(D10A)-UGI核苷酸序列。
3.根据权利要求2中所述的碱基编辑器，其特征在于，所述PX459重组质粒的核苷酸序列如SEQ ID NO.1所示。
4.如权利要求1至3任意一项所述的碱基编辑器的制备方法，其特征在于，所述制备方法的具体步骤为：
(I)用带有双酶切位点的引物对PX459质粒进行PCR扩增，回收PCR产物经双酶切获得的PX459-U6-sgRNA-CBh-T2A-PURO载体骨架；
(II)用带有双酶切位点的引物对YE1-BE4max-NG质粒进行PCR扩增，回收PCR产物经双酶切获得的rAPOBEC1(W90Y+R126E；YE1)-SpCas9n(D10A)-UGI核苷酸序列；
(III)利用DNA连接酶将PX459-U6-sgRNA-CBh-T2A-PURO载体骨架和rAPOBEC1(W90Y+R126E；YE1)-SpCas9n(D10A)-UGI核苷酸序列连接，获得所述PX459重组质粒。
5.根据权利要求4中所述的碱基编辑器的制备方法，其特征在于，进行双酶切所使用的酶为AgeI限制性内切酶和SalI限制性内切酶。
6.如权利要求书1至3任意一项所述的碱基编辑器在基因编辑中的应用，其特征在于，所述应用的方法为：
(1)确定待编辑基因的靶位点；
(2)根据基因的靶位点合成gRNA引物，然后将gRNA引物进行高温退火形成引物二聚体；
(3)利用限制性内切酶将所述PX459重组质粒进行线性化，然后与引物二聚体混合后进行连接，获得连接产物；
(4)将连接产物进行转化感受态细胞，扩增培养后回收纯化质粒；
(5)将回收的纯化质粒进行细胞转染，进行传代培养；
(6)加入抗生素进行细胞的抗性筛选；
(7)撤除抗生素继续进行细胞培养，直至长出单细胞克隆。
7.根据权利要求6中所述的碱基编辑器在基因编辑中的应用，其特征在于，所述步骤(3)中限制性内切酶为BbsI限制性内切酶。
8.根据权利要求6中所述的碱基编辑器在基因编辑中的应用，其特征在于，所述步骤(5)中细胞转染是采用Lipofectamine 3000试剂进行细胞转染，优选的所述细胞转染的时间为6h。
9.根据权利要求6中所述的碱基编辑器在基因编辑中的应用，其特征在于，所述步骤(5)中细胞传代培养的密度为1万/35mm直径培养皿，优选的所述细胞传代培养的时间为2天。
10.根据权利要求6中所述的碱基编辑器在基因编辑中的应用，其特征在于，所述步骤(6)中抗生素为嘌呤霉素；优选的所述嘌呤霉素的使用量为1μg/mL；优选的所述细胞抗性筛选的时间为72h。
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| CN114196702A (zh) * | 2021-12-06 | 2022-03-18 | 深圳市人民医院 | 一种利用单碱基编辑器构建长qt疾病干细胞的方法 |
| CN114561392A (zh) * | 2022-03-22 | 2022-05-31 | 绍兴市妇幼保健院 | 一种基于碱基编辑技术关闭靶基因清除HBV e抗原的方法 |
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