CN105861485B - 一种提高基因置换效率的方法 - Google Patents
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Abstract
本发明属于生物医学领域,涉及一种利用Cas9核酸酶的aptamer提高基因置换效率的方法,包括以下步骤:(1)双功能DNA单链的合成:合成具有Cas9特异结合能力的aptamer和DNA供体的序列;所述aptamer序列为:5′‑AGTCCATGGTAAACCCACCTTGGGGT‑GACT‑3′;(2)质粒的制备:构建Cas9‑gRNA质粒,使其能表达Cas9蛋白和转录靶位点附近的gRNA;(3)把所得双功能DNA单链与质粒共转染到要进行基因置换的细胞或组织。本发明采用高亲和度的特异DNA aptamer,不干扰Cas9‑gRNA复合物的形成与稳定,使同源性重组和基因修复的比例达到最高。
Description
技术领域
本发明属于生物医学领域,涉及一种基因置换方法,尤其涉及一种利用Cas9核酸酶的aptamer(适配体)提高基因置换效率的方法,可用于基因治疗。
背景技术
生物医学中的基因置换技术对基因功能研究、药物筛选和基因治疗有着不可估量的重要性。细胞内基因置换是通过同源性重组实现的,而同源性重组在绝大多数细胞中的效率极低,因此提高同源性重组效率是生物学家面临的巨大挑战。若能提高同源性重组的效率,基因置换和基因治疗的成功率就会极大地提高。
目前,现有基因置换技术主要存在以下问题:(1)同源性重组在绝大多数细胞中的效率极低。(2)Cas9-gRNA复合物只能定位到靶位点进行基因切断,不能完成基因置换。(3)基因治疗过程中的靶定向特异性低,置换、修复效率低。
发明内容
针对上述问题,发明人进行了大量试验,积累的研究表明,先切断某个基因片段会大大提高这个基因置换的频率(几个数量级),进而得到一种aptamer介导的提高基因置换效率的方法,采用高亲和度的特异DNA aptamer,不干扰Cas9-gRNA复合物的形成与稳定,使同源性重组和基因修复的比例达到最高。
为解决上述问题,本发明通过以下技术方案实现:
设计一种提高基因置换效率的方法,包括以下步骤:
(1)双功能DNA单链的合成:采用DNA合成仪经常规方法合成包含Cas9特异结合能力的aptamer和DNA供体的单链序列;所述aptamer序列为:5′-AGTCCATGGTAAACCCACCTTGGGGTGACT-3′;
(2)质粒的制备:常规方法构建Cas9-gRNA质粒,使其能表达Cas9蛋白和转录靶位点附近的gRNA;
(3)把所得双功能DNA单链与质粒共转染到要进行基因置换的细胞或组织。
优选的,将所述双功能DNA单链与质粒以摩尔比1:(15~25)共转染到要进行基因置换的细胞或组织。更优选的,将双功能DNA单链、Cas9-gRNA质粒以摩尔比1:20于Lipofectamine试剂盒中室温混合15~25min,再加入具有细胞培养液的6孔板中,培养两天后,筛选细胞株验证置换效果。所述细胞培养液浓度为60wt%。
本发明的优势表现在:
在本发明的技术方案中,将具有Cas9特异结合能力的单链DNA aptamer和单链DNA供体(donor)连在一块,被一个结合在靶位点的Cas9核酸酶富集到靶位点,来对被Cas9核酸酶切断后的靶位点进行基因置换;即利用Cas9-aptamer把aptamer-donor富集到靶位点。
(1)本发明采用高亲和度的特异DNA aptamer,不干扰Cas9-gRNA复合载体的形成与稳定。
(2)Cas9-gRNA质粒,以及aptamer-donor单链之间的摩尔比例得到优化,同源性重组和基因修复的比例可达到最高(>15%)。
附图说明
图1为Cas9-gRNA质粒的构建流程示意图;
图2为Cas9-gRNA质粒的剪切活性试验结果;
图3为aptamer与Cas9-gRNA结合能力关系图;
图4为aptamer与Cas9-gRNA剪切活性关系图;
图5为靶基因修复率验证效果图。
具体实施方式
以下结合具体实施例进一步阐述本发明。下述实施例中所涉及的试验方法或分析方法,如无特别说明,均为常规方法,所用试剂如无特别说明,均为市售。
本实施例以用野生型抗癌基因p53置换突变致病性p53(SEQ ID No:3)为例,提供一种基因置换方法,具体分为以下几个步骤实施:
(1)合成双功能DNA单链:采用DNA合成仪用常规方法将Cas9aptamer(SEQ ID No:1)和野生型p53序列(SEQ ID No:2)连成一起的DNA单链(SEQ ID No:4)。
(2)合成Cas9-gRNA双功能-双表达质粒:常规方法构建Cas9-gRNA质粒,使其能表达Cas9蛋白和转录p53附近位点gRNA;具体构建方法见图1,首先合成寡核苷酸片段,用作PCR模板,其中含有定向到p53的gRNA2和gRNA1序列,被两个BbsI内切酶位点隔开,便于PCR后通过Gibson连接,加上gRNA1的转录启动子U6,再通过BbsI加入到表达Cas9的质粒上构成Cas9-gRNA双功能-双表达质粒。
完成构建后对Cas9-gRNA双功能-双表达质粒进行验证,结果见图2,其中,图2a是Cas9-gRNA双功能-双表达质粒中的gRNA2、gRNA1和Cas9编码序列的位置图;图2b表明Cas9在有gRNA1或gRNA2、gRNA1帮助下有剪切活性,而在只有gRNA2时不能定向到靶位点进行剪切;图2c和2d进一步证实了这一点。
从图3可以看出,在有无10nM(M=mol/L)aptamer存在的情况下,持续增加gRNA1的浓度,Cas9与gRNA1的结合都会增加,并不受这个Cas9特异性aptamer的影响,说明这个aptamer与gRNA1可同时与Cas9结合发挥各自的功能,aptamer并不干扰gRNA与Cas9结合。
从图4可以看出,在有无aptamer存在的情况下,并不影响Cas9的剪切活性。
(3)共转染:将双功能DNA单链、Cas9-gRNA质粒以摩尔比1:20于Lipofectamine试剂盒中,室温混合20min,再加到有细胞培养液的6孔板中,培养两天;
(4)筛选基因被置换的细胞株,基因组DNA提取后,对靶基因进行PCR扩增,扩增产物测序验证基因置换。将有p53突变的细胞株培养在48孔板上,Cas9-gRNA双功能-双表达质粒与p53-WT-Cy5(p53野生型片段、单链、Cy5荧光标记)或aptamer-p53-WT-Cy5共转染,数字荧光显微镜计数;几天后,收集细胞群落,分别提取DNA测序,得到统计数字,结果见图5,数据表明,有aptamer的供体DNA提高基因修复率十几倍。
SEQUENCE LISTING
<110> 上海伊丽萨生物科技有限公司
<120> 一种提高基因置换效率的方法
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Claims (5)
1.一种提高基因置换效率的方法,其特征在于,包括以下步骤:
(1)双功能DNA单链的合成:合成包含Cas9特异结合能力的aptamer和DNA供体的单链序列;
(2)质粒的制备:构建Cas9-gRNA质粒,使其能表达Cas9蛋白和转录靶位点附近的gRNA;
(3)把所得双功能DNA单链与质粒共转染到要进行基因置换的细胞或组织。
2.根据权利要求1所述提高基因置换效率的方法,其特征在于:所述aptamer序列为:5′-AGTCCATGGTAAACCCACCTTGGGGT-GACT-3′。
3.根据权利要求1所述提高基因置换效率的方法,其特征在于:步骤(3)中所述双功能DNA单链与质粒以摩尔比1:(15~25)共转染。
4.根据权利要求3所述提高基因置换效率的方法,其特征在于:步骤(3)中将双功能DNA单链、质粒以摩尔比1:20于Lipofectamine试剂盒中室温混合15~25min,再加入具有细胞培养液的6孔板中,培养两天后,筛选细胞株验证置换效果。
5.根据权利要求4所述提高基因置换效率的方法,其特征在于:所述细胞培养液浓度为60wt%。
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