CN110115770A - 一种防治丙型病毒性肝炎的新靶点及其CRISPR-Cas9靶向系统与应用 - Google Patents

一种防治丙型病毒性肝炎的新靶点及其CRISPR-Cas9靶向系统与应用 Download PDF

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CN110115770A
CN110115770A CN201910282353.8A CN201910282353A CN110115770A CN 110115770 A CN110115770 A CN 110115770A CN 201910282353 A CN201910282353 A CN 201910282353A CN 110115770 A CN110115770 A CN 110115770A
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张旭
王宏
盖作启
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Guangzhou Aidi Gene Technology Co Ltd
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Abstract

本发明公开了一种防治丙型病毒性肝炎的新靶点及其CRISPR‑Cas9靶向系统与应用。本发明研究发现一种新的丙型病毒性肝炎防治靶点，即miR‑615‑3p，该基因与ApoB和ApoE具有正向调节关系，敲除miR‑615‑3p后可抑制ApoB和ApoE基因的表达，从而防治丙型病毒性肝炎。同时本发明还构建了一种特异性靶向miR‑615‑3p基因的CRISPR‑Cas9系统，利用该系统可以高效特异性敲除miR‑615‑3p基因，靶向性提高、操作技术简单，在丙型病毒性肝炎防治方面具有很好的应用前景，同时，本发明的成果有助于进一步揭示丙型病毒性肝炎的防治的分子机制，在新的治疗方案的研发中具有十分重要的意义。

Description

一种防治丙型病毒性肝炎的新靶点及其CRISPR-Cas9靶向系 统与应用
技术领域
本发明属于生物医药技术领域。更具体地，涉及一种防治丙型病毒性肝炎的新靶点及其CRISPR-Cas9靶向系统与应用。
背景技术
丙型病毒性肝炎是一种由丙型肝炎病毒(hepatitis C virus，HCV)感染引起的病毒性肝炎，可通过母婴、输血、性接触等传播。全世界约有1.8亿人感染了HCV，导致肝纤维化和肝硬化，严重时会发展成肝癌，已成为全球重要的公共卫生问题之一。
早期治疗HCV主要通过聚乙二醇干扰素联合利巴韦林，但副作用较大，治疗周期较长，且对不同基因型疗效不同。近几年直接抗病毒药物(Direct antiviralagents，DAA)得到长足发展，其主要是针对HCV生命周期中特定病毒蛋白的靶向药物，能有效提高患者的持续病毒应答率(SVR)。但是，这种治疗方案同样会产生药物副作用，并且治疗过程中伴有耐药突变、费用高昂的缺陷。此外，HCV暂无在病毒侵入前的预防性药物或治疗手段，晚期丙肝患者在进行肝移植手术后的再次感染率非常高。
另外，现有的技术中，CN103215269A公布了一种利用靶向干扰磷脂酰肌醇4-激酶(PI4K)基因双链核糖核酸(dsRNA)用于治疗HCV病毒感染；CN10135095A公布了一种靶向HCV的内部核糖体进入位点(IRES)序列的短发夹核糖核酸(shRNA)和双链小干扰核糖核酸(siRNA)构件用于抑制HCV的表达或活性。CN102257140A公布了通过miRNA-196降低Bach1表达，同时上调HMOX1表达，进而治疗患有HCV感染的哺乳动物。以上结果表明，通过基因干扰可以实现治疗丙型病毒性肝炎。
在基因干扰技术方面，CRISPR(clustered regularly interspaced shortpalindromic repeats)是一种来自细菌抵御入侵的病毒或其它外源DNA的免疫机制。其中CRISPR/Cas9系统是应用最为广泛的基因编辑技术，它利用可与基因序列配对的单链sgRNA引导Cas9核酸酶靶向并切割特定基因组位点，形成的双链DNA断裂激发内源性DNA修复机制引入基因突变。CRISPR与内源性同源重组(Homologous recombination,HR)结合可实现简单高效的精确遗传操作。从2013年问世以来已被应用到药物筛选、癌症治疗、农作物和家畜育种等多个领域。与ZFN和siRNA相比，CRISPR-Cas9具有快速、高效、特异性靶向敲除基因的优势。
因此，不断寻求新的靶点以及基因干扰手段是本领域的研究热点。
发明内容
本发明的第一个目的是研究筛选获得了一种新的丙型病毒性肝炎防治靶点，即miR-615-3p(基因前体序列如SEQ ID NO.1所示)。本发明研究发现，miR-615-3p与ApoB和ApoE具有正向调节关系，敲除miR-615-3p后可抑制ApoB和ApoE基因的表达，从而防治丙型病毒性肝炎。
本发明另一目的是提供一种靶向敲除miR-615-3p基因的CRISPR-Cas9系统，利用该系统可以实现高效特异性敲除miR-615-3p基因的目的。
本发明上述目的通过以下技术方案实现：
基于上述本发明的研究，以下应用均应在本发明的保护范围之内：
miR-615-3p基因在作为丙型病毒性肝炎的防治靶点方面的应用。
miR-615-3p基因在制备丙型病毒性肝炎的防治试剂中的应用。
miR-615-3p基因在作为下调ApoB基因和/或ApoE基因表达的靶点方面的应用。
miR-615-3p基因在制备下调ApoB基因和/或ApoE基因表达的试剂中的应用。
同时基于上述研究成果及其应用，本发明还提供了：
一种特异性靶向miR-615-3p基因的CRISPR-Cas9系统，包括但不限于：特异性靶向敲除miR-615-3p基因的sgRNA和包含sgRNA的载体。
其中，所述sgRNA的序列如SEQ ID NO.2所示，具有很好的特异性敲除miR-615-3p的效果。
优选地，为了实现更高的细胞转染效率，易于筛选阳性克隆，所述包含sgRNA的载体为质粒lentiCRISPR-v2-sgRNA-miR-615-3p，序列包含用于启动sgRNA表达的U6promoter基因启动子、sgRNA序列、用于启动Cas编码基因表达的EF-1α、Cas9编码基因；具体序列如SEQ ID NO.5所示。
另外，上述CRISPR-Cas9系统在制备miR-615-3p基因敲除的细胞模型或miR-615-3p基因敲除试剂盒中的应用，也应在本发明的保护范围之内。
同时，一种miR-615-3p基因敲除的细胞模型也应在本发明的保护范围之内，该细胞模型敲除了miR-615-3p基因，具体优选是采用本文上述CRISPR-Cas9系统敲除了miR-615-3p基因。
一种miR-615-3p基因敲除试剂盒也应在本发明的保护范围之内，该试剂盒包括如下(1)-(3)中的任一种或几种：
(1)上述靶向敲除miR-615-3p基因的sgRNA；
(2)上述载体lentiCRISPR-v2-sgRNA-miR-615-3p；
(3)上述miR-615-3p基因敲除的细胞模型。
本发明具有以下有益效果：
本发明明确了miR-615-3p基因表达与丙型病毒性肝炎的关系，提供了一种新的丙型病毒性肝炎防治靶点。本发明通过构建miR-615-3p基因敲除的细胞模型研究发现，敲除miR-615-3p基因能抑制脂蛋白合成从而防治丙型病毒性肝炎，同时该模型为研究丙型病毒性肝炎等疾病特别是靶向miR-615-3p的药物发现与筛选提供了有力工具。
本发明还构建了一种特异性靶向miR-615-3p基因的CRISPR-Cas9系统，利用该系统可以实现高效特异性敲除miR-615-3p基因的目的，基因敲除效率得到显著提高，实现了在基因组水平对基因的沉默作用，降低了传统技术siRNA或蛋白水平的沉默不彻底或者无法沉默基因表达的缺陷。而且靶向性提高、操作技术简单。
同时，本发明的成果有助于进一步揭示丙型病毒性肝炎的防治的分子机制，在新的治疗方案的研发中具有十分重要的意义。
附图说明
图1为miR-615-3p基因敲除的载体lentiCRISPR-v2-sgRNA-miR-615-3p示意图。
图2为miR-615-3p基因敲除前后载脂蛋白ApoB和ApoE的mRNA表达水平和外泌载脂蛋白含量图。
图3为建细胞模型前后HCV拷贝数含量图。
具体实施方式
以下结合具体实施例来进一步说明本发明，但实施例并不对本发明做任何形式的限定。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明，以下实施例所用试剂和材料均为市购。
实施例1 sgRNA设计
sgRNA序列的设计与合成：
针对miR-615-3p基因前体序列设计sgRNA，共设计了8条sgRNA：
(1)ctcgggaggggcgggaggg
(2)aggggcgggaggggggtccc
(3)ggaagagggagacccaggct
(4)ccccggtgctcggatctcga
(5)tctcgagggtgcttattgtt
(6)ttattgttcggtccgagcct
(7)tggggggaagagggagaccc
(8)ggaagagggagacccaggct
经过优化筛选，选择了第(8)条。
实施例2构建miR-615-3p基因的sgRNA表达载体
1、sgRNA序列DNA片段的制备
(1)合成上述实施例1第(8)条sgRNA的正向和反向磷酸化寡核苷酸序列：
F(5’-3’)(SEQ ID NO.3)：CACCG ggaagagggagacccaggct
R(5’-3’)(SEQ ID NO.4)：AAACagcctgggtctccctcttccC
将对应的正向和反向寡核苷酸序列退火、复性，形成具有粘性末端的双链DNA片段。
反应(10μL)体系如下所示：
(2)将上述反应体系放入PCR仪，并按以下程序进行反应。
反应程序：
95℃ 5min
95-25℃ 5℃/min
反应产物于-20℃储存。
2、构建sgRNA表达载体
(1)利用BsmBI限制性内切酶酶切目标载体lentiCRISPR-v2质粒。
按照以下反应体系进行配制：
(2)将上述反应体系放入PCR仪，并按以下程序进行反应。
反应程序：
(3)转化大肠杆菌
反应结束后将连接混合物转化大肠杆菌DH5α菌株：向大肠杆菌DH5α感受态细胞中加入连接混合物，冰上孵育30min；将混合物放入42℃水浴，热激90s后放在冰上冷却；向混合物加入100μL LB培养基，涂布Amp LB平板，37℃过夜培养。
(4)鉴定正确的转化克隆
从Amp LB平板上挑选若干菌落进行扩大培养，提取质粒进行酶切鉴定。挑选可能正确的克隆进行测序，通过测序，发现插入序列正确。对于正确的lentiCRISPR-v2-sgRNA-miR-615-3p载体克隆进行保种，并且提取相应的质粒(载体lentiCRISPR-v2-sgRNA-miR-615-3p的示意图如图1所示)。
实施例3慢病毒系统构建Huh.7.5稳定细胞株
1、病毒包装
通过培养293FT细胞，按照核心质粒：包装质粒：包膜质粒＝4:3:2的比例进行转染，sgRNA组核心质粒为：lentiCRISPR-v2-sgRNA-miR-615-3p，以lentiCRISPR-v2作为对照组。在转染后24h、48h和72h收集病毒液，将收集细胞的三次病毒液混匀，并进行超滤浓缩和病毒滴度测定。
2、细胞转染
提前一天将Huh.7.5细胞铺板，转染时细胞密度达到40-60％。转染前移去培养基，用新鲜培养基稀释病毒浓缩液及转染试剂处理细胞，感染96h后用1.0μg/mL嘌呤霉素进行筛选，筛选时间是7天。经嘌呤霉素筛选后用终浓度为0.6μg/mL的嘌呤霉素维持Huh.7.5稳定细胞株。
3、单克隆细胞有限稀释法筛选敲除miR-615-3p基因的Huh.7.5细胞株
通过胰蛋白酶消化得到细胞后采用有限稀释法稀释至100μL培养基0.5个细胞，按每孔100μL细胞稀释液加入96孔板。接种后5-7天用荧光显微镜观察细胞生长状态并初步筛选单克隆，待细胞长满后，消化转移至24孔板。待其长满收集细胞，一部分用于传代留种，一部分用于提取基因组DNA，筛选靶向敲除miR-615-3p基因成功的Huh.7.5细胞。
实施例4构建敲除miR-615-3p的Huh.7.5细胞株在研究丙型肝炎中的应用
1、miR-615-3p基因敲除的Huh.7.5细胞株中载脂蛋白ApoB和ApoE的mRNA表达水平和外泌载脂蛋白含量检测
提取miR-615-3p基因敲除的Huh.7.5细胞株RNA，反转录成cDNA，之后利用实时定量PCR检测载脂蛋白ApoB和ApoE基因表达水平，以未进行基因敲除的细胞为对照。如图2中A图所示，与对照相比，miR-615-3p基因敲除的Huh.7.5细胞的ApoB和ApoE基因表达水平显著下降，分别降低到27.3％和41.9％。另通过收集细胞培养基上清，通过Elisa检测载脂蛋白ApoB和ApoE含量，以未进行基因敲除的细胞培养基上清为对照。如图2中B图所示，与对照相比，miR-615-3p基因敲除的Huh.7.5细胞株分泌的ApoB和ApoE含量显著降低，分别降低到74.8％和44.8％。结果表面，miR-615-3p基因敲除的Huh.7.5细胞株，可以有效降低载脂蛋白的相关基因表达和分泌，进而抑制丙型肝炎病毒感染。
2、构建细胞模型前后HCV拷贝数含量检测
利用日本重症肝炎1型(JFH-1)基因质粒获得HCV病毒液转染基因miR-615-3p基因敲除Huh-7.5细胞，培养4h后更换培养基，继续培养48h，收集细胞用于检测病毒拷贝数。提取细胞总RNA，反转录成cDNA，之后利用实时定量PCR检测HCV 5’-UTR表达水平，以为进行基因敲除的细胞为对照。如图3所示，与对照相比，miR-615-3p基因敲除的Huh.7.5细胞HCV颗粒显著降低，表明miR-615-3p基因敲除的细胞株可以有效降低HCV感染。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
SEQUENCE LISTING
<110> 广州艾迪基因科技有限责任公司
<120> 一种防治丙型病毒性肝炎的新靶点及其CRISPR-Cas9靶向系统与应用
<130>
<160> 5
<170> PatentIn version 3.3
<210> 1
<211> 96
<212> DNA
<213> miR-615-3p基因前体序列
<400> 1
ctcgggaggg gcgggagggg ggtccccggt gctcggatct cgagggtgct tattgttcgg 60
tccgagcctg ggtctccctc ttccccccaa cccccc 96
<210> 2
<211> 20
<212> DNA
<213> 靶向miR-615-3p基因前体序列的sgRNA
<400> 2
ggaagaggga gacccaggct 20
<210> 3
<211> 25
<212> DNA
<213> sgRNA正向寡核苷酸序列F
<400> 3
caccgggaag agggagaccc aggct 25
<210> 4
<211> 25
<212> DNA
<213> sgRNA反向寡核苷酸序列R
<400> 4
aaacagcctg ggtctccctc ttccc 25
<210> 5
<211> 13013
<212> DNA
<213> 质粒lentiCRISPR-v2-sgRNA-miR-615-3p的序列
<400> 5
gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 60
catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 120
acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 180
ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 240
aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 300
ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 360
tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc 420
ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt 480
ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa 540
tgggcggtag gcgtgtacgg tgggaggtct atataagcag cgcgttttgc ctgtactggg 600
tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg 660
cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt 720
gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt 780
ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga 840
ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa 900
aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc 960
gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat 1020
ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg 1080
gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc 1140
agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc 1200
atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa 1260
acaaaagtaa gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat 1320
atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta 1380
ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga 1440
ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca 1500
atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat 1560
ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag 1620
cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg 1680
atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg 1740
agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa 1800
attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa 1860
aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac 1920
ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt 1980
ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca 2040
ttatcgtttc agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa 2100
gaagaaggtg gagagagaga cagagacaga tccattcgat tagtgaacgg atcggcactg 2160
cgtgcgccaa ttctgcagac aaatggcagt attcatccac aattttaaaa gaaaaggggg 2220
gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac 2280
taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag 2340
cagagatcca gtttggttaa ttaaggtacc gagggcctat ttcccatgat tccttcatat 2400
ttgcatatac gatacaaggc tgttagagag ataattagaa ttaatttgac tgtaaacaca 2460
aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt 2520
ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat 2580
ttcttggctt tatatatctt gtggaaagga cgaaacaccg ggaagaggga gacccaggct 2640
gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt 2700
ggcaccgagt cggtgctttt ttgaattcgc tagctaggtc ttgaaaggag tgggaattgg 2760
ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca gtccccgaga agttgggggg 2820
aggggtcggc aattgatccg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga 2880
tgtcgtgtac tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt 2940
agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc agaacacagg accggttcta 3000
gagcgctgcc accatggaca agaagtacag catcggcctg gacatcggca ccaactctgt 3060
gggctgggcc gtgatcaccg acgagtacaa ggtgcccagc aagaaattca aggtgctggg 3120
caacaccgac cggcacagca tcaagaagaa cctgatcgga gccctgctgt tcgacagcgg 3180
cgaaacagcc gaggccaccc ggctgaagag aaccgccaga agaagataca ccagacggaa 3240
gaaccggatc tgctatctgc aagagatctt cagcaacgag atggccaagg tggacgacag 3300
cttcttccac agactggaag agtccttcct ggtggaagag gataagaagc acgagcggca 3360
ccccatcttc ggcaacatcg tggacgaggt ggcctaccac gagaagtacc ccaccatcta 3420
ccacctgaga aagaaactgg tggacagcac cgacaaggcc gacctgcggc tgatctatct 3480
ggccctggcc cacatgatca agttccgggg ccacttcctg atcgagggcg acctgaaccc 3540
cgacaacagc gacgtggaca agctgttcat ccagctggtg cagacctaca accagctgtt 3600
cgaggaaaac cccatcaacg ccagcggcgt ggacgccaag gccatcctgt ctgccagact 3660
gagcaagagc agacggctgg aaaatctgat cgcccagctg cccggcgaga agaagaatgg 3720
cctgttcgga aacctgattg ccctgagcct gggcctgacc cccaacttca agagcaactt 3780
cgacctggcc gaggatgcca aactgcagct gagcaaggac acctacgacg acgacctgga 3840
caacctgctg gcccagatcg gcgaccagta cgccgacctg tttctggccg ccaagaacct 3900
gtccgacgcc atcctgctga gcgacatcct gagagtgaac accgagatca ccaaggcccc 3960
cctgagcgcc tctatgatca agagatacga cgagcaccac caggacctga ccctgctgaa 4020
agctctcgtg cggcagcagc tgcctgagaa gtacaaagag attttcttcg accagagcaa 4080
gaacggctac gccggctaca ttgacggcgg agccagccag gaagagttct acaagttcat 4140
caagcccatc ctggaaaaga tggacggcac cgaggaactg ctcgtgaagc tgaacagaga 4200
ggacctgctg cggaagcagc ggaccttcga caacggcagc atcccccacc agatccacct 4260
gggagagctg cacgccattc tgcggcggca ggaagatttt tacccattcc tgaaggacaa 4320
ccgggaaaag atcgagaaga tcctgacctt ccgcatcccc tactacgtgg gccctctggc 4380
caggggaaac agcagattcg cctggatgac cagaaagagc gaggaaacca tcaccccctg 4440
gaacttcgag gaagtggtgg acaagggcgc ttccgcccag agcttcatcg agcggatgac 4500
caacttcgat aagaacctgc ccaacgagaa ggtgctgccc aagcacagcc tgctgtacga 4560
gtacttcacc gtgtataacg agctgaccaa agtgaaatac gtgaccgagg gaatgagaaa 4620
gcccgccttc ctgagcggcg agcagaaaaa ggccatcgtg gacctgctgt tcaagaccaa 4680
ccggaaagtg accgtgaagc agctgaaaga ggactacttc aagaaaatcg agtgcttcga 4740
ctccgtggaa atctccggcg tggaagatcg gttcaacgcc tccctgggca cataccacga 4800
tctgctgaaa attatcaagg acaaggactt cctggacaat gaggaaaacg aggacattct 4860
ggaagatatc gtgctgaccc tgacactgtt tgaggacaga gagatgatcg aggaacggct 4920
gaaaacctat gcccacctgt tcgacgacaa agtgatgaag cagctgaagc ggcggagata 4980
caccggctgg ggcaggctga gccggaagct gatcaacggc atccgggaca agcagtccgg 5040
caagacaatc ctggatttcc tgaagtccga cggcttcgcc aacagaaact tcatgcagct 5100
gatccacgac gacagcctga cctttaaaga ggacatccag aaagcccagg tgtccggcca 5160
gggcgatagc ctgcacgagc acattgccaa tctggccggc agccccgcca ttaagaaggg 5220
catcctgcag acagtgaagg tggtggacga gctcgtgaaa gtgatgggcc ggcacaagcc 5280
cgagaacatc gtgatcgaaa tggccagaga gaaccagacc acccagaagg gacagaagaa 5340
cagccgcgag agaatgaagc ggatcgaaga gggcatcaaa gagctgggca gccagatcct 5400
gaaagaacac cccgtggaaa acacccagct gcagaacgag aagctgtacc tgtactacct 5460
gcagaatggg cgggatatgt acgtggacca ggaactggac atcaaccggc tgtccgacta 5520
cgatgtggac catatcgtgc ctcagagctt tctgaaggac gactccatcg acaacaaggt 5580
gctgaccaga agcgacaaga accggggcaa gagcgacaac gtgccctccg aagaggtcgt 5640
gaagaagatg aagaactact ggcggcagct gctgaacgcc aagctgatta cccagagaaa 5700
gttcgacaat ctgaccaagg ccgagagagg cggcctgagc gaactggata aggccggctt 5760
catcaagaga cagctggtgg aaacccggca gatcacaaag cacgtggcac agatcctgga 5820
ctcccggatg aacactaagt acgacgagaa tgacaagctg atccgggaag tgaaagtgat 5880
caccctgaag tccaagctgg tgtccgattt ccggaaggat ttccagtttt acaaagtgcg 5940
cgagatcaac aactaccacc acgcccacga cgcctacctg aacgccgtcg tgggaaccgc 6000
cctgatcaaa aagtacccta agctggaaag cgagttcgtg tacggcgact acaaggtgta 6060
cgacgtgcgg aagatgatcg ccaagagcga gcaggaaatc ggcaaggcta ccgccaagta 6120
cttcttctac agcaacatca tgaacttttt caagaccgag attaccctgg ccaacggcga 6180
gatccggaag cggcctctga tcgagacaaa cggcgaaacc ggggagatcg tgtgggataa 6240
gggccgggat tttgccaccg tgcggaaagt gctgagcatg ccccaagtga atatcgtgaa 6300
aaagaccgag gtgcagacag gcggcttcag caaagagtct atcctgccca agaggaacag 6360
cgataagctg atcgccagaa agaaggactg ggaccctaag aagtacggcg gcttcgacag 6420
ccccaccgtg gcctattctg tgctggtggt ggccaaagtg gaaaagggca agtccaagaa 6480
actgaagagt gtgaaagagc tgctggggat caccatcatg gaaagaagca gcttcgagaa 6540
gaatcccatc gactttctgg aagccaaggg ctacaaagaa gtgaaaaagg acctgatcat 6600
caagctgcct aagtactccc tgttcgagct ggaaaacggc cggaagagaa tgctggcctc 6660
tgccggcgaa ctgcagaagg gaaacgaact ggccctgccc tccaaatatg tgaacttcct 6720
gtacctggcc agccactatg agaagctgaa gggctccccc gaggataatg agcagaaaca 6780
gctgtttgtg gaacagcaca agcactacct ggacgagatc atcgagcaga tcagcgagtt 6840
ctccaagaga gtgatcctgg ccgacgctaa tctggacaaa gtgctgtccg cctacaacaa 6900
gcaccgggat aagcccatca gagagcaggc cgagaatatc atccacctgt ttaccctgac 6960
caatctggga gcccctgccg ccttcaagta ctttgacacc accatcgacc ggaagaggta 7020
caccagcacc aaagaggtgc tggacgccac cctgatccac cagagcatca ccggcctgta 7080
cgagacacgg atcgacctgt ctcagctggg aggcgacaag cgacctgccg ccacaaagaa 7140
ggctggacag gctaagaaga agaaagatta caaagacgat gacgataagg gatccggcgc 7200
aacaaacttc tctctgctga aacaagccgg agatgtcgaa gagaatcctg gaccgaccga 7260
gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc cccagggccg tacgcaccct 7320
cgccgccgcg ttcgccgact accccgccac gcgccacacc gtcgatccgg accgccacat 7380
cgagcgggtc accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa 7440
ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga 7500
agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct 7560
ggccgcgcag caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg 7620
gttcctggcc accgtcggag tctcgcccga ccaccagggc aagggtctgg gcagcgccgt 7680
cgtgctcccc ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc 7740
cgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga 7800
ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag cccggtgcct gaacgcgtta 7860
agtcgacaat caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta 7920
tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc 7980
ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga 8040
ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac 8100
ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc 8160
cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc 8220
tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg 8280
gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc 8340
ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc 8400
gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcgtcg 8460
actttaagac caatgactta caaggcagct gtagatctta gccacttttt aaaagaaaag 8520
gggggactgg aagggctaat tcactcccaa cgaagacaag atctgctttt tgcttgtact 8580
gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca 8640
ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg 8700
tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc 8760
agggcccgtt taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt 8820
tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc 8880
ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg 8940
tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga 9000
tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc 9060
ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac 9120
cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc 9180
cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt 9240
tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg 9300
gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag 9360
tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt 9420
ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt 9480
taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc 9540
ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag 9600
tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc 9660
atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct 9720
ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct 9780
gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc 9840
ccgggagctt gtatatccat tttcggatct gatcagcacg tgttgacaat taatcatcgg 9900
catagtatat cggcatagta taatacgaca aggtgaggaa ctaaaccatg gccaagttga 9960
ccagtgccgt tccggtgctc accgcgcgcg acgtcgccgg agcggtcgag ttctggaccg 10020
accggctcgg gttctcccgg gacttcgtgg aggacgactt cgccggtgtg gtccgggacg 10080
acgtgaccct gttcatcagc gcggtccagg accaggtggt gccggacaac accctggcct 10140
gggtgtgggt gcgcggcctg gacgagctgt acgccgagtg gtcggaggtc gtgtccacga 10200
acttccggga cgcctccggg ccggccatga ccgagatcgg cgagcagccg tgggggcggg 10260
agttcgccct gcgcgacccg gccggcaact gcgtgcactt cgtggccgag gagcaggact 10320
gacacgtgct acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa 10380
tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct 10440
tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca 10500
caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca 10560
tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat 10620
ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag 10680
ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg 10740
cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa 10800
tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca 10860
ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg 10920
taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc 10980
agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc 11040
cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac 11100
tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc 11160
tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata 11220
gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc 11280
acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca 11340
acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag 11400
cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta 11460
gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg 11520
gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc 11580
agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt 11640
ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa 11700
ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat 11760
atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga 11820
tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac 11880
gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg 11940
ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg 12000
caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt 12060
cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct 12120
cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat 12180
cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta 12240
agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca 12300
tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat 12360
agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac 12420
atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa 12480
ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt 12540
cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg 12600
caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat 12660
attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt 12720
agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg 12780
acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc 12840
cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc 12900
gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct 12960
tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gtt 13013

Claims (10)

1.miR-615-3p基因在作为丙型病毒性肝炎的防治靶点方面的应用。
2.miR-615-3p基因在制备丙型病毒性肝炎的防治试剂中的应用。
3.miR-615-3p基因在作为下调ApoB基因和/或ApoE基因表达的靶点方面的应用。
4.miR-615-3p基因在制备下调ApoB基因和/或ApoE基因表达的试剂中的应用。
5.一种特异性靶向miR-615-3p基因的CRISPR-Cas9系统，其特征在于，包括特异性靶向敲除miR-615-3p基因的sgRNA和包含sgRNA的载体。
6.根据权利要求5所述的CRISPR-Cas9系统，其特征在于，所述sgRNA的序列如SEQ IDNO.2所示。
7.根据权利要求5所述的CRISPR-Cas9系统，其特征在于，所述包含sgRNA的载体为质粒lentiCRISPR-v2-sgRNA-miR-615-3p，其序列如SEQ ID NO.5所示。
8.权利要求5-7任一所述CRISPR-Cas9系统在制备miR-615-3p基因敲除的细胞模型或miR-615-3p基因敲除试剂盒中的应用。
9.一种miR-615-3p基因敲除细胞模型，其特征在于，该细胞模型敲除了miR-615-3p基因。
10.一种miR-615-3p基因敲除试剂盒，其特征在于，包括如下(1)-(3)中的任一种或几种：
(1)权利要求5-7任一中所述sgRNA；
(2)权利要求5或8中所述载体；
(3)权利要求9所述细胞模型。
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