CN107488649A - 一种Cpf1和p300核心结构域的融合蛋白、相应的DNA靶向激活系统和应用 - Google Patents
一种Cpf1和p300核心结构域的融合蛋白、相应的DNA靶向激活系统和应用 Download PDFInfo
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Abstract
本发明公开了一种Cpf1和p300核心结构域的融合蛋白、相应的DNA靶向激活系统和应用。本发明将具有酶切割活性的CRISPR/Cpf1突变为无酶切割活性有靶向基因识别特性,并融和p300蛋白转录激活作用,达到在哺乳动物细胞内靶向基因激活的目的,具有简单、高效、高特异性。脱靶率低、节约成本等优点。本发明可与现有的基于CRISPR/Cas9的靶向基因激活方法相互补充,拓宽了CRISPR/Cas系统的基因编辑范围,具有良好的应用前景。
Description
技术领域
本发明涉及基因工程和生物技术领域,具体涉及利用CRISPR/Cpf1及其变体和p300融和蛋白对哺乳动物细胞进行靶向基因激活。
背景技术
CRISPR/Cas(Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-Associated systems),全名是常间回文重复序列丛集/常间回文重复序列丛集关联蛋白系统,是细菌及古生菌的一种获得性免疫系统。2010年, Garneau等发现,在嗜热链球菌的CRISPRⅡ型系统中Cas9是唯一能够介导DNA 切割的Cas核酸酶。2013年,张锋等科学家首次报道了CRISPR-Cas9系统在哺乳动物基因组编辑中的应用,开启了CRISPR-Cas9强大的基因编辑功能成为生命科学领域的新星,获得广泛研究和应用。
尽管CRISPR比之前的基因编辑方法要简单得多,但是张锋认为仍有改进的余地。他和他的团队搜索了整个细菌王国,找到了一种替代实验室中常用Cas9 酶的选择。2015年,他们在金黄色葡萄球菌中发现了一个更小版本的Cas9即 Cpf1,全新基因编辑系统CRISPR/Cpf1酶更容易进入成熟细胞,可能将克服 CRISPR-Cas9系统应用中的一些限制。CRISPR/Cpf1归类于CRISPRⅡ型系统,其系统包括Cpf1蛋白和单一向导RNA,后者由19bp具种属特异性的保守重复序列(或scaffold骨架序列)和23bp的间隔区(Spacer)构成,Cpf1与其向导 RNA形成的蛋白-核酸复合物由向导RNA引导,识别具有5’T-rich PAM(原型间隔序列毗邻基序)靶序列,随后Cpf1蛋白中的核酸酶活性结构域对靶向DNA 双链进行切割,产生双粘性末端。
与Cas9相比,Cpf1有以下四大优势:第一,大小不同。Cas9剪切DNA需要两个小RNA分子,而Cpf1只需要一个。Cpf1酶比标准的SpCas9要小,更容易进入组织和细胞。第二,剪切方式不同。Cas9是在同一个位置同时剪切DNA 分子的双链,最后形成平末端;而Cpf1剪切后形成黏性末端,平末端通常不容易处理,粘性末端让DNA插入更可控。第三,剪切位置不同。Cpf1剪切时离识别位点很远,这让研究人员在编辑位置的选择上有了更多的选项。第四,Cpf1 系统在目标位置的选择上提供了灵活性,Cpf1复合物识别的5’T-rich PAM序列,而Cas9识别G-rich PAM。
在已知16种来源于不同菌属的Cpf1家族蛋白中,来自氨基酸球菌属(Acidominococcus)的AsCpf1亚型和来自毛螺旋菌科(Lachnospiraceae)的 LbCpf1亚型被证明在人源细胞系中作用活跃,具高效基因编辑潜质。目前对 CRISPR/Cpf1系统的认识和探究尚处于初始阶段,其成为新晋基因编辑工具的优良潜质具有极大的开发和应用空间。
p300蛋白具有组蛋白乙酰转移酶活性,使染色质结构重构,暴露基因的启动子和或增强子,同时富集转录因子于此区域,起到调控基因的高表达的作用.有报导指出p300蛋白的基因激活作用效果优于VP64等相似功能蛋白,但其在基因修饰领域的应用仍少于VP64等传统作用因子。
发明内容
本发明的目的在于:针对已有基因激活方法靶向性低,激活效率低,涉及作用原件繁多作用系统复杂等问题,将具有酶切割活性的CRISPR/Cpf1突变为无酶切割活性有靶向基因识别特性,并融和p300蛋白转录激活作用,提供一种可在哺乳动物细胞内靶向基因激活的融合蛋白、表达载体和相应的DNA靶向激活系统。
为了实现上述发明目的,本发明提供了一种融合蛋白,其含有两个异源多肽结构域,其中一个多肽结构域包含Cpf1蛋白,另一个多肽结构域包含具有转录激活活性的p300核心结构域;所述Cpf1蛋白包含AsCpf1亚型和LbCpf1亚型;且所述AsCpf1亚型中含有D908A单位点和/或E993A单位点氨基酸突变(突变后的AsCpf1蛋白为丧失核酸酶活性且保留特异性识别、结合目标DNA功能的变体d AsCpf1);所述LbCpf1亚型中含有D832A单位点和/或E925A单位点氨基酸突变(突变后的LbCpf1蛋白为丧失核酸酶活性且保留特异性识别、结合目标DNA功能的变体d LbCpf1)。
为了实现上述发明目的,本发明还提供了一种表达载体,用于表达上述融合蛋白。
作为本发明所述表达载体的一种优选技术方案,所述表达载体的启动子为 CAG,剪切肽为P2A,入核信号为NLS和SV40NLS,Cpf1蛋白和p300核心结构域之间插入NLS信号和3xHA标签。
为了实现上述发明目的,本发明还提供了一种DNA靶向激活系统,其包含上述融合蛋白和至少一种向导RNA;所述向导RNA为针对目的基因启动子或增强子区域设计的一段长度为23bp的序列,其5’端为19bp的骨架序列,其中, AsCpf1的骨架序列如SEQ ID NO:1所示;LbCpf1的骨架序列如SEQ ID NO:2 所示。
作为本发明所述DNA靶向激活系统的一种优选技术方案,所述启动子或增强子区域位于MyoD、IL1RN和/或OCT4。
作为本发明所述DNA靶向激活系统的一种优选技术方案,所述DNA靶向激活系统含有四种不同的向导RNA。
本发明DNA靶向激活系统可对哺乳动物细胞进行靶向基因激活,所述哺乳动物细胞包括293T、U2OS和MCF7。
具体地,可以包括如下步骤:
(1)构建含有至少一种氨基酸突变的Cpf1表达载体;
(2)构建表达特定向导RNA的载体;
(3)将步骤(1)所述Cpf1表达载体和步骤(2)所述表达特定向导RNA 的载体混合,通过脂质体转染试剂聚乙烯亚胺(PEI)共转染到哺乳动物细胞,在细胞内表达的融合蛋白与其特定向导RNA结合,将p300核心蛋靶向目标基因区。
之后,可通过转染培养48h,提取mRNA,检查目标基因mRNA表达水平;再经全转录水平mRNA测序,分析靶向激活结果和特异性水平。
相对于现有技术,本发明具有如下优点和有益效果:
本发明所利用的CRISPR/Cpf1系统特异性高,脱靶率低,所需向导RNA序列较Cas9简单,具有易于合成,节约成本的优点;与p300核心区结合成的融合蛋白具有激活效率高,特异性高(图26)的特点。此外,因Cpf1向导RNA 识别的PAM序列有异于Cas9,本发明方法可与已有的基于CRISPR/Cas9的靶向基因激活方法相互补充,拓宽CRISPR/Cas系统的基因编辑范围。本发明方法还具有操作简便,涉及作用原件少、作用效果优良等优势,具备成为高效高特异性的靶向基因激活方法的潜质。
附图说明
图1是AsCpf1原始质粒图谱。
图2是LbCpf1原始质粒图谱。
图3是AsCpf1-3xHATag质粒图谱。
图4是LbCpf1-3xHATag质粒图谱。
图5是p2U6-pCAG-dCas9-mCherry质粒图谱。
图6是p2U6-pCAG-AsCpf1-mCherry质粒图谱。
图7是p2U6-pCAG-LbCpf1-mCherry质粒图谱。
图8是p1U6-Asscaf-pCAG-AsCpf1-mCherry质粒图谱。
图9是p1U6-Lbscaf-pCAG-LbCpf1-mCherry质粒图谱。
图10是p1U6-Asscaf-dAsD908A-mCherry质粒图谱。
图11是p1U6-Asscaf-dAsE993A–mCherry质粒图谱。
图12是dAsCpf1酶切割活性检验PAGE胶电泳结果。
图13是dLbCpf1酶切割活性检验PAGE胶电泳结果。
图14是T7EIdAs/dLbCpf1酶切割活性检验电泳结果。
图15是p1U6-As scaf-pCAG-dAsCpf1D908A-p300-mCherry质粒图谱。
图16是p1U6-As scaf-pCAG-dAsCpf1E993A-p300-mCherry质粒图谱。
图17是p1U6-As scaf-pCAG-dLbCpf1D832A-p300-mCherry质粒图谱。
图18是p1U6-As scaf-pCAG-dLbCpf1E925A-p300-mCherry质粒图谱。
图19是pCAG-dAsCpf1(D908A)-p300-mCherry质粒图谱。
图20是pCAG-dAsCpf1(E993A)-p300-mCherry质粒图谱。
图21是pCAG-dLbCpf1(D832A)-p300-mCherry质粒图谱。
图22是pCAG-dLbCpf1(E925A)-p300-mCherry质粒图谱。
图23是pBlu2SKP质粒图谱。
图24是pBlu2SKP-pU6-As质粒图谱。
图25是western blot检测Cpf1-p300融合蛋白表达结果图。
图26是a是Cpf1-P300介导基因激活模式图,b是Cpf1图突变体模式图, c是在293T细胞中,靶向基因MyoD、IL1RN和OCT4启动子处,激活两者情况的Real-Time PCR柱状结果图,d是通过全转录水平mRNA测序,MyoD激活水平散点图。
图27是在细胞系U2OS和MCF7中靶向基因MyoD和IL1RN启动子处,激活两者情况的Real-Time PCR柱状结果图。
图28是在293T中,靶向基因MyoD和IL1RN增强子处,激活两者情况的 Real-TimePCR柱状结果图。
具体实施方式
为了使本发明的目的、技术方案和有益技术效果更加清晰,以下结合实施例,对本发明进行进一步详细说明。应当理解的是,本说明书中描述的实施例仅仅是为了解释本发明,并非为了限定本发明,实施例的参数、比例等可因地制宜做出选择而对结果并无实质性影响。
实施例
As/LbCpf1-3xHATag(图3~4)载体构建:
由商业公司合成一段含BamHI-3xHATag-XohI-AGC-(Ser)-AgeI的单链DNA 片段Oligo-F和Oligo-R,其碱基序列如SEQ ID NO:3~4所示。
两条部分互补配对单链DNA片段合成双链的DNA片段。具体步骤如下:
10ul 100uM Oligo-F和10ul 100uM Oligo-R预混于1.5ml EP管中,用烧杯煮沸800ml的蒸馏水,将1.5ml EP管置于沸水中5分钟,取出1.5ml EP管室温放置过夜。
用BamHI和NdeI切割由商业公司合成的AsCpf1/LbCpf1原始质粒(图 1~2),用0.8%琼脂糖凝胶电泳分析酶产物,分别切胶回收4015bp条带,并利用 NanoDrop测定回收片段浓度,将线性化的AsCpf1/LbCpf1原始质粒与上述双链的DNA片段通过NEB购买的T4DNA连接酶连接,然后将连接产物转化到 TOP10大肠杆菌中,涂在含有100μg/mL氨苄青霉素的LB固体平板,37℃培养过夜,然后挑取单个克隆,37℃250rpm摇菌后提取质粒进行DNA测序,由此筛选到构建的载体,命名为As/LbCpf1-3xHATa(图3~4)。
T4连接体系10μL,具体包括:
T4连接酶1μL,10x T4ligase buffer1μL,稀释15倍的双链的DNA片段2μL,线性化AsCpf1/LbCpf1原始质粒2μL,ddH2O4μL,反应条件:25℃水浴1h。
p2U6-pCAG-As/LbCpf1-mCherry载体构建(图6~7):
用EcoRI和AgeI切割As/LbCpf1-3xHATag(图3~4)和由商业公司合成的p2U6-pCAG-Cas9-mCherry(图5)质粒,用0.8%琼脂糖凝胶电泳分析酶产物,切胶分别回收4000bp和6000bp条带,并利用NanoDrop测定浓度.上述两个片段通过NEB购买的T4DNA链接酶连接,然后将连接产物转化到TOP10大肠杆菌中,涂在含有100μg/mL氨苄青霉素的LB固体平板,37℃培养过夜,然后挑取单个克隆,37℃250rpm摇菌后提取质粒进行DNA测序,由此筛选到构建的载体,得到质粒命名为p2U6-pCAG-As/LbCpf1-mCherry(图6~7).
T4连接体系10μL,包括:
T4连接酶1μL,10x T4ligase buffer1μL,线性化As/LbCpf1-3xHATag: 2μL,线性化p2U6-pCAG-Cas9-mCherry质粒2μL,ddH2O4μL,反应条件:25℃水浴1h。
p1U6-pCAG-As/LbCpf1-scaffold-mCherry(图8~9)载体构建“”
由商业公司合成AsCpf1和LbCpf1向导RNA的骨架序列,序列如下:
AsCpf1-scaffold-OLIGO F如SEQ ID NO:5所示,AsCpf1-scaffold-OLIGO R 如SEQ ID NO:6所示,LbCpf1-scaffold-OLIGO F如SEQ ID NO:7所示, LbCpf1-scaffold-OLIGO R如SEQ ID NO:8所示。
AsCpf1/LbCpf1两条互补配对的单链DNA骨架序列分别合成双链的 AsCpf1/LbCpf1DNA骨架片段.具体步骤如下:
10ul 100uM F和10ul 100uM R预混于1.5ml EP管中,用烧杯煮沸800ml 的蒸馏水,将1.5ml EP管置于沸水中5分钟,取出1.5ml EP管室温放置过夜。
用BaeⅠ和MluⅠ切割p2U6-pCAG-As/LbCpf1-mCherry(图6~7)载体,用0.8%琼脂糖凝胶电泳分析酶产物,切胶回收条带,并利用NanoDrop测定浓度.上述片段分别与AsCpf1/LbCpf1骨架序片段通过NEB购买的T4DNA链接酶连接,然后将连接产物转化到TOP10大肠杆菌中,涂在含有100μg/mL氨苄青霉素的LB固体平板,37℃培养过夜,然后挑取单个克隆,37℃250rpm摇菌后提取质粒进行DNA测序,由此筛选到构建的载体得到质粒命名为 p1U6-As/Lbscaf-pCAG-As/LbCpf1-mCherry。(图8~9)。
T4连接体系10μL,包括:
T4连接酶1μL,10x T4ligase buffer1μL,稀释15倍的双链的AsCpf1/LbCpf1 骨架片段2μL,线性化p2U6-pCAG-As/LbCpf1-mCherry质粒2μL,ddH2O4μL,反应条件:25℃水浴1h。
核酸酶活性沉默型dAs/LbCpf1突变载体构建
具体突变位点包括AsCpf1的D908A和E993A中的至少一个,LbCpf1的 D832A和E925A中的至少一个。具体步骤如下:
shuttle-As/LbCpf1载体质粒构建:
用EcoR I和BamH I切割shuttle质粒和p2U6-pCAG-As/LbCpf1-mCherry(图 6,7)质粒,用0.8%琼脂糖凝胶电泳分析酶产物,切胶分别回收2521bp和4000bp 条带,并利用NanoDrop测定浓度,上述两者通过NEB购买的T4DNA连接酶连接,然后将连接产物转化到TOP10大肠杆菌中,涂在含有100μg/mL氨苄青霉素的LB固体平板,37℃培养过夜,然后挑取单个克隆,37℃250rpm摇菌后提取质粒进行DNA测序,由此筛选到构建的载体质粒命名为shuttle-As/LbCpf1。
shuttle-dAs/LbCpf1载体质粒构建:
用Apa1和BamH1切割shuttle-As/LbCpf1质粒,同时以shuttle-As/LbCpf1 为模版PCR,所用引物序列如下表1,具体反应体系如下:
表1
PCR体系如下:模版质粒:1μL(50ng),前向引物:1μL(10μM),后向引物:1μL(10μM),2ⅹTaq酶混合物:25μL,ddH2O:至50μL。
PCR程序如下:①95℃:2min,②95℃:30s,③58℃:30s,④72℃:30s,②③④循环39次,⑤72℃2min,⑥16℃保存。
用0.8%琼脂糖凝胶电泳分析酶切和PCR产物,切胶分别回收,使用从NEB 公司购买的连接试剂盒Gibson Assembly将上述回收的两个片段连接,将连接产物转化到TOP10大肠杆菌中,涂在含有100μg/mL氨苄青霉素的LB固体平板, 37℃培养过夜,挑取单个克隆,37℃250rpm摇菌后提取质粒进行DNA测序,所得载体包括四个突变体AsCpf1D908A/E993A和LbCpf1D832A/E925A。由此筛选所得到的载体质粒命名为:shuttle-dAsD908A Cpf1/shuttle-dAsE993A Cpf1/ shuttle-dLbD832A Cpf1/shuttle-dLb E925A Cpf1。
p1U6-As/Lbscaf-dAs/LbCpf1-mCherry(图10~11)载体构建:
用EcoRⅠ和BamHⅠ分别切割shuttle-dAs/Lb Cpf1和p1U6-As/Lb scaf-pCAG-As/LbCpf1-mCherry质粒,用0.8%琼脂糖凝胶电泳分析酶产物,切胶分别回收4000bp和5471bp条带,两者通过NEB购买的T4DNA连接酶连接,产物转化到TOP10大肠杆菌中,涂在含有100μg/mL氨苄青霉素的LB固体平板, 37℃培养过夜,挑取单个克隆,37℃250rpm摇菌后提取质粒进行DNA测序,筛选所得到载体质粒命名为:p1U6-Asscaf-dAsD908A-mCherry(图10) /p1U6-Asscaf-dAsE993A–mCherry(图11)/p1U6-Lbscaf-dLbD832A–mCherry/ p1U6-Lbscaf-dLb E925A-mCherry。
选取DNMT-1为靶基因位点,构建p1U6-DNMTgRNA-dAs/LbCpf1-mCherry 载体。
设计合成gRNA,其序列如下:
DNMT-1-3-F如SEQ ID NO:20所示,DNMT-1-3-R如SEQ ID NO:21所示。
两条部分互补配对单链DNA片段合成双链的DNA片段。
用BaeI切割p1U6-As/Lb scaf-dAs/LbCpf1-mCherry(图10~11)质粒,与上述合成的双链DNA片段通过NEB购买的T4DNA连接酶连接,产物转化到TOP10 大肠杆菌中,涂在含有100μg/mL氨苄青霉素的LB固体平板,37℃培养过夜,挑取单个克隆,37℃250rpm摇菌后提取质粒进行DNA测序,所筛选得到的载体质粒的命名为p1U6-DNMTgRNA-dAs/LbCpf1-mCherry。
核酸酶活性沉默dCpf1功能鉴定,包括AsCpf1D908A/E993A和LbCpf1 D832A/E925A。
于24孔板铺1.5*105细胞每孔,置于37℃,5%CO2孵箱培养18~24小时,至细胞密度为65~70%,换为无血清的基础培养基DMEM,以待质粒转染,使用脂质体转染试剂聚乙烯亚胺(PEI),具体方法如下:
制备PEI(10ⅹ):1mg/ml,pH7.0,制备HEBS:20Mm Hepes,150mM NaCl, pH7.4。
转染条件:DNA500ng每孔,PEI:total DNA=3:1(m/m)。
先将待转染质粒预混于30μl HEBS中,室温静置5min,再与PEI混合,室温静置25min后均匀的加入24孔板孔中,置于37℃,5%CO2孵箱培养,8h后换为全培基,48h后胰酶消化后收取细胞,用十二烷基肌氨酸钠缓冲溶液提取细胞基因组DNA,具体方法如下:
制备:十二烷基肌氨酸钠缓冲溶液:0.5%十二烷基肌氨酸钠,10Mm Tris,10MmEDTA,10Mm NaCL,1%蛋白酶K(现配现加)。加入200μl十二烷基肌氨酸钠缓冲溶液重悬细胞,置于55℃水浴中过夜,加入20%饱和NaCl,再加入 2倍体积的无水乙醇,析出白色沉淀,12000rpm,离心10min,去上清液,再加入1mL70%的乙醇清洗沉淀,12000rpm,离心10min,反复两次。最后挥干乙醇后,加入适量的ddH2O溶解,利用NanoDrop测定浓度。
设计引物在目标基因DNMT-1切割位点两侧,PCR产物300bp,用5%聚丙烯酰胺凝胶电泳(Polyacrylamide Gel Electrophoresis Based Genotyping/PAGE) 进行切割与否鉴定,结果如图12、图13所示,相比于野生AsCpf1(WT),AsCpf1 单突变体D908A(M908)、单突变体E993A(M993)和双突变体 AsCpf1D908A/E993A(DM)无核酸酶切割,同样突变体LbCpf1D832A(M832)和或E925A(M925)无核酸酶切割,可见各突变体DNA酶切割活性明显降低或丧失。PAGE所用引物序列如下:
DNMT1-3-PAGE-F如SEQ ID NO:22所示,DNMT1-3-PAGE-R如SEQ ID NO:23所示。
PCR体系如下:模版质粒:1μL(30ng),前向引物:0.6μL(10μM),后向引物:0.6μL(10μM),2ⅹTaq酶混合物:10μL,ddH2O:至20μL。
PCR-PAGE程序如下:①95℃:2min,②95℃:30s,③58℃:30s,④72℃:30s,②③④循环35次,⑤72℃2min,⑥95℃5min,⑦95℃-85℃1℃/s,⑧16℃保存。
T7EI验证dLbCpf1核酸酶切割活性,具体操作如下:
使用引物DNMT1-3-PAGE-F和DNMT1-3-PAGE-R进行T7EI实验,两引物位于目标基因DNMT-1切割位点两侧,产物大小400bp,经过T7核酸内切酶切割可得到120bp和280bp条带,具体过程如下:
PCR体系如下:模版质粒:1μL(100ng),前向引物:2.5μL(10μM),后向引物:2.5μL(10μM),2ⅹTaq酶混合物:25μL,ddH2O:至50μL。
PCR程序如下:①95℃:2min,②95℃:30s,③58℃:30s,④72℃:40s,②③④循环40次,⑤72℃2min,⑥16℃保存。
使用PCR纯化试剂盒纯化PCR产物,利用NanoDrop测定浓度.取400ngPCR 产物加入2uL 10xNEB缓冲液2,补水至20uL,然后退火,再加0.4uLT7核酸内切酶,37℃,2小时,用2%琼脂糖凝胶电泳分析切割产物,结果如图14,退火具体过程如下:①95℃2min,②-2℃/s to85℃,③-0.1℃/s to 25℃,④16℃保存。
dAs/LbCpf1-p300融合蛋白表达载体构建:
以p1U6-dCas9-p300mutant-iRFP670质粒为模板,通过PCR获得p300Core 蛋白序列片段,并引入XhoI和AgeI酶切位点,所用引物序列如下:P300PCR Primer-F如SEQ ID NO:24所示,P300PCR Primer-R如SEQ ID NO:25所示。
用Age I和Xho I切割PCR所得p300片段和p1U6-As/Lb scaf-pCAG-dAs/LbCpf1-mCherry(图11,12)质粒,用0.8%琼脂糖凝胶电泳分析酶产物,切胶分别回收条带,两者通过NEB购买的T4DNA连接酶连接,产物转化到TOP10大肠杆菌中,涂在含有100μg/mL氨苄青霉素的LB固体平板,37℃培养过夜,然后挑取单个克隆,37℃250rpm摇菌后进行DNA测序,筛选得到载体质粒命名为:p1U6-As/Lbscaf-pCAG-dAs/LbCpf1-p300-mCherry质粒(图 15~18)。
用MfeI和MluI双酶切割,经Klenow Fragment修饰和NEB购买的T4DNA 连接酶连接,产物转化到TOP10大肠杆菌中,涂在含有100μg/mL氨苄青霉素的LB固体平板,37℃培养过夜,然后挑取单个克隆,37℃250rpm摇菌后进行 DNA测序,筛选所得到的载体质粒命名为:pCAG-dAs/LbCpf1-p300-mCherry融合蛋白表达终质粒(图19~22)。
pBlu2SKP-As/Lbscaffold载体质粒构造:
以p1U6-As/Lb scaf-pCAG-As/LbCpf1-p300-mCherry为模板,通过PCR合成含U6-scaffold片段,并引入SalI酶切位点,所用引物序列如下:U6PCR-F如 SEQ ID NO:26所示,As scaffold-R如SEQ ID NO:27所示,Lb scaffold-R如SEQ ID NO:28所示。
用Sal I和EcoR V切割购买的质粒pBlu2SKP(Agilent Technologies:212205)(图23)和上述PCR产物,经过NEB购买的T4DNA连接酶连接,所筛选得到载体质粒的命名为pBlu2SKP-As/Lbscaffold载体(图24)。
pBlu2SKP-gRNA-As/Lbscaffold载体质粒构造:
用EcoR V和Xma I切割上述所得pBlu2SKP-As/Lbscaffold载体,通过 Benchling网站设计gRNA,由商业公司合成gRNA见表2,并两条部分互补配对单链DNA片段合成双链的DNA片段,将上述线性化载体和双链的DNA片段通过T4连接酶连接,筛选所得的载体质粒命名为:pBlu2SKP-gRNA-As/Lbscaffold。
表2
dCpf1-p300融合蛋白功能鉴定,包括dLbCpf1D832A/E925A
于12孔板铺3*105细胞每孔,置于37℃,5%CO2孵箱培养18~24小时,至细胞密度为65-70%,换为无血清的基础培养基DMEM,以待质粒转染,使用脂质体转染试剂聚乙烯亚胺(PEI),具体方法如下:
制备PEI(10ⅹ)1mg/ml,pH7.0,制备HEBS20Mm Hepes,150mM NaCl, pH7.4。
转染条件:DNA:1000ng每孔,PEI:total DNA=4:1(g/g)。
先将待转染质粒包括pCAG-dLbCpf1-p300-mCherry和 pBlu2SKP-gRNA-Lbscaffold,预混于30μlHEBS中,室温静置5min,再与PEI混合,室温静置25min后均匀的加入12孔板孔中,置于37℃,5%CO2孵箱培养,8h后换为全培基,培养48h后,一半细胞量提取细胞全蛋白质进行western blot检测,结果显示dLbCpf1在哺乳动物细胞中表达(图25),一半细胞量使用QIAGEN RNA 提取试剂盒提取总RNA,实验步骤参见(Qiagen’s RNA MiniKit:74106说明书),设计引物序列,具体如表3所示,进行Real-Time PCR实验,实验步骤参见 (TaKaRa:RR036Q说明书中Biosystems 7500RT-Time PCR System,StepOnePlus), 结果显示,在人肾上皮细胞系293T中,目标基因MyoD、IL1RN、OCT4表达量明显升高,同样在乳腺癌细胞系MCF7和人骨肉瘤细胞系U2OS两种不类别细胞系中MyoD、IL1RN表达量也明显升高,结果如图26~28所示,因此本发明构建的Cpf1-p300靶向激活系统具有明显的广谱性。
表3
Real-timePCR引物 | 序列(5’-3’) |
GAPDH-qPCR-F | SEQ ID NO:61 |
GAPDH-qPCR-R | SEQ ID NO:62c |
MyoD-qPCR-F | SEQ ID NO:63 |
MyoD-qPCT-R | SEQ ID NO:64 |
IL1RN-qPCR-F | SEQ ID NO:65 |
IL1RN-qPCR-R | SEQ ID NO:66 |
OCT-4-qPCR-F | SEQ ID NO:67 |
OCT-4-qPCR-R | SEQ ID NO:68 |
将收取RNA送往公司,进行全转录水平mRNA水平测序,结果如图26中d,显示MYOD基因表达水平明显高于对照组,说明成功构建基因组靶向激活系统,同时为了验证Cpf1-p300特异性水平,进行了特异性分析,结果显示如表4、5、6,实验组dLbCpf1(M832)单基因突变体相比于对照组dLbCpf1(C832)激活MyoD 基因中,分析测序结果显示,转录水平较高的前5位基因中,MyoD水平明显高于其它基因,同样,实验组dLbCpf1(M925)单基因突变体和实验组dLbCpf1(DM) 相比于对照组dLbCpf1(C925)单基因突变体和dLbCpf1(CDM),MyoD基因激活水平也明显高于其它基因,因此说明本发明所构建的Cpf1-p300靶向激活系统特异性高,具有很好的应用前景。
表4
表5
表6
序列表
<110> 南方医科大学
<120> 一种Cpf1和p300核心结构域的融合蛋白、相应的DNA靶向激活系统和应用
<160> 68
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> AsCpf1的骨架序列(Artificial Sequence)
<400> 1
taatttctac tcttgtagat 20
<210> 2
<211> 20
<212> DNA
<213> LbCpf1的骨架序列(Artificial Sequence)
<400> 2
aatttctact aagtgtagat 20
<210> 3
<211> 101
<212> DNA
<213> Oligo-F(Artificial Sequence)
<400> 3
gatcctaccc atacgatgtt ccagattacg cttatcccta cgacgtgcct gattatgcat 60
acccatacga tgtccccgac tatgccctcg agagcaccgg t 101
<210> 4
<211> 99
<212> DNA
<213> Oligo-R(Artificial Sequence)
<400> 4
taaccggtgc tctcgagggc atagtcgggg acatcgtatg ggtatgcata atcaggcacg 60
tcgtagggat aagcgtaatc tggaacatcg tatgggtag 99
<210> 5
<211> 56
<212> DNA
<213> AsCpf1-scaffold-OLIGO F(Artificial Sequence)
<400> 5
taatttctac tcttgtagat atcaccgcct acgtcagtac ctacaagctt ttttta 56
<210> 6
<211> 65
<212> DNA
<213> AsCpf1-scaffold-OLIGO R(Artificial Sequence)
<400> 6
cgcgtaaaaa aagcttgtag gtactgacgt aggcggtgat atctacaaga gtagaaatta 60
cggtg 65
<210> 7
<211> 56
<212> DNA
<213> LbCpf1-scaffold-OLIGO F(Artificial Sequence)
<400> 7
aatttctact aagtgtagat atcaccgcct acgtcagtac ctacaagctt ttttta 56
<210> 8
<211> 65
<212> DNA
<213> LbCpf1-scaffold-OLIGO R(Artificial Sequence)
<400> 8
cgcgtaaaaa aagcttgtag gtactgacgt aggcggtgat atctacactt agtagaaatt 60
cggtg 65
<210> 9
<211> 29
<212> DNA
<213> AsCpf1-Mut-D908A-F(Artificial Sequence)
<400> 9
cgacctgtct gatgaggcca gggccctgc 29
<210> 10
<211> 29
<212> DNA
<213> AsCpf1-Mut-D908A- Rm(Artificial Sequence)
<400> 10
ctctcgcccc gggcgatgcc gatgatagg 29
<210> 11
<211> 27
<212> DNA
<213> AsCpf1-Mut-D908A- Fm(Artificial Sequence)
<400> 11
atcggcatcg cccggggcga gagaaac 27
<210> 12
<211> 27
<212> DNA
<213> AsCpf1-Mut-D908A- R(Artificial Sequence)
<400> 12
aaagtggcac cgagtcggtg cggatcc 27
<210> 13
<211> 27
<212> DNA
<213> AsCpf1-Mut-E993A- Rm(Artificial Sequence)
<400> 13
attcaggttg gctagcacca ccacggc 27
<210> 14
<211> 27
<212> DNA
<213> AsCpf1-Mut-E993A- Fm(Artificial Sequence)
<400> 14
ggtggtgcta gccaacctga atttcgg 27
<210> 15
<211> 36
<212> DNA
<213> LbCpf1-Mut-D832A-Fm(Artificial Sequence)
<400> 15
aacccctatg tgatcggcat cgctaggggc gagcgc 36
<210> 16
<211> 33
<212> DNA
<213> LbCpf1-Mut-D832A- R(Artificial Sequence)
<400> 16
cttgaaaaag tggcaccgag tcggtgcgga tcc 33
<210> 17
<211> 32
<212> DNA
<213> LbCpf1-Mut-E925A-F(Artificial Sequence)
<400> 17
acgataaccc ctatgtgatc ggcatcgata gg 32
<210> 18
<211> 32
<212> DNA
<213> LbCpf1-Mut-E925A- Rm(Artificial Sequence)
<400> 18
agttcaggtc ggctagcgcg atcacggcat cg 32
<210> 19
<211> 32
<212> DNA
<213> LbCpf1-Mut-E925A- Fm(Artificial Sequence)
<400> 19
ccgtgatcgc gctagccgac ctgaactctg gc 32
<210> 20
<211> 28
<212> DNA
<213> DNMT-1-3-F(Artificial Sequence)
<400> 20
ctgatggtcc atgtctgtta ctcttttt 28
<210> 21
<211> 28
<212> DNA
<213> DNMT-1-3-R(Artificial Sequence)
<400> 21
gagtaacaga catggaccat cagatcta 28
<210> 22
<211> 20
<212> DNA
<213> DNMT1-3-PAGE-F(Artificial Sequence)
<400> 22
caagtgctta gagcaggcgt 20
<210> 23
<211> 20
<212> DNA
<213> DNMT1-3-PAGE-R(Artificial Sequence)
<400> 23
gtgacgggag ggcagaacta 20
<210> 24
<211> 26
<212> DNA
<213> P300 PCR Primer-F(Artificial Sequence)
<400> 24
gcgcctcgag attttcaaac cagaag 26
<210> 25
<211> 27
<212> DNA
<213> P300 PCR Primer-R(Artificial Sequence)
<400> 25
atataccggt gtcctggctc tgcgtgt 27
<210> 26
<211> 27
<212> DNA
<213> U6PCR-F(Artificial Sequence)
<400> 26
tatagtcgac aaggtcgggc aggaaga 27
<210> 27
<211> 21
<212> DNA
<213> As scaffold-R(Artificial Sequence)
<400> 27
cgcggatatc tacaagagta g 21
<210> 28
<211> 21
<212> DNA
<213> Lb scaffold-R(Artificial Sequence)
<400> 28
cgcggatatc tacacttagt a 21
<210> 29
<211> 31
<212> DNA
<213> MyoD-P 1-F(Artificial Sequence)
<400> 29
ggctactacg gataaatagc ccattttttt c 31
<210> 30
<211> 35
<212> DNA
<213> MyoD-P 1-R(Artificial Sequence)
<400> 30
ccgggaaaaa aatgggctat ttatccgtag tagcc 35
<210> 31
<211> 31
<212> DNA
<213> MyoD-P 2-F(Artificial Sequence)
<400> 31
tccgtagtag cctaaacgcc ccgttttttt c 31
<210> 32
<211> 35
<212> DNA
<213> MyoD-P 2-R(Artificial Sequence)
<400> 32
ccgggaaaaa aacggggcgt ttaggctact acgga 35
<210> 33
<211> 31
<212> DNA
<213> MyoD-P 3-F(Artificial Sequence)
<400> 33
gaaagggcgt gccggagagc caattttttt c 31
<210> 34
<211> 35
<212> DNA
<213> MyoD-P 3-R(Artificial Sequence)
<400> 34
ccgggaaaaa aattggctct ccggcacgcc ctttc 35
<210> 35
<211> 31
<212> DNA
<213> MyoD-P 4-F(Artificial Sequence)
<400> 35
ccgcggatac agcagtcggg tgtttttttt c 31
<210> 36
<211> 35
<212> DNA
<213> MyoD-P 4-R(Artificial Sequence)
<400> 36
ccgggaaaaa aaacacccga ctgctgtatc cgcgg 35
<210> 37
<211> 31
<212> DNA
<213> MyoD-DRR-1-F(Artificial Sequence)
<400> 37
gcgcccccca cctcccggcc agattttttt c 31
<210> 38
<211> 35
<212> DNA
<213> MyoD-DRR-1-R(Artificial Sequence)
<400> 38
ccgggaaaaa aatctggccg ggaggtgggg ggcgc 35
<210> 39
<211> 31
<212> DNA
<213> MyoD-DRR-2-F(Artificial Sequence)
<400> 39
tatatatagc ctctggaaac ccattttttt c 31
<210> 40
<211> 35
<212> DNA
<213> MyoD-DRR-2-F(Artificial Sequence)
<400> 40
ccgggaaaaa aatgggtttc cagaggctat atata 35
<210> 41
<211> 31
<212> DNA
<213> MyoD-DRR-3-F(Artificial Sequence)
<400> 41
ccagggagca agtttgtcag gggttttttt c 31
<210> 42
<211> 35
<212> DNA
<213> MyoD-DRR-3-R(Artificial Sequence)
<400> 42
ccgggaaaaa aacccctgac aaacttgctc cctgg 35
<210> 43
<211> 31
<212> DNA
<213> MyoD-DRR-4-F(Artificial Sequence)
<400> 43
cagaggccag ctctccattt atattttttt c 31
<210> 44
<211> 35
<212> DNA
<213> MyoD-DRR-4-R(Artificial Sequence)
<400> 44
ccgggaaaaa aatataaatg gagagctggc ctctg 35
<210> 45
<211> 29
<212> DNA
<213> OCT4pro-1-F(Artificial Sequence)
<400> 45
gcccagtaga tcgaggctac atttttttc 29
<210> 46
<211> 33
<212> DNA
<213> OCT4pro-1-R(Artificial Sequence)
<400> 46
ccgggaaaaa aatgtagcct cgatctactg ggc 33
<210> 47
<211> 31
<212> DNA
<213> OCT4pro-2-F(Artificial Sequence)
<400> 47
tgtgggggac ctgcactgag gtcttttttt c 31
<210> 48
<211> 35
<212> DNA
<213> OCT4pro-2-R(Artificial Sequence)
<400> 48
ccgggaaaaa aagacctcag tgcaggtccc ccaca 35
<210> 49
<211> 31
<212> DNA
<213> OCT4pro-3-F(Artificial Sequence)
<400> 49
cctaatggtg gtggcaatgg tgtttttttt c 31
<210> 50
<211> 35
<212> DNA
<213> OCT4pro-3-R(Artificial Sequence)
<400> 50
ccgggaaaaa aaacaccatt gccaccacca ttagg 35
<210> 51
<211> 31
<212> DNA
<213> OCT4pro-4-F(Artificial Sequence)
<400> 51
tccccccacc tccctctcct ccattttttt c 31
<210> 52
<211> 35
<212> DNA
<213> OCT4pro-4-R(Artificial Sequence)
<400> 52
ccgggaaaaa aatggaggag agggaggtgg gggga 35
<210> 53
<211> 31
<212> DNA
<213> OCT4PE-1-F(Artificial Sequence)
<400> 53
ggccccctcc actatggaac ctgttttttt c 31
<210> 54
<211> 35
<212> DNA
<213> OCT4PE-1-R(Artificial Sequence)
<400> 54
ccgggaaaaa aacaggttcc atagtggagg gggcc 35
<210> 55
<211> 31
<212> DNA
<213> OCT4PE-2-F(Artificial Sequence)
<400> 55
gggttagagc tgccccctct gggttttttt c 31
<210> 56
<211> 35
<212> DNA
<213> OCT4PE-2-R(Artificial Sequence)
<400> 56
ccgggaaaaa aacccagagg gggcagctct aaccc 35
<210> 57
<211> 31
<212> DNA
<213> OCT4PE-3-F(Artificial Sequence)
<400> 57
gcgtctctga aggggattct gtgttttttt c 31
<210> 58
<211> 35
<212> DNA
<213> OCT4PE-3-R(Artificial Sequence)
<400> 58
ccgggaaaaa aacacagaat ccccttcaga gacgc 35
<210> 59
<211> 31
<212> DNA
<213> OCT4PE-4-F(Artificial Sequence)
<400> 59
ccaacctttg ctgaaacaga gtgttttttt c 31
<210> 60
<211> 35
<212> DNA
<213> OCT4PE-4-R(Artificial Sequence)
<400> 60
ccgggaaaaa aacactctgt ttcagcaaag gttgg 35
<210> 61
<211> 20
<212> DNA
<213> GAPDH-qPCR-F(Artificial Sequence)
<400> 61
agaaggctgg ggctcatttg 20
<210> 62
<211> 20
<212> DNA
<213> GAPDH-qPCR-R(Artificial Sequence)
<400> 62
aggggccatc cacagtcttc 20
<210> 63
<211> 20
<212> DNA
<213> MyoD-qPCR-F(Artificial Sequence)
<400> 63
tccctctttc acggtctcac 20
<210> 64
<211> 20
<212> DNA
<213> MyoD-qPCT-R(Artificial Sequence)
<400> 64
aacacccgac tgctgtatcc 20
<210> 65
<211> 20
<212> DNA
<213> IL1RN-qPCR-F(Artificial Sequence)
<400> 65
ggaatccatg gagggaagat 20
<210> 66
<211> 20
<212> DNA
<213> IL1RN-qPCR-R(Artificial Sequence)
<400> 66
tgttctcgct caggtcagtg 20
<210> 67
<211> 30
<212> DNA
<213> OCT-4-qPCR-F(Artificial Sequence)
<400> 67
cgaaagagaa agcgaaccag tatcgagaac 30
<210> 68
<211> 27
<212> DNA
<213> OCT-4-qPCR-R(Artificial Sequence)
<400> 68
cgttgtgcat agtcgctgct tgatcgc 27
Claims (9)
1.一种融合蛋白,其特征在于,含有两个异源多肽结构域,其中一个多肽结构域包含Cpf1蛋白,另一个多肽结构域包含具有转录激活活性的p300核心结构域;所述Cpf1蛋白包含AsCpf1亚型和LbCpf1亚型;且所述AsCpf1亚型中含有D908A单位点和/或E993A单位点氨基酸突变;所述LbCpf1亚型中含有D832A单位点和/或E925A单位点氨基酸突变。
2.一种表达载体,其特征在于,该表达载体用于表达权利要求1所述的融合蛋白。
3.根据权利要求2所述的表达载体,其特征在于,所述表达载体的启动子为CAG,剪切肽为P2A,入核信号为NLS和SV40NLS,Cpf1蛋白和p300核心结构域之间插入NLS信号和3xHA标签。
4.一种DNA靶向激活系统,其特征在于,包含权利要求1所述的融合蛋白和至少一种向导RNA;所述向导RNA为针对目的基因启动子或增强子区域设计的一段长度为23bp的序列。
5.根据权利要求4所述的DNA靶向激活系统,其特征在于,所述启动子或增强子区域位于MyoD、IL1RN和/或OCT4。
6.根据权利要求4所述的DNA靶向激活系统,其特征在于,所述DNA靶向激活系统含有四种不同的向导RNA。
7.权利要求4~6中任意一条权利要求所述DNA靶向激活系统对细胞进行靶向基因激活中的应用。
8.根据权利7所述的DNA靶向激活系统对细胞进行靶向基因激活中的应用,其特征在于,所述细胞包括293T、U2OS和MCF7。
9.根据权利要求7或8所述的DNA靶向激活系统对细胞进行靶向基因激活中的应用,其特征在于,包括如下步骤:
(1)构建含有至少一种氨基酸突变的Cpf1表达载体;
(2)构建表达特定向导RNA的载体;
(3)将步骤(1)所述Cpf1表达载体和步骤(2)所述表达特定向导RNA的载体混合,通过脂质体转染试剂聚乙烯亚胺共转染到哺乳动物细胞,在细胞内表达的融合蛋白与其特定向导RNA结合,将p300核心蛋白靶向目标基因区。
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US11912985B2 (en) | 2020-05-08 | 2024-02-27 | The Broad Institute, Inc. | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
CN112159822A (zh) * | 2020-09-30 | 2021-01-01 | 扬州大学 | 一种PS转座酶与CRISPR/dCpf1融合蛋白表达载体及其介导的定点整合方法 |
CN114441772A (zh) * | 2022-01-29 | 2022-05-06 | 北京大学 | 用于检测细胞内能够与rna结合的靶分子的方法和试剂 |
CN114441772B (zh) * | 2022-01-29 | 2023-03-21 | 北京大学 | 用于检测细胞内能够与rna结合的靶分子的方法和试剂 |
CN114958910A (zh) * | 2022-05-13 | 2022-08-30 | 南方医科大学 | DropCRISPRa高效靶向相分离基因激活系统及其构建方法和应用 |
CN114958910B (zh) * | 2022-05-13 | 2024-03-01 | 南方医科大学 | DropCRISPRa高效靶向相分离基因激活系统及其构建方法和应用 |
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