CN114317601A - 一种基于SviCas3的碱基编辑方法 - Google Patents
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Abstract
本发明公开了一种基于SviCas3的碱基编辑方法,首次实现了Cas3对生物细胞染色体DNA的碱基编辑。该方法中碱基编辑模板同时作为DNA向导和碱基置换模板,通过SviCas3在DNA靶位点的切割,引发细胞同源重组机制进行修复,从而实现碱基编辑。该方法具有载体分子量小、任意靶序列选择、可多位点同时碱基置换、无脱靶、操作简单高效等优点。该方法有望应用于治疗单核苷酸突变导致的遗传病。
Description
本发明涉及生物技术领域中的基因组工程技术,确切地说是一种基于 SviCas3的碱基编辑方法。
背景技术
大约三分之二的人类遗传病由单核苷酸突变(单核苷酸多态性:singlenucleotide polymorphisins/SNPs)所导致(Luke W Koblan,Jordan L Doman,Christopher Wilson,Jonathan M Levy,Tristan Tay,Gregory A Newby,Juan PabloMaianti,Aditya Raguram&David R Liu(2018)Improving cytidine and adenine baseeditors by expression optimization and ancestral reconstruction.Naturebiotechnology 36,843-846)。碱基编辑器可以纠正生物细胞基因组DNA中的单核苷酸突变,使得对罕见遗传病的修复成为可能(Yanhong Wang,Lifang Zhou,Nan Liu andShaohua Yao(2019)BE-PIGS:a base-editing tool with deaminases inlaid into Cas9PI domain significantly expanded the editing scope.Signal Transduction andTargeted Therapy,4,36),故而成为当今生命技术领域的研究热点。
碱基置换分为转换(嘌呤与嘌呤置换,或嘧啶与嘧啶置换)与颠换(嘌呤与嘧啶置换,或嘧啶与嘌呤置换)两类。先期的碱基编辑工具(仅实现转换)是基于sgRNA导向(形成R-loop)的dCas9和脱氨酶构成的碱基编辑器(即:胞嘧啶碱基编辑器cytosine base editor/CBE和腺嘌呤碱基编辑器adenine base editor /ABE)(Shuai Jin,Yuan Zong,Qiang Gao,Zixu Zhu,Yanpeng Wang,Peng Qin, Chengzhi Liang,Daowen Wang,Jin-Long Qiu,FengZhang and Caixia Gao(2019) Cytosine,but not adenine,base editors inducegenome-wide off-target mutations in rice.Science,364(6437):292-295),当前的碱基编辑器(Prime Editor/PE)(可实现转换与颠换)需要pegRNA与nCas9(H840A)定位、切口及逆转录酶复制修复(Andrew V Anzalone,Peyton B Randolph,Jessie R Davis,Alexander A Sousa, Luke W Koblan,Jonathan M Levy,Peter J Chen,ChristopherWilson,Gregory A Newby,Aditya Raguram,David R Liu(2019)Search-and-replacegenome editing without double-strand breaks or donor DNA.Nature,576(7785):149-157),它们均为RNA导向的碱基编辑器。基于RNA导向的碱基编辑技术存在载体分子量大、靶序列选择严紧、难以实现多位点同时碱基置换、操作复杂等缺点。本实验室已报道SviCas3能通过DNA导向进行基因编辑(童望宇,唐严严,夏婷婷,一种基于I-B型CRISPR-Cas系统基因cas3的基因编辑方法,申请号:201710847193.8)。迄今为止,基于Cas3的碱基编辑器未见报道,更未见有基于DNA导向的碱基编辑器的报道。
发明公开一种DNA引导的SviCas3的碱基编辑方法,该方法中tb-DNA同时作为向导(形成D-loop)和DNA编辑模板,通过SviCas3在D-loop中的内切,进而引发细胞同源重组机制以tb-DNA为模板进行修复,从而实现碱基编辑。本方法具有载体分子量小、任意靶序列选择、可多位点同时碱基置换、无脱靶、操作简单高效等优点。该方法能方便地修复哺乳细胞染色体DNA中的点突变,从而有望治疗单核苷酸突变导致的基因遗传病。
发明内容
本发明要解决的技术问题是提供一种基于SviCas3的碱基编辑方法,通过构建靶标碱基编辑载体并导入靶细胞、得到并验证碱基编辑重组子,实现修复哺乳细胞染色体DNA中的点突变。
为达此目的,本发明所采用的技术方案是:
一种基于SviCas3的碱基编辑方法,其特征在于:所述的碱基编辑方法中含有一个碱基编辑载体(BE-Vector-Svicas3-tb-DNA),导入宿主细胞后,可对细胞基因组进行碱基编辑。
所述的一种基于SviCas3的碱基编辑方法,其特征在于:碱基编辑载体中含有源于维吉尼亚链霉菌IBL14(Streptomyces virginiae IBL14)的I-B SviCas系统的SviCas3和所需碱基置换的碱基编辑模板(template DNA for base editing/ tb-DNA)(附图1)。
所述的一种基于SviCas3的碱基编辑方法,其特征在于:包括以下步骤:
(1)构建碱基编辑载体
设计引物(附表1),将化学合成的tb-DNA与Svicas3基因序列通过 overlap-PCR扩增连接,并对连接产物与载体通过限制性酶进行酶切、连接酶连接和转化验证得到碱基编辑载体BE-Vector-Svicas3-tb-DNA;
(2)构建并验证碱基编辑重组子
制备目标真核生物细胞,将按步骤(1)得到的BE-Vector-Svicas3-tb-DNA导入目标生物细胞中,得到碱基置换的碱基编辑重组子,对重组子染色体靶DNA 序列进行PCR扩增和基因测序,以确证编辑后的目的重组子。
所述的一种基于SviCas3的碱基编辑方法,其特征在于:所述宿主细胞指哺乳真核生物细胞。
所述的一种基于SviCas3的碱基编辑方法,其特征在于:所述碱基编辑指对生物细胞基因组DNA中的碱基进行转换与颠换。
有益效果
本发明所建立的是一种基于维吉尼亚链霉菌IBL14中I-B SviCas亚型系统中SviCas3的碱基编辑器方法,首次公开了一种基于DNA引导的核酸内切酶对哺乳细胞染色体DNA的碱基编辑。该方法中碱基编辑模板同时作为向导和碱基核苷酸置换模板,通过SviCas3在DNA靶位点的切割,引发细胞同源重组机制进行修复,从而实现碱基编辑。该方法具有载体分子量小、任意靶序列选择、可多位点同时碱基置换、无脱靶、操作简单高效等优点。应用该方法能有效地修复哺乳细胞染色体DNA中的点突变,从而治疗单核苷酸突变导致的基因遗传病。
附图说明
图1、碱基编辑载体BE-AIO-Svicas3-tb2-CAMKMT构建示意图。Ori/origin:DNA 复制起始位点,f1 ori:f1噬菌体起源的复制起始位,nucleoplasmin NLS:核定位信号,mCherry:红色荧光染料基因,AmpR/ampicillin resistance:氨苄青霉素抗性,T3promoter:T3启动子,起始DNA转录,U6promoter:U6启动子。
图2、碱基编辑重组子的验证。(A)碱基编辑靶序列PCR产物的凝胶电泳图(L: DNA分子量大小标准品,1:空载体,2:宿主细胞,3:水,4-6:碱基编辑重组子)。(B)模板DNA及碱基编辑重组子靶序列的测序结果(4个被编辑的碱基请见蓝色大写字母:T12005→A12005/颠换,G12011→A12011/转换,C12170→A12170/颠换和T12172→C12172/转换)。
具体实施方式
为了更充分理解本发明的技术内容,下面结合具体实施例对本发明的技术方案作进一步介绍和说明,旨在更好的解释本发明的内容,以下实施例不限制本发明的保护范围。此外,在所列实施例中如无特别说明均采用如下材料:
1)菌株与质粒
大肠杆菌Escherichia coli Top 10/EC Top 10,Escherichia coli DH5α/ECDH5α,人胚肾细胞human embryonic kidney 293 cells/HEK293,小鼠胚胎成纤维细胞mouse embryo fibroblast 3T3 cells/NIH3T3,质粒AIO-mCherry(缩写为: AIO)。
2)缓冲液与培养基
LiAc/DTT/TE缓冲液(400ml)
4.088g 0.1M LiAc·H2O,0.484g 10mM Tris(pH7.5),0.117g 1mM EDTA,加入300ml双蒸水溶解,用HCl调pH至7.5,定容至400ml,115℃/30min灭菌;0.617g DTT过滤灭菌后加入缓冲液中。
1M山梨醇
18.217g山梨醇,加双蒸水至100ml,115℃/30min灭菌。
50mg/ml氨苄青霉素
称取0.5g的氨苄青霉素置于10ml离心管中,加入4ml双蒸水,混匀后,定容至10ml,用0.2μm的无菌水系滤膜过滤后,分装到1.5ml的EP管中,放于-20℃冰箱中保存。
HEPES缓冲液
称取0.8g NaCl,0.037g KCl,0.0135g Na2HPO4·H2O,0.5g葡萄糖,0.5g HEPES,加90ml双蒸水溶解,NaOH调pH至7.5,定容至100ml。
50mg/ml G418
称取0.5g G418置于10ml离心管中,加入4ml HEPES缓冲液,混匀后,定容至10ml,用0.2μm的无菌水系滤膜过滤后,分装到1.5ml的EP管中,放于-20℃冰箱中保存。
LB培养基
酵母粉5g,蛋白胨10g,NaCl 10g,加入适量自来水溶解后,定容至1L, pH调至7.0~7.2,分装包扎后,121℃/20min灭菌,如制平板加入20g琼脂。
SCCM培养基(1 liter DMEM)
100ml牛胎儿血清,100mg青霉素,100mg链霉素,10g谷胺酰胺。
高糖DMEM培养基(GibcoTM)
购自Thermo Fisher Scientific Inc,168 Third Avenue,Waltham,MA USA02451。
所用试剂均为市售品。
实施例1、HEK293T-CAMKMT-b2的碱基编辑
(1)蛋白表达载体BE-AIO-Svicas3的构建
基于蛋白SviCas3(附表1)碱基优化的cDNA/Svicas3序列(Svicas3由滁州通用生物公司合成)及载体AIO的DNA序列信息,设计与载体AIO互补的基因Svicas3的特异性正向引物AIO-Svicas3-F和反向引物AIO-Svicas3-R(表1)。经常规PCR扩增[50μl PCR反应体系:5μl 10×Pfu buffer,5μl dNTPs(2.5mM each),1μl 10μM AIO-cas3-F,1μl 10μM AIO-cas3-R,5μl DMSO,0.5μl Pfu DNA Polymerase,0.5μl SV IBL14基因组DNA,32μl无菌水(nuclease-free),反应条件:95℃5min,94℃60±30s,55±3℃30s,72℃90±30s,2.5±0.5U DNA 聚合酶(TransStrart FastPfu DNA Ploymerase),30个循环,72℃10min],1%琼脂糖凝胶电泳检测,试剂盒回收,得纯化的cas3全长基因产物。将纯化的cas3 与载体AIO一步克隆连接,经EC DH5α转化、扩增、纯化后得蛋白表达载体BE-AIO-Svicas3。
(2)编辑载体BE-AIO-Svicas3-tb2-CAMKMT的构建
根据HEK293基因组CAMKMT基因2号外显子及所需碱基置换信息(四对碱基置换:T→A,G→A,C→A,T→C),设计合成同源臂引物(含XbaI限制性内切酶酶切位点与BamHI限制性内切酶酶切位点)tb2-CAMKMT-F和 tb2-CAMKMT-R(表1),PCR合成得tb2-CAMKMT碱基编辑模板(PCR扩增条件同实施例1-1)。取经1.5%琼脂糖电泳检测、试剂盒纯化回收的碱基编辑模板 tb2-CAMKMT,通过BamHI限制性内切酶酶、XbaI限制性内切酶切出粘性末端,然后通过全式金生物技术有限公司生产的T4连接酶将其连接到BE-AIO-Svicas3 载体上,得碱基编辑载体BE-AIO-Svicas3-tb2-CAMKMT。
(3)编辑重组子HEK293T-CAMKMT-b2的构建与验证
用灭菌过的牙签从平板上挑取Top10单克隆于30ml LB液体培养基中, 37±3℃,200±100rpm过夜培养;500±100μl过夜培养液转接至新的30ml LB 液体培养基中,37±3℃,200±100rpm培养约3±2h,至菌液OD600值为0.4-0.6;取1ml上述菌液至1.5ml EP管中,4℃下3000rpm离心5min,去除上清液,用50μl冰上预冷过的DEPC处理水将菌体沉淀吹打均匀,即得Top10的感受态细胞,-80±20℃下保存备用(此过程全程在冰上进行)。将5μl载体BE-AIO-Svicas3-tb2-CAMKMT加入到50μlEC Top 10感受态中,混合均匀,于冰上20±5min,45±3℃水浴90s,再于冰上15±5min,均匀涂布在LB固体培养基中,30±5℃培养24-48h,经PCR、电泳和DNA测序验证得含载体 BE-AIO-Svicas3-tb2-CAMKMT的EC Top 10转化子。
将HEK293细胞接种于含DMEM培养基的6孔板中,37℃培养,待细胞汇合率达80%时,加入vector-buffer混合液(6μg BE-AIO-Svicas3-tb2-CAMKMT和 100μl电穿孔缓冲液),通过电转仪(CTX-1500A-EX)转染HEK293T,3天后将转染的HEK293T细胞六孔板在接种到大的细胞培养皿中(1×106个/皿),待细胞培养长满于培养皿上时,提取基因组,经PCR,1%琼脂糖电泳及测序得正确的碱基编辑重组子HEK293T-CAMKMT-b2(图2)。
实施例2、NIH3T3-Lepr-b3的碱基编辑
(1)蛋白表达载体BE-AIO-Svicas3的构建
构建步骤同实施例1步骤(1)
(2)编辑载体BE-AIO-Svicas3-tb3-Lepr的构建
除根据所需碱基置换信息(四对碱基置换:C→G,A→G,G→C,G→T)设计合成相应同源臂引物外(表1),BE-AIO-Svicas3-tb3-Lepr的构建步骤同实施例1步骤(2)。
(3)编辑重组子NIH3T3-Lepr-b3的构建与验证
构建含载体BE-AIO-Svicas3-tb3-Lepr的EC Top 10转化子步骤同实施例1步骤(3)。
将NIH3T3细胞接种于含DMEM培养基的6孔板中,37℃培养,待细胞汇合率达70%时,将构建好的载体BE-AIO-Svicas3-tb3-Lepr转染NIH3T3细胞 (ExFect转染试剂),6小时后更换新鲜细胞培养液,3天后将转染的NIH3T3 细胞接种到大的细胞培养皿中(1×106个/皿),待细胞培养长满于培养皿上时,提取基因组进行PCR,经1%琼脂糖电泳检测,提取基因组,经PCR,1%琼脂糖电泳及测序得正确的碱基编辑重组子NIH3T3-Lepr-b3。
实施例3、HEK293T-ΔCAMKMT::egfp-g1/b2的基因及碱基编辑
(1)蛋白表达载体BE-AIO-Svicas3的构建
构建步骤同实施例1步骤(1)
(2)编辑载体BE-AIO-Svicas3-tg1/tb2-ΔCAMKMT::egfp的构建
除根据碱基置换信息不同(C→T,A→C,A→T,T→G)设计合成相应同源臂引物外(表1),tb2-CAMKMT2的构建步骤同实施例1步骤(2)。
根据HEK293基因组CAMKMT基因1号外显子及增强绿色荧光蛋白 (Enhanced GreenFluorescent Protein)的基因序列信息,设计在上、下游同源臂之间可插入绿色荧光蛋白(Enhanced Green Fluorescent Protein)基因egfp的上游同源臂引物tg1-CAMKMT-F(含XbaI限制性内切酶酶切位点)和tg1-CAMKMT-UR、下游同源臂引物tg1-CAMKMT-DF和tg1-CAMKMT-R(表1),PCR合成得 tg1-ΔCAMKMT::egfp基因编辑模板。
取经1.5%琼脂糖电泳检测、试剂盒纯化回收的碱基编辑模板tb2-CAMKMT2 和基因编辑模板tg1-ΔCAMKMT::egfp,通过overlap PCR扩增得基因碱基编辑模板tg1/tb2-ΔCAMKMT::egfp(overlap PCR扩增条件:25μl反应体系,模板各0.5μl,94℃5min,94℃1min,62℃30s,72℃1min,一个循环后加入引物 tg1-CAMKMT-F和tb2-CAMKMT-R2各1μl,继续PCR,反应条件为:94℃5min, 94℃30s,62℃30s,72℃1min,进行30个循环,72℃10min)。将电泳检测、纯化回收获得的tg1/tb2-ΔCAMKMT::egfp通过BamHI限制性内切酶酶、XbaI 限制性内切酶切出粘性末端,然后通过全式金生物技术有限公司生产的T4连接酶将其连接到BE-AIO-Svicas3载体上,得基因碱基编辑载体 BE-AIO-Svicas3-tg1/tb2-ΔCAMKMT::egfp。
(3)编辑重组子HEK293T-ΔCAMKMT::egfp-g1/b2的构建与验证
构建与验证步骤同实施例1步骤(3)。
以上所述仅以实施例来进一步说明本发明的技术内容,以便于读者更容易理解,但不代表本发明的实施方式仅限于此,任何依本发明所做的技术延伸或再创造,均受本发明的保护。
Claims (5)
1.一种基于SviCas3的碱基编辑方法,其特征在于:所述的碱基编辑方法中含有一个碱基编辑载体(BE-Vector-Svicas3-tb-DNA),导入宿主细胞后,可对细胞基因组进行碱基编辑。
2.根据权利要求1所述的一种基于SviCas3的碱基编辑方法,其特征在于:碱基编辑载体中含有源于维吉尼亚链霉菌IBL14(Streptomyces virginiae IBL14)的I-B SviCas系统的SviCas3和所需碱基置换的碱基编辑模板(template DNA for base editing/tb-DNA)(附图1)。
3.根据权利要求1所述的一种基于SviCas3的碱基编辑方法,其特征在于:包括以下步骤:
(1)构建碱基编辑载体
设计引物(附表1),将化学合成的tb-DNA与Svicas3基因序列通过overlap-PCR扩增连接,并对连接产物与载体通过限制性酶进行酶切、连接酶连接和转化验证得到碱基编辑载体BE-Vector-Svicas3-tb-DNA;
(2)构建并验证碱基编辑重组子
制备目标真核生物细胞,将按步骤(1)得到的BE-Vector-Svicas3-tb-DNA导入目标生物细胞中,得到碱基置换的碱基编辑重组子,对重组子染色体靶DNA序列进行PCR扩增和基因测序,以确证编辑后的目的重组子。
4.根据权利要求1所述的一种基于SviCas3的碱基编辑方法,其特征在于:所述宿主细胞指哺乳真核生物细胞。
5.根据权利要求1所述的一种基于SviCas3的碱基编辑方法,其特征在于:所述碱基编辑指对生物细胞基因组DNA中的碱基进行转换与颠换。
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