CN116200431A - 一种在哺乳动物体内介导a到c突变或t到g突变的新型基因编辑系统及其应用 - Google Patents

一种在哺乳动物体内介导a到c突变或t到g突变的新型基因编辑系统及其应用 Download PDF

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CN116200431A
CN116200431A CN202111447109.6A CN202111447109A CN116200431A CN 116200431 A CN116200431 A CN 116200431A CN 202111447109 A CN202111447109 A CN 202111447109A CN 116200431 A CN116200431 A CN 116200431A
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陈亮
李大力
黄一帆
汝高盟
刘明耀
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East China Normal University
Bioray Laboratories Inc
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Abstract

本发明涉及一种在哺乳动物体内介导A到C突变或T到G突变的新型基因编辑系统及其应用。所述基因编辑系统为：将人工进化的小鼠来源3‑甲基腺嘌呤糖苷酶、单体腺苷脱氨酶Tad‑8e、催化活性受损的Cas9n融合，构建而成的融合蛋白。所述在哺乳动物体内介导A到C突变或T到G突变的方法包括如下步骤：(1)在体外制备针对靶基因组编辑位点的gRNA序列；(2)在体外制备单碱基编辑器ACBE2Q的mRNA；(3)将所述单碱基编辑器系统和gRNA导入目标真核细胞；(4)筛选正确编辑的真核细胞。

Description

一种在哺乳动物体内介导A到C突变或T到G突变的新型基因编 辑系统及其应用
技术领域
本发明属于生物技术领域，具体的，涉及一种在哺乳动物体内介导A到C突变或T到G突变的新型基因编辑系统及其应用。
背景技术
人类遗传病发生的本质是由于基因突变，60％左右的遗传疾病由单个碱基突变引起，传统的利用基因组编辑技术介导的同源重组进行纠正这类遗传病非常低效(0.1％-5％)[1,2]。基于CRISPR系统衍生出来的单碱基编辑器是近年来新兴的高效碱基编辑技术,因不产生DNA双链断裂、无须重组模板、高效编辑等优势使其在基础研究和临床疾病治疗展示了巨大的应用前景。
经典的碱基编辑器主要分为胞嘧啶碱基编辑器(CBE)和腺嘌呤碱基编辑器(ABE)，前者由活性受损的来源于酿脓链球菌(Streptococcus pyogenes)spCas9n、大鼠来源的胞嘧啶脱氨酶rAPOBEC1和尿嘧啶糖苷酶抑制剂组成，其中Cas9蛋白以NGG作为PAM识别并特异结合DNA，紧接着在脱氨酶以及DNA修复的作用下，最终在NGG(21-23位)上游靶向序列20bp范围实现C·G-T·A的替换,编辑窗口主要位于4-8位[2]，有望纠正14％人类致病性点突变；后者则是将细菌来源的TadA与spCas9融合，在定向进化和蛋白质工程化改造技术的辅助下，经历7轮进化最终获得可作用于单链DNA的腺嘌呤碱基编辑器ABE7.10，活性编辑区域主要位于4-7位，该系统在人类细胞中引起A·T-G·C的平均编辑效率约为53％，远高于利用同源重组介导碱基突变的效率，其产物纯度高达99.9％以及极低的indels(插入和缺失)发生[3]，更重要的是人类致病性点突变约47％是由C·G突变为T·A所形成，而腺嘌呤碱基编辑器有望纠正近一半的病原性点突变[1]，展现出其在突变碱基修改以及遗传病治疗的巨大潜力，目前ABE已广泛应用于动物模型制备[4-8]和基因治疗[9-14]。
无论是CBE还是ABE均只能实现碱基的转换，科学家在开发CBE的早期过程中，发现敲除细胞内尿嘧啶糖苷酶(UNG)或者去除胞嘧啶糖苷酶抑制剂(UGI)会产生C·G-to-G·C和C·G-to-A·T编辑副产物，即发生基于C的颠换[3,15]。近期，科学家根据之前CBE产生的编辑副产物现象，将去除UGI的CBE融合不同类型UNG，DNA损伤修复蛋白或者跨损伤聚合酶等，开发出CGBE系列[16-19]，有望治疗11％G·C to C·G的致病性点突变。
然而没有已报道的酶可直接将基因组DNA中的腺嘌呤(A)催化为胞嘧啶(C)，反链即为胞嘧啶(T)到鸟嘌呤(G)，而需要A到C或者T到G来纠正的人类致病点突变(SNV)占占比16％，C到A或者G到T病原性点突变同时也是第二大最常见的致病性SNV，这超出了经典CBE可以覆盖的疾病范围[1]。
[参考文献]
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[2]Komor AC,Kim YB,Packer MS,et al.Programmable editing of a targetbase in genomic DNA without double-stranded DNA cleavage.Nature,2016,533:420-424
[3]Gaudelli NM,Komor AC,Rees HA,et al.Programmable base editing of a*t to g*c in genomic DNA without DNA cleavage.Nature,2017,551:464-471
[4]Liang P,Sun H,Zhang X,et al.Effective and precise adenine baseediting in mouse zygotes.Protein Cell,2018,9:808-813
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[9]Suh S,Choi EH,Leinonen H,et al.Restoration of visual function inadult mice with an inherited retinal disease via adenine base editing.NatBiomed Eng,2021,5:169-178
[10]Rothgangl T,Dennis MK,Lin PJC,et al.In vivo adenine base editingof pcsk9 in macaques reduces ldl cholesterol levels.Nat Biotechnol,2021,
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[14]Musunuru K,Chadwick AC,Mizoguchi T,et al.In vivo crispr baseediting of pcsk9 durably lowers cholesterol in primates.Nature,2021,593:429-434
[15]Komor AC,Zhao KT,Packer MS,et al.Improved base excision repairinhibition and bacteriophage mu gam protein yields c:G-to-t:A base editorswith higher efficiency and product purity.Sci Adv,2017,3:eaao4774
[16]Koblan LW,Arbab M,Shen MW,et al.Efficient c*g-to-g*c base editorsdeveloped using crispri screens,target-library analysis,and machinelearning.Nat Biotechnol,2021,
[17]Chen L,Park JE,Paa P,et al.Programmable c:G to g:C genome editingwith crispr-cas9-directed base excision repair proteins.Nat Commun,2021,12:1384
[18]Zhao D,Li J,Li S,et al.Glycosylase base editors enable c-to-a andc-to-g base changes.Nat Biotechnol,2021,39:35-40
[19]Kurt IC,Zhou R,Iyer S,et al.Crispr c-to-g base editors forinducing targeted DNA transversions in human cells.Nat Biotechnol,2021,39:41-46。
发明内容
本发明涉及一种体外编辑真核细胞基因组单碱基的方法，所述方法包括如下步骤：
(1)在体外制备针对靶基因组编辑位点的gRNA序列；
(2)在体外制备单碱基编辑器ACBE2Q的mRNA；
(3)将所述单碱基编辑器和gRNA导入目标真核细胞；
(4)筛选正确编辑的真核细胞。
所述的真核细胞包括但不限于：体细胞、生殖细胞；优选的，所述的真核细胞为卵细胞；
步骤(2)所述的单碱基编辑器ACBE2Q为：将人工进化的小鼠来源3-甲基腺嘌呤糖苷酶(3-methyladenine glycosylase，Aag)与单体腺苷脱氨酶Tad-8e(或其功能突变体)、催化活性受损的Cas9n融合，构建而成的融合蛋白，并增加核定位信号，其mRNA编码SEQ IDNO.1所示的多肽；
步骤(3)所述的导入目标真核细胞的方法包括但不限于，载体转染、显微注射、转染、脂质转染、热休克、电穿孔、基因枪、DEAE-葡聚糖介导的转染中的一种或任几种组合，优选的，使用显微注射的方法；
步骤(4)所述的筛选为通过基因测序的手段，筛选编辑结果正确的目标真核细胞。
优选的，步骤(3)所述的导入目标真核细胞的方法为显微注射法，
具体的，对单细胞进行显微注射，细胞注射液的配方为：
使用无核酸酶水配置注射混合物，注射混合物总体积为20μL；
其中含有所述ACBE2Q融合蛋白mRNA的浓度为1-1000ng/μL，优选的，10-600ng/μL，更优选的，50-150ng/μL；其中含有所述sgRNA的浓度为1-1000ng/μL，优选的，10-600ng/μL，更优选的，150-250ng/μL。
最优选的，总体积为20μL的注射混合物中，含有终浓度为100ng/μL的所述ACBE2Q融合蛋白mRNA与终浓度为200ng/μL的所述sgRNA。
本发明还涉及一种单碱基编辑系统的如下应用：
(1)制备基因编辑产品；
(2)制备治疗和/或预防疾病的药品或生物制品；
(3)制备动物模型或植物新品种；
所述的单碱基编辑系统包含：编码所述单碱基编辑器ACBE2Q的核酸，和/或包含编码所述单碱基编辑器ACBE2Q的核酸的重组载体，和/或包含所述单碱基编辑器ACBE2Q的核酸的重组细胞或重组菌。
所述的ACBE2Q融合蛋白的氨基酸序列如SEQ ID NO.1所示。
SEQ ID NO.1：
MKRTADGSEFESPKKKRKVDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSSGGSSGGSSGSETPGTSESATPESSGGSSGGSPARGGSARPGRGALKPVSVTLLPDTEQPPFLGRARRPGNARAGSLVTGYHEVGQMPAPLSRKIGQKKQRLADSEQQQTPKERLLSTPGLRRSIYFSSPEDHSGRLGPEFFDQPAVTLARAFLGQVLVRRLADGTELRGRIVETEAYLGPEDEAAHSRGGRQTPENRGMFMKPGTLYVFLIYGMYFCLNVSSQGAGACVLLRALEPLEGLETMRQLRNSLRKSTVGRSLKDRELCSGPSKLCQALAIDKSFDQRDLAQDDAVWLEHGPLESSSPAVVVAAARIGIGHAGEWTQKPLRFYVQGSPWVSVVDRVAEQMDQPQQTACSEGLLIVQKSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRQSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSINSGGSSGGSSGSETPGTSESATPESSGGSSGGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDMKRTADGSEFESPKKKRKV
本发明的有益效果在于：
我们将人工进化的小鼠来源3-甲基腺嘌呤糖苷酶(3-methyladenineglycosylase)与单体腺苷脱氨酶Tad-8e、催化活性受损的Cas9n融合构建ACBE，并在小鼠体内实现A到C或T到G的碱基颠换，该技术如进一步应用于临床，有望治疗16％C·G到A·T疾病相关的SNP；也将极大地促进人类疾病模型制作，作物遗传育种等方面的应用。
附图说明
图1、靶向杜氏肌营养不良(DMD)基因剪接受体位点示意图。
图2、ACBE2Q系统的mRNA显微注射生殖细胞后，产生F0代高通量测序结果的比对。
图3、DMD产生A到C突变的小鼠在剪接位点的编辑效率。
图4、F0小鼠进行免疫荧光染色检测抗肌萎缩蛋白(Dystrophin)的表达，图中，DAPI为核染料、层粘连蛋白(Laminin)为对照组蛋白样本，Merge为DAPI和Dystrophin/Laminin的荧光叠加照片，标尺为50μm。
具体实施方式
根据DNA碱基切除修复机制，我们将小鼠来源3-甲基腺嘌呤糖苷酶(Aag)变体和与单体腺苷脱氨酶Tad-8e和催化活性受损的Cas9n融合，构建ACBE2Q，具体的融合蛋白的结构及设计见申请人同日申请的中国发明专利，发明名称为：“一种介导A到C突变或T到G突变的新型基因编辑系统及其应用”。
显然，本领域技术人员能够理解，下述实施例仅用于展示本发明的实质，而非限定本发明的保护范围。更进一步的，使用本发明的方法，能够对包括生殖细胞、体细胞、干细胞在内的各种哺乳动物细胞就行单碱基A到C(正义链)或T到G(反义链)的碱基颠换。
根据DNA碱基切除修复机制，我们将小鼠来源3-甲基腺嘌呤糖苷酶(Aag)变体和与单体腺苷脱氨酶Tad-8e和催化活性受损的Cas9n融合，构建ACBE2Q，并应用其注射小鼠胚胎，体内编辑DMD基因的剪接受体位点(SAS，图1)。以下实施例中，构建动物模型使用的小鼠品系为C57/BL6小鼠。
实施例1、ACBE单碱基编辑器融合蛋白的mRNA与靶点sgRNA转录模板的构建及体外转录
1、工作系统mRNA与靶点sgRNA转录模板的构建
使用引物对IVT-T7-ACBE2Q-F和IVT-T7-ACBE2Q-R(表2)将T7启动子通过PCR引入ACBE2Q的mRNA模板。
表1、所用靶点序列及鉴定引物
Figure BDA0003385091230000041
表2、IVT所用PCR引物
引物名称 引物序列(5’-3’)
IVT-T7-ACBE2Q-F GCCGCGATCACTAATACGACTCACTATAGGGAGAGCCGC
IVT-T7-ACBE2Q-R CTAGACTTTCCTCTTCTTCTTGGGCTCGAATTCG
ACBE2Q序列单碱基编辑器的融合蛋白的结构及序列见本申请人同日申请的中国 发明专利，发明名称为：“一种介导A到C突变或T到G突变的新型基因编辑系统及其应用”。
2、sgRNA(DMD-splice sg7)体外转录
在NCBI上下载小鼠相关基因序列，如图1所示，在目标位点(抗肌萎缩蛋白基因即DMD基因第27个外显子5’端的剪接受体位点区域)设计并订购化学修饰合成的sgRNA(靶序列见表1)(订购于金斯瑞生物科技)。
sgRNA:UUUUCAGAGAGCUAAAGAAGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU。
3、工作系统mRNA(ACBE2Q)的转录
利用体外RNA转录试剂盒(mMESSAGE
Figure BDA0003385091230000051
Ultra Kit)对PCR(表2)获得的ACBE2Q的T7模板进行体外转录得到工作系统mRNA，并对其进行纯化。
4、显微注射混合物的制备
用无核酸酶的水配置注射混合物，获得总体积为20μL，终浓度为100ng/μL的工作系统mRNA(含有(ACBE2Q的mRNA)与终浓度为200ng/μL的sgRNA(DMD-splice sg7)的混合液。
实施例2、体外编辑生殖细胞
1、细胞期胚胎的收集
(1)第一天：于下午1-2点间向6-8周龄的供体母鼠腹腔注射100μL(5IU)的PMSG工作液。
(2)第三天：于下午2-4点间向注射过PMSG的母鼠腹腔注射100μL(5IU)的hCG工作液，注射后，将经激素处理后的母鼠与10-14周齡的公鼠一对一合笼。同时，将处于发情期未经激素处理的雌鼠与结扎的雄鼠在下午4点左右交配用于假孕母鼠的准备。
(3)第四天：早上9点前，检查与结扎公鼠合笼的受体母鼠是否有孕栓，将有孕栓的母鼠集中于新的笼子内用于下午的胚胎移植实验。
(4)通过二氧化碳窒息法将超排卵的供体母鼠处死取出输卵管，放置于平皿中，平皿中加入预热的M2培养基。
(5)将输卵管放置于另一个新的平皿中，平皿内加入预热的M2培养基与透明质酸，M2培养基与透明质酸的体积比为9:1。在体式显微镜下，用摄子拉扯输卵管壶腹部，使胚胎从输卵管释放至平皿中。把胚胎一直孵育在有透明质酸的M2培养基中直到卵丘细胞掉落。在去除卵丘细胞后，将胚胎转移至一个新的平皿中，平皿内加入无透明质酸的M2培养基，反复用M2培养基冲洗胚胎使透明质酸和卵丘细胞均被冲洗干净。
(6)将冲洗干净的胚胎转移至一个新的平皿中，平皿内先分散加入几滴KSOM培养基，然后再缓慢向平皿加入矿物油，使KSOM培养基被矿物油隔开与覆盖。一般来说，在一个35毫米的平皿中，可加6个点的KSOM培养基，每个点为50μL。将50个胚胎作为一组首先放于中间的KSOM培养基点进行漂洗，然后转移至一个新的培养基点。在显微注射前，把取出的胚胎孵育在细胞培养箱的M2培养基中。
2、显微注射与胚胎移植
(1)准备固定针，注射针以及硅化的玻璃载玻片，往载玻片中间滴一滴被矿物油覆盖的M2培养基。
(2)使注射针依靠毛细管作用自动吸入并充满实施例1的步骤4制备的显微注射混合液，将注射针装载至显微注射仪的固定柄上。
(3)转移50个胚胎至载玻片的M2培养基内，移动固定针靠近胚胎，使胚胎依靠负压作用被固定在固定针上。胚胎被固定后在高倍镜下找到细胞质，推动注射针的尖端穿过透明带，细胞膜，将混合液注射到胚胎的细胞质中。
(4)将注射过的胚胎细胞转移至新的M2培养基点中。重复步骤(3)和(4)直至所有的胚胎被注射完。注射完一组实验组后，将胚胎转移至新的KSOM培养基中，将胚胎放置于细胞培养箱中培养1-2小时或过夜。待所有胚胎均被注射后，排除因机械力损伤致死的胚胎，将健康的胚胎转移至新的KSOM培养基中。
(5)向假孕母鼠腹腔注射600μL阿佛丁，将假孕母鼠麻醉。使用剃毛器将母鼠背部的毛剔除。使用70％乙醇擦拭剃毛后的皮肤。
(6)在卵巢位置处剪开一个小口，使用钝头镊子牵拉着卵巢的脂肪垫从而将卵巢拉出，并同时用止血钳将卵巢固定于外侧，使用钝头镊子找到位于卵巢囊下侧的输卵管漏斗状口。
(7)使转移针依次吸入M2培养基，两个小气泡，约15个胚胎，气泡是为了便于观察胚胎在转移针中的位置。
(8)轻轻地剖开卵巢的囊，用镊子定位输卵管的漏斗状口，将转移针伸向卵巢的开口处，然后将转移针内的胚胎打出，轻轻地将转移针撤回。
(9)释放固定卵巢脂肪垫的止血钳，将卵巢放回原来的腔洞，用缝合线分别缝合肌肉开口处与皮肤开口处。
(10)将手术后的小鼠放置于恒温37度的保温台上，待小鼠恢复知觉后，将其转移至饲养笼具中饲养，等待胚胎发育直至分娩。一般来说，移植成功的母鼠在3周后生出小鼠。
实施例3、单碱基编辑效果的鉴定
1、小鼠基因组鉴定
实施例2移植成功后出生的小鼠在出生10-15天左右，剪其脚趾进行基因组的鉴定，具体步骤如下：
1.1、基因组提取
①剪其脚趾装入1.5mL离心管中，每管加入500μL按蛋白酶K：组织裂解液＝1：500的比例配制的脚趾消化液，55℃水浴过夜；
②取出过夜消化的脚趾，室温放置10-15分钟，充分颠倒混匀，13000rpm离心15分钟。
③每管吸出400μL上清，加入等体积氯仿，充分混匀待DNA析出后，12000rpm离心10分钟。
④每管加入提前在-20℃冰箱预冷的75％酒精200μL，轻柔地混匀，12000rpm 4℃离心5min，弃上清，在洁净工作台中晾干。
⑤根据DNA量加入50-100μL去离子超纯水，55℃溶解2小时即可作为PCR模板。
1.2、基因组的鉴定
按照表1中靶点DMD-splice sg7的引物对F/R获得含有靶点的DNA片段，先通过一代测序确认有双峰出现，后续再进行高通量深度测序得出编辑效率。根据高通量结果显示，ACBE2Q处理组共产生32只F0小鼠(图2)，有30只F0发生突变，21只F0在剪接位点发生A到C的突变，数量占突变F0比例为70％，在剪接位点产生的A到C突变平均效率为56.1％(图3)。
1.3、DMD突变小鼠的表型鉴定
取来自5周大的野生型小鼠(空白对照)以及前述经鉴定的在DMD基因剪接位点产生的A到C突变小鼠(#D02)，按如下方法进行免疫组织化学检测：
取小鼠的胫骨前肌，用酒精、PBS漂洗干净。将其放入铺有OTC胶的小正方体盒中，置于异戊烷烧杯，在液氮中冷冻，约30s取出，放入-20℃保存，之后进行冰冻切片。将制备好的切片用PBST洗3次/5min，对准组织位置画油圈，加入封闭液进行封闭，封闭1h后，分别用层粘连蛋白(Laminin)一抗或抗肌萎缩蛋白(Dystrophin)一抗(用1：500稀释后的一抗兔多克隆抗体(Abcam，ab11575)或一抗兔多克隆抗体(Abcam，ab15277))进行过夜孵育。用PBST洗3次/5min，然后以1：1000的抗兔稀释液二抗孵育2h，PBST洗3次/5min后，加入以1:100稀释的DAPI孵育10min，PBST洗3次/5min，滴加防猝灭剂，加盖玻片，指甲油封片，最后用荧光显微镜观察切片的荧光。
结果表明：与野生型小鼠对比，ACBE2Q处理组(#D02)影响抗肌萎缩蛋白的表达(图4)，这也证明DMD动物疾病模型构建成功。
最后需要说明的是，以上实施例仅用于帮助本领域技术人员理解本发明的实质，不用于限定本发明的保护范围。
SEQUENCE LISTING
<110> 华东师范大学&上海邦耀生物科技有限公司
<120> 一种在哺乳动物体内介导A到C突变或T到G突变的新型基因编辑系统及其应用
<130> BYP2021-0008
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1967
<212> PRT
<213> 人工序列（Artificial Sequence）
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Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn
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Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg
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Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp
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Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp
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Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val
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Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu
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Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln
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Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val
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Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu
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Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe
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Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser
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Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr
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Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr
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Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu
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Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser
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Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu
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His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys
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Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp
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His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr
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Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile
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Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe
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Leu Lys Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys
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Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys
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Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln
900 905 910
Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu
915 920 925
Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln
930 935 940
Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys
945 950 955 960
Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu
965 970 975
Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys
980 985 990
Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn
995 1000 1005
Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu
1010 1015 1020
Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys
1025 1030 1035
Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys
1040 1045 1050
Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile
1055 1060 1065
Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr
1070 1075 1080
Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe
1085 1090 1095
Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val
1100 1105 1110
Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile
1115 1120 1125
Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp
1130 1135 1140
Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala
1145 1150 1155
Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys
1160 1165 1170
Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu
1175 1180 1185
Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys
1190 1195 1200
Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys
1205 1210 1215
Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala
1220 1225 1230
Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser
1235 1240 1245
Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu
1250 1255 1260
Lys Gly Ser Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu
1265 1270 1275
Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly
1280 1285 1290
Ser Ser Gly Gly Ser Pro Ala Arg Gly Gly Ser Ala Arg Pro Gly
1295 1300 1305
Arg Gly Ala Leu Lys Pro Val Ser Val Thr Leu Leu Pro Asp Thr
1310 1315 1320
Glu Gln Pro Pro Phe Leu Gly Arg Ala Arg Arg Pro Gly Asn Ala
1325 1330 1335
Arg Ala Gly Ser Leu Val Thr Gly Tyr His Glu Val Gly Gln Met
1340 1345 1350
Pro Ala Pro Leu Ser Arg Lys Ile Gly Gln Lys Lys Gln Arg Leu
1355 1360 1365
Ala Asp Ser Glu Gln Gln Gln Thr Pro Lys Glu Arg Leu Leu Ser
1370 1375 1380
Thr Pro Gly Leu Arg Arg Ser Ile Tyr Phe Ser Ser Pro Glu Asp
1385 1390 1395
His Ser Gly Arg Leu Gly Pro Glu Phe Phe Asp Gln Pro Ala Val
1400 1405 1410
Thr Leu Ala Arg Ala Phe Leu Gly Gln Val Leu Val Arg Arg Leu
1415 1420 1425
Ala Asp Gly Thr Glu Leu Arg Gly Arg Ile Val Glu Thr Glu Ala
1430 1435 1440
Tyr Leu Gly Pro Glu Asp Glu Ala Ala His Ser Arg Gly Gly Arg
1445 1450 1455
Gln Thr Pro Glu Asn Arg Gly Met Phe Met Lys Pro Gly Thr Leu
1460 1465 1470
Tyr Val Phe Leu Ile Tyr Gly Met Tyr Phe Cys Leu Asn Val Ser
1475 1480 1485
Ser Gln Gly Ala Gly Ala Cys Val Leu Leu Arg Ala Leu Glu Pro
1490 1495 1500
Leu Glu Gly Leu Glu Thr Met Arg Gln Leu Arg Asn Ser Leu Arg
1505 1510 1515
Lys Ser Thr Val Gly Arg Ser Leu Lys Asp Arg Glu Leu Cys Ser
1520 1525 1530
Gly Pro Ser Lys Leu Cys Gln Ala Leu Ala Ile Asp Lys Ser Phe
1535 1540 1545
Asp Gln Arg Asp Leu Ala Gln Asp Asp Ala Val Trp Leu Glu His
1550 1555 1560
Gly Pro Leu Glu Ser Ser Ser Pro Ala Val Val Val Ala Ala Ala
1565 1570 1575
Arg Ile Gly Ile Gly His Ala Gly Glu Trp Thr Gln Lys Pro Leu
1580 1585 1590
Arg Phe Tyr Val Gln Gly Ser Pro Trp Val Ser Val Val Asp Arg
1595 1600 1605
Val Ala Glu Gln Met Asp Gln Pro Gln Gln Thr Ala Cys Ser Glu
1610 1615 1620
Gly Leu Leu Ile Val Gln Lys Ser Glu Val Glu Phe Ser His Glu
1625 1630 1635
Tyr Trp Met Arg His Ala Leu Thr Leu Ala Lys Arg Ala Arg Asp
1640 1645 1650
Glu Arg Glu Val Pro Val Gly Ala Val Leu Val Leu Asn Asn Arg
1655 1660 1665
Val Ile Gly Glu Gly Trp Asn Arg Ala Ile Gly Leu His Asp Pro
1670 1675 1680
Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln Gly Gly Leu Val
1685 1690 1695
Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr Val Thr Phe
1700 1705 1710
Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser Arg Ile
1715 1720 1725
Gly Arg Val Val Phe Gly Val Arg Gln Ser Lys Arg Gly Ala Ala
1730 1735 1740
Gly Ser Leu Met Asn Val Leu Asn Tyr Pro Gly Met Asn His Arg
1745 1750 1755
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu
1760 1765 1770
Leu Cys Asp Phe Tyr Arg Met Pro Arg Gln Val Phe Asn Ala Gln
1775 1780 1785
Lys Lys Ala Gln Ser Ser Ile Asn Ser Gly Gly Ser Ser Gly Gly
1790 1795 1800
Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro
1805 1810 1815
Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser Pro Glu Asp Asn Glu
1820 1825 1830
Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr Leu Asp Glu
1835 1840 1845
Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala
1850 1855 1860
Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg
1865 1870 1875
Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe
1880 1885 1890
Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp
1895 1900 1905
Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu
1910 1915 1920
Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr
1925 1930 1935
Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp Met Lys Arg Thr Ala
1940 1945 1950
Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys Arg Lys Val
1955 1960 1965

Claims (7)

1.一种体外编辑真核细胞基因组单碱基的方法，所述方法包括如下步骤：
(1)在体外制备针对靶基因组编辑位点的gRNA序列；
(2)在体外制备单碱基编辑器ACBE2Q的mRNA；
(3)将所述单碱基编辑器和gRNA导入目标真核细胞；
(4)筛选正确编辑的真核细胞。
所述的真核细胞包括但不限于：体细胞、生殖细胞；优选的，所述的真核细胞为卵细胞。
2.根据权利要求1所述的方法，其特征在于，
步骤(2)所述的单碱基编辑器ACBE2Q的mRNA编码由SEQ ID NO.1所示的多肽。
3.根据权利要求1所述的方法，其特征在于，
步骤(3)所述的导入目标真核细胞的方法包括但不限于，载体转染、显微注射、转染、脂质转染、热休克、电穿孔、基因枪、DEAE-葡聚糖介导的转染中的一种或任几种组合；
步骤(4)所述的筛选为通过基因测序的手段，筛选编辑结果正确的目标真核细胞。
4.根据权利要求1-3任一所述的方法，其特征在于，步骤(3)所述的导入目标真核细胞的方法为显微注射法。
5.根据权利要求4所述的方法，其特征在于，对单细胞进行所述显微注射时，使用细胞注射液的配方为：
使用无核酸酶水配置注射混合物，注射混合物总体积为10-50μL；其中含有：
(1)所述ACBE2Q的mRNA的浓度为1-1000ng/μL，优选的浓度为，10-600ng/μL，更优选的浓度为，50-150ng/μL；
(2)所述sgRNA的浓度为1-1000ng/μL，优选的浓度为，10-600ng/μL，更优选的浓度为，150-250ng/μL。
6.根据权利要求5所述的方法，其特征在于，注射混合物总体积为20μL；
其中含有终浓度为100ng/μL的所述ACBE2Q的mRNA与终浓度为200ng/μL的所述sgRNA。
7.一种单碱基编辑系统的如下应用：
(1)制备基因编辑产品；
(2)制备治疗和/或预防疾病的药品或生物制品；
(3)制备动物模型或植物新品种；
所述的单碱基编辑系统包含：编码所述单碱基编辑器ACBE2Q的核酸，和/或包含编码所述单碱基编辑器ACBE2Q的核酸的重组载体，和/或包含所述单碱基编辑器ACBE2Q的核酸的重组细胞或重组菌。
所述的ACBE2Q融合蛋白的氨基酸序列如SEQ ID NO.1所示。
CN202111447109.6A 2021-11-30 2021-11-30 一种在哺乳动物体内介导a到c突变或t到g突变的新型基因编辑系统及其应用 Pending CN116200431A (zh)

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