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CN104726449A - 一种用于预防和/或治疗HIV的CRISPR-Cas9系统及其制备方法和用途 - Google Patents

一种用于预防和/或治疗HIV的CRISPR-Cas9系统及其制备方法和用途 Download PDF

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CN104726449A
CN104726449A CN201510127880.3A CN201510127880A CN104726449A CN 104726449 A CN104726449 A CN 104726449A CN 201510127880 A CN201510127880 A CN 201510127880A CN 104726449 A CN104726449 A CN 104726449A
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蒋兴宇
刘野
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National Center for Nanosccience and Technology China
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Abstract

本发明提供一种用于预防和/或治疗HIV的CRISPR-Cas9系统及其制备方法和用途。所述CRISPR-Cas9系统包括特异性靶向HIV基因组上的特定基因位点的sgRNA，所述HIV基因组上的特定基因位点选自Gag、Env、Pol、Tat、Nef、Vif、Vpr、Vpu、5’LTR、3’LTR和Rev。所述CRISPR-Cas9系统的制备方法包括通过PCR扩增获得sgRNA片段的步骤。本发明还提供所述CRISPR-Cas9系统在制备用于治疗和/或预防HIV感染的药物的用途。本发明采用高效基因递送系统，将CRISPR-Cas9系统递送进入HIV感染细胞，高效抑制HIV的产生，抑制率高达96％，达到了和肽类抗HIV药物相当的效果。

Description

一种用于预防和/或治疗HIV的CRISPR-Cas9系统及其制备方法和用途
技术领域
本发明涉及基因治疗药物领域，特别涉及HIV的基因治疗领域，尤其涉及一种用于预防和/或治疗HIV的CRISPR-Cas9系统及其制备方法和用途。
背景技术
人类免疫缺陷病毒(Human Immunodeficiency Virus或HIV)属逆转录病毒，是一种感染人类免疫系统细胞的慢病毒，能造成人类免疫系统缺陷，使人体失去抵抗力，易产生各种疾病甚至癌症。其导致的疾病——艾滋病至今无有效疗法，具有致命性。
预防传染病的常规手段是人为干预和疫苗，人为干预(如避孕套等)无法真正意义上消除HIV，仅作为限制HIV的一种物理性强制手段；而相关疫苗虽然经历了三十多年的探索和开发，至今尚未有成熟的产品问世。因此，现行的HIV主要疗法为高效联合抗逆转录病毒法，但其同样只能延缓HIV的发展。与以上疗法不同的是，基因治疗一直被视为HIV治疗的最理想模式；因其理论上可以达到预防和消除HIV的效果。
近年来，CRISPR-Cas9技术因其高效性和简便性被迅速推广和应用。它是细菌和古细菌在长期演化过程中形成的一种适应性免疫防御系统，可用来对抗入侵的病毒及外源DNA。CRISPR-Cas9系统通过将入侵噬菌体和质粒DNA的片段整合到CRISPR，并利用相应的CRISPR RNAs(sgRNAs)来指导同源序列的降解。该系统的主要组成部分为：(1)sgRNA序列，负责靶向特异性基因位点；(2)Cas9酶，负责对靶向位点的DNA进行修饰切割。目前，这一技术主要应用于基因修饰动物模型的构建，而将其开发为HIV预防性或治疗性的药物则很少被涉及。在公开发表的论文“RNA-directed Gene Editing SpecificallyEradicates Latent and Prevents New HIV-1Infection”中，刘文辉等根据HIV基因的两个片段序列构建了一个长度为20bp的sgRNA，其能靶向位于HIV序列5’LTR的HIV-1U3位点；由于位点单一等原因，该系统仅能说明其对于HIV的抑制具有关联性，但抑制的效果十分有限。
发明内容
本发明的目的在于提供一种用于预防和/或治疗HIV的CRISPR-Cas9系统及其制备方法和用途。
为达到以上目的，本发明采用以下技术方案：
第一方面，本发明提供一种用于预防和/或治疗HIV的CRISPR-Cas9系统，其包括特异性靶向HIV基因组上的特定基因位点的sgRNA，所述HIV基因组上的特定基因位点选自Gag、Env、Pol、Tat、Nef、Vif、Vpr、Vpu、5’LTR、3’LTR和Rev。
优选地，所述HIV基因组上的特定基因位点包括Gag、Env、Pol、Tat、Nef、Vif、Vpr、Vpu、5’LTR、3’LTR和Rev。
优选地，特异性靶向每个基因位点的sgRNA为1条以上，例如可以是1条、2条、3条、4条、5条、6条、7条、8条、9条、10条或11条，进一步优选为4条。
进一步优选地，每条sgRNA的长度为16-22bp，例如可以是16bp、17bp、18bp、19bp、20bp、21bp或22bp。
优选地，所述CRISPR-Cas9系统还包括Cas9。
优选地，所述Cas9和所述特异性靶向HIV基因组上的特定基因位点的sgRNA分别存在于质粒中。
第二方面，本发明提供第一方面所述药物的制备方法，PCR扩增所用引物选自Cas9-5'LTR-1引物对、Cas9-5'LTR-2引物对、Cas9-5'LTR-3引物对、Cas9-5'LTR-4引物对、Cas9-Pol-1引物对、Cas9-Pol-2引物对、Cas9-Pol-3引物对、Cas9-Pol-4引物对、Cas9-vif-1引物对、Cas9-vif-2引物对、Cas9-vif-3引物对、Cas9-vif-4引物对、Cas9-vpr-1引物对、Cas9-vpr-2引物对、Cas9-vpr-3引物对、Cas9-tat-1引物对、Cas9-tat-2引物对、Cas9-tat-3引物对、Cas9-tat-4引物对、Cas9-rev2-1引物对、Cas9-rev2-2引物对、Cas9-rev2-3引物对、Cas9-vpu-1引物对、Cas9-vpu-2引物对、Cas9-env-1引物对、Cas9-env-2引物对、Cas9-env-3引物对、Cas9-env-4引物对、Cas9-nef-1引物对、Cas9-nef-2引物对、Cas9-nef-3引物对、Cas9-nef-4引物对、Cas9-3'LTR-1引物对、Cas9-3'LTR-2引物对、Cas9-3'LTR-3引物对、Cas9-Gag-1引物对、Cas9-Gag-2引物对、Cas9-Gag-3引物对和Cas9-Gag-4引物对；
所述引物对的序列按5'-3'方向如下：
Cas9-5'LTR-1引物对：
gacaagatatccttggtttt，和CAAGGATATCTTGTCcggtg；
Cas9-5'LTR-2引物对：
cctatgagcctgcagtttt，和TGCAGGCTCATAGGcggtg；
Cas9-5'LTR-3引物对：
gctttttgcctgtagtttt，和TACAGGCAAAAAGCcggtg；
Cas9-5'LTR-4引物对：
gacccttttagtcagtggtttt，和CACTGACTAAAAGGGTCcggtg；
Cas9-Pol-1引物对：
aataccacatcccgcagtttt，和TGCGGGATGTGGTATTCcggtg；
Cas9-Pol-2引物对：
ccacagggatggaagtttt，和TTCCATCCCTGTGGcggtg；
Cas9-Pol-3引物对：
gtcagatttatgcgtttt，和GCATAAATCTGACcggtg；
Cas9-Pol-4引物对：
cctggattcctgaatgtttt，和ATTCAGGAATCCAGGcggtg；
Cas9-vif-1引物对：
tcagaagtacacatcccag，和GATGTGTACTTCTGAcggtg；
Cas9-vif-2引物对：
acatattggggtctgcatacgtttt，和CAGACCCCAATATGTcggtg；
Cas9-vif-3引物对：
gccagggagtctccatagaagtttt，和TTCTATGGAGACTCCcggtg；
Cas9-vif-4引物对：
gatctctacaatactgtttt，和AGTATTGTAGAGATCcggtg；
Cas9-vpr-1引物对：
atggctccatagcttgtttt，和AAGCTATGGAGCCATcggtg；
Cas9-vpr-2引物对：
ggagatacttggacgtttt，和GTCCAAGTATCTCCcggtg；
Cas9-vpr-3引物对：
tgccaacatagcagaatgtttt，和ATTCTGCTATGTTGGCAcggtg；
Cas9-tat-1引物对：
agccctggaagcatccgtttt，和GGATGCTTCCAGGGCTcggtg；
Cas9-tat-2引物对：
ccaggaagtcagcctgtttt，和AGGCTGACTTCCTGGcggtg；
Cas9-tat-3引物对：
atggcaggaagaaggtttt，和CTTCTTCCTGCCATAcggtg；
Cas9-tat-4引物对：
cgaaggaatcgaagagtttt，和TCTTCGATTCCTTCGcggtg；
Cas9-rev2-1引物对：
tcttagcactgttctgtttt，和AGAACAGTGCTAAGAcggtg；
Cas9-rev2-2引物对：
acttactcttgattgtagcggtttt，和ACAATCAAGAGTAAGTcggtg；
Cas9-rev2-3引物对：
ggtgggaagtcctcatgtttt，和ATATTTGAGGACTTCCCACCcggtg；
Cas9-vpu-1引物对：
caataatagcaatagttatagtttt，和TATAACTATTGCTATTATTGcggtg；
Cas9-vpu-2引物对：
ccatagtattaataaaatatgtttt，和ATATTTTATTAATACTATGGcggtg；
Cas9-env-1引物对：
catggtagaccagatgcatggtttt，和CATGCATCTGGTCTACCATGcggtg；
Cas9-env-2引物对：
tgctctttcaatatcaccacgtttt，和GTGGTGATATTGAAAGAGCAcggtg；
Cas9-env-3引物对：
gtctagcagaagaaggtttt，和CTTCTTCTGCTAGACcggtg；
Cas9-env-4引物对：
ctgctattaacaagagagtttt，和TCTCTTGTTAATAGCAGcggtg；
Cas9-nef-1引物对：
aatgggatggcctgctgtaagtttt，和TTACAGCAGGCCATCCCATTcggtg；
Cas9-nef-2引物对：
gagctgagccagcagcagatgtttt，和ATCTGCTGCTGGCTCAGCTCcggtg；
Cas9-nef-3引物对：
gggagcagcatctagagaccgtttt，和GGTCTCTAGATGCTGCTCCCcggtg；
Cas9-nef-4引物对：
tgcctggctagaagcacaaggtttt，和CTTGTGCTTCTAGCCAGGCAcggtg；
Cas9-3'LTR-1引物对：
gatttccactgacctttggagtttt，和TCCAAAGGTCAGTGGAAATCcggtg；
Cas9-3'LTR-2引物对：
cggagaaagaagtgttagtggtttt，和CACTAACACTTCTTTCTCCGcggtg；
Cas9-3'LTR-3引物对：
ctttccgctggggactttccgtttt，和GGAAAGTCCCCAGCGGAAAGcggtg；
Cas9-Gag-1引物对：
gtcagtattaagtgcgtttt，和GCACTTAATACTGACcggtg；
Cas9-Gag-2引物对：
cacaggaaaaggcagccgtttt，和GGCTGCCTTTTCCTGTGcggtg；
Cas9-Gag-3引物对：
gaacgatttgcagtcaagtttt，和TTGACTGCAAATCGTTCcggtg；
Cas9-Gag-4引物对：
ggaagctttagagaagtttt，和TTCTCTAAAGCTTCCcggtg。
本领域的技术人员可以根据以上提供的引物对确定所扩增的相应的sgRNA的核苷酸序列，所述sgRNA可以特异性靶向所述HIV基因组上的特定基因位点。
优选地，所述PCR扩增的退火温度为：
Gag：63℃；
Env：63℃；
Pol：58℃；
Tat：51℃；
Vif：51℃；
Nef：64℃；
Vpu：60℃；
Vpr：67℃；
3’LTR：67℃；
5’LTR：58℃；
Rev：51℃。
优选地，所述制备方法还包括将所扩增的各sgRNA片段分别构建入质粒载体的步骤。
第三方面，本发明提供如第一方面所述的CRISPR-Cas9系统在制备用于治疗和/或预防HIV感染的药物的用途。
与现有技术相比，本发明至少具有以下有益效果：
本发明的CRISPR-Cas9系统，优化设计针对HIV基因组上11个基因位点(Gag、Env、Pol、Tat、Nef、Vif、Vpr、Vpu、5’LTR、3’LTR、Rev)的sgRNAs，采用高效基因递送系统，将CRISPR-Cas9系统递送进入HIV感染细胞，高效抑制HIV的产生，抑制率高达96％，单从抑制率来讲，达到了和肽类抗HIV药物相当的效果。
附图说明
图1为本发明的CRISPR-Cas9系统的作用示意图。
具体实施方式
下面通过具体实施方式来进一步说明本发明的技术方案。本领域技术人员应该明了，所述实施例仅仅用于帮助理解本发明，而不应视为对本发明的具体限制。
实施例1
(1)sgRNA基因的获得
sgRNAs引物序列直接在invitrogen公司订购获得，相关序列如表1所示。
表1：sgRNA引物序列
(2)将sgRNA构建至表达载体
a)：sgRNA的PCR扩增
反应体系：无菌ddH2O：37.5μL，10×PCR Buffer(含MgCl2)：5μL，2.5mMdNTP：4μL，相应的上、下游引物各1μL(引物浓度50pmol/μL)，模板DNA(50ng/μL)：1μL，PyrobestTM DNA Polymerase：0.5μL。温和震荡混匀后，按表2所示条件进行PCR扩增。
表2：PCR扩增条件
b)载体构建及鉴定
用Quigen生产的PCR回收试剂盒按照常规方法进行PCR扩增产物的回收，然后用SalI单酶切PCR片段，SalI和EcoRV双酶切质粒载体p1.0，两者进行连接，转化Top10或DH5α感受态，以SalI和EcoRV酶切筛选出阳性克隆并送Invitrogen公司测序。
(3)HIV抑制测试
选用TZM-BL细胞株，预先用HIV感染6小时，再用纳米载体系统将本发明的CRISPR-Cas9系统递送进入细胞，引导CRISPR-Cas9在细胞内的表达。培养细胞2天后，检测HIV的产量。将单独只感染HIV的细胞组设为阴性对照，既不加HIV也不加CRISPR-Cas9的细胞组设为空白对照；加HIV和已上市的抗HIV药物T20的细胞组设为阳性对照。测试结果如表3所示。
表3：HIV抑制测试结果
CRISPR-Cas9系统 抑制率
CRISPR-Cas9(Env) 0.96
CRISPR-Cas9(Pol) 0.96
CRISPR-Cas9(Gag) 0.96
CRISPR-Cas9(3’LTR) 0.92
CRISPR-Cas9(5’LTR) 0.93
CRISPR-Cas9(Vif) 0.96
CRISPR-Cas9(Rev) 0.95
CRISPR-Cas9(Tat) 0.96
CRISPR-Cas9(Nef) 0.90
CRISPR-Cas9(Vpr) 0.88
CRISPR-Cas9(Vpu) 0.73
阳性对照 0.90
阴性对照 0
空白对照 0
从以上结果可以看出，本发明的CRISPR-Cas9系统对于HIV的抑制率为73％～96％，说明本发明的CRISPR-Cas9系统能抑制HIV在细胞内的产生；阳性对照是已上市的抗HIV药物，其抑制率为90％。单从抑制率来讲，本发明的CRISPR-Cas9系统达到了和市售的抗HIV药物以及肽类抗HIV药物相当的效果。
申请人声明，本发明通过上述实施例来说明本发明的工艺方法，但本发明并不局限于上述工艺步骤，即不意味着本发明必须依赖上述工艺步骤才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明所选用原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。

Claims (10)

1.一种用于预防和/或治疗HIV的CRISPR-Cas9系统，其包括特异性靶向HIV基因组上的特定基因位点的sgRNA，所述HIV基因组上的特定基因位点选自Gag、Env、Pol、Tat、Nef、Vif、Vpr、Vpu、5’LTR、3’LTR和Rev。
2.根据权利要求1所述的CRISPR-Cas9系统，其特征在于，所述HIV基因组上的特定基因位点包括Gag、Env、Pol、Tat、Nef、Vif、Vpr、Vpu、5’LTR、3’LTR和Rev。
3.根据权利要求1或2所述的CRISPR-Cas9系统，其特征在于，特异性靶向每个基因位点的sgRNA为1条以上，优选为4条；
进一步优选地，每条sgRNA的长度为16-22bp。
4.根据权利要求1-3任一项所述的CRISPR-Cas9系统，其特征在于，所述CRISPR-Cas9系统还包括Cas9。
5.根据权利要求1-4任一项所述的CRISPR-Cas9系统，其特征在于，所述Cas9和所述特异性靶向HIV基因组上的特定基因位点的sgRNA分别存在于质粒中。
6.如权利要求1-5任一项所述CRISPR-Cas9系统的制备方法，包括通过PCR扩增获得sgRNA片段的步骤。
7.如权利要求6所述的制备方法，其特征在于，PCR扩增所用引物选自Cas9-5'LTR-1引物对、Cas9-5'LTR-2引物对、Cas9-5'LTR-3引物对、Cas9-5'LTR-4引物对、Cas9-Pol-1引物对、Cas9-Pol-2引物对、Cas9-Pol-3引物对、Cas9-Pol-4引物对、Cas9-vif-1引物对、Cas9-vif-2引物对、Cas9-vif-3引物对、Cas9-vif-4引物对、Cas9-vpr-1引物对、Cas9-vpr-2引物对、Cas9-vpr-3引物对、Cas9-tat-1引物对、Cas9-tat-2引物对、Cas9-tat-3引物对、Cas9-tat-4引物对、Cas9-rev2-1引物对、Cas9-rev2-2引物对、Cas9-rev2-3引物对、Cas9-vpu-1引物对、Cas9-vpu-2引物对、Cas9-env-1引物对、Cas9-env-2引物对、Cas9-env-3引物对、Cas9-env-4引物对、Cas9-nef-1引物对、Cas9-nef-2引物对、Cas9-nef-3引物对、Cas9-nef-4引物对、Cas9-3'LTR-1引物对、Cas9-3'LTR-2引物对、Cas9-3'LTR-3引物对、Cas9-Gag-1引物对、Cas9-Gag-2引物对、Cas9-Gag-3引物对和Cas9-Gag-4引物对；
所述引物对碱基序列按5'-3'方向如下：
Cas9-5'LTR-1引物对：
gacaagatatccttggtttt，和CAAGGATATCTTGTCcggtg；
Cas9-5'LTR-2引物对：
cctatgagcctgcagtttt，和TGCAGGCTCATAGGcggtg；
Cas9-5'LTR-3引物对：
gctttttgcctgtagtttt，和TACAGGCAAAAAGCcggtg；
Cas9-5'LTR-4引物对：
gacccttttagtcagtggtttt，和CACTGACTAAAAGGGTCcggtg；
Cas9-Pol-1引物对：
aataccacatcccgcagtttt，和TGCGGGATGTGGTATTCcggtg；
Cas9-Pol-2引物对：
ccacagggatggaagtttt，和TTCCATCCCTGTGGcggtg；
Cas9-Pol-3引物对：
gtcagatttatgcgtttt，和GCATAAATCTGACcggtg；
Cas9-Pol-4引物对：
cctggattcctgaatgtttt，和ATTCAGGAATCCAGGcggtg；
Cas9-vif-1引物对：
tcagaagtacacatcccag，和GATGTGTACTTCTGAcggtg；
Cas9-vif-2引物对：
acatattggggtctgcatacgtttt，和CAGACCCCAATATGTcggtg；
Cas9-vif-3引物对：
gccagggagtctccatagaagtttt，和TTCTATGGAGACTCCcggtg；
Cas9-vif-4引物对：
gatctctacaatactgtttt，和AGTATTGTAGAGATCcggtg；
Cas9-vpr-1引物对：
atggctccatagcttgtttt，和AAGCTATGGAGCCATcggtg；
Cas9-vpr-2引物对：
ggagatacttggacgtttt，和GTCCAAGTATCTCCcggtg；
Cas9-vpr-3引物对：
tgccaacatagcagaatgtttt，和ATTCTGCTATGTTGGCAcggtg；
Cas9-tat-1引物对：
agccctggaagcatccgtttt，和GGATGCTTCCAGGGCTcggtg；
Cas9-tat-2引物对：
ccaggaagtcagcctgtttt，和AGGCTGACTTCCTGGcggtg；
Cas9-tat-3引物对：
atggcaggaagaaggtttt，和CTTCTTCCTGCCATAcggtg；
Cas9-tat-4引物对：
cgaaggaatcgaagagtttt，和TCTTCGATTCCTTCGcggtg；
Cas9-rev2-1引物对：
tcttagcactgttctgtttt，和AGAACAGTGCTAAGAcggtg；
Cas9-rev2-2引物对：
acttactcttgattgtagcggtttt，和ACAATCAAGAGTAAGTcggtg；
Cas9-rev2-3引物对：
ggtgggaagtcctcatgtttt，和ATATTTGAGGACTTCCCACCcggtg；
Cas9-vpu-1引物对：
caataatagcaatagttatagtttt，和TATAACTATTGCTATTATTGcggtg；
Cas9-vpu-2引物对：
ccatagtattaataaaatatgtttt，和ATATTTTATTAATACTATGGcggtg；
Cas9-env-1引物对：
catggtagaccagatgcatggtttt，和CATGCATCTGGTCTACCATGcggtg；
Cas9-env-2引物对：
tgctctttcaatatcaccacgtttt，和GTGGTGATATTGAAAGAGCAcggtg；
Cas9-env-3引物对：
gtctagcagaagaaggtttt，和CTTCTTCTGCTAGACcggtg；
Cas9-env-4引物对：
ctgctattaacaagagagtttt，和TCTCTTGTTAATAGCAGcggtg；
Cas9-nef-1引物对：
aatgggatggcctgctgtaagtttt，和TTACAGCAGGCCATCCCATTcggtg；
Cas9-nef-2引物对：
gagctgagccagcagcagatgtttt，和ATCTGCTGCTGGCTCAGCTCcggtg；
Cas9-nef-3引物对：
gggagcagcatctagagaccgtttt，和GGTCTCTAGATGCTGCTCCCcggtg；
Cas9-nef-4引物对：
tgcctggctagaagcacaaggtttt，和CTTGTGCTTCTAGCCAGGCAcggtg；
Cas9-3'LTR-1引物对：
gatttccactgacctttggagtttt，和TCCAAAGGTCAGTGGAAATCcggtg；
Cas9-3'LTR-2引物对：
cggagaaagaagtgttagtggtttt，和CACTAACACTTCTTTCTCCGcggtg；
Cas9-3'LTR-3引物对：
ctttccgctggggactttccgtttt，和GGAAAGTCCCCAGCGGAAAGcggtg；
Cas9-Gag-1引物对：
gtcagtattaagtgcgtttt，和GCACTTAATACTGACcggtg；
Cas9-Gag-2引物对：
cacaggaaaaggcagccgtttt，和GGCTGCCTTTTCCTGTGcggtg；
Cas9-Gag-3引物对：
gaacgatttgcagtcaagtttt，和TTGACTGCAAATCGTTCcggtg；
Cas9-Gag-4引物对：
ggaagctttagagaagtttt，和TTCTCTAAAGCTTCCcggtg。
8.根据权利要求7所述的制备方法，其特征在于，所述PCR扩增的退火温度为：
Gag：63℃；
Env：63℃；
Pol：58℃；
Tat：51℃；
Vif：51℃；
Nef：64℃；
Vpu：60℃；
Vpr：67℃；
3’LTR：67℃；
5’LTR：58℃；
Rev：51℃。
9.根据权利要求6-8任一项所述的制备方法，其特征在于，还包括将所扩增的各sgRNA片段分别构建入质粒载体的步骤。
10.如权利要求1-5任一项所述的CRISPR-Cas9系统在制备用于治疗和/或预防HIV感染的药物的用途。
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