CN108546718A - crRNA介导的CRISPR/Cas13a基因编辑系统在肿瘤细胞中的应用 - Google Patents
crRNA介导的CRISPR/Cas13a基因编辑系统在肿瘤细胞中的应用 Download PDFInfo
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Abstract
本发明公开了一种crRNA介导的CRISPR/Cas13a基因编辑系统在肿瘤细胞中的应用。本发明的Cas13a蛋白在U87胶质瘤细胞系中,可以由单链的crRNA介导到互补的目的RNA上,并进行切割。同时,Cas13a蛋白还能够在真核生物细胞中触发连带剪切的效应,即在开始切割第一条目的RNA之后,Cas13a蛋白对其他遇到的RNA,进行无目的的随机切割,从而起到降低肿瘤细胞指数,抑制小鼠成瘤率和肿瘤大小等抑制和杀伤肿瘤的作用。本发明提供了一种抑制或杀伤肿瘤细胞的新方法,为CRISPR‑Cas13系统随机剪切效应在真核细胞中的应用奠定了基础。
Description
技术领域
本发明涉及DNA重组技术,更具体地是一种crRNA介导的CRISPR/Cas13a基因编辑系统在肿瘤细胞中的应用。
背景技术
短回文重复序列(Clustered regularly interspaced shot palindromicrepeat,CRISPR)和CRISPR相关蛋白(CRISPR-associated proteins,Cas)是古生菌和细菌的获得性免疫系统。Cas蛋白主要分为两大类:获得性模块(adaptation module)和效应模块(effector module)。获得性模块可以将外源核酸信息导入CRISPR序列中,并生成CRISPRRNAs(crRNAs),作为系统的向导。效应模块在crRNA的介导下,剪切外源入侵的核酸。在CRISPR-Cas系统中,获得性模块相似度较高,主要包括Cas1和Cas2。相比之下,效应系统的种类较多,根据亚基的数量,主要分为1级系统(Class 1systems,C1)和2级系统(Class2systems,C2)。C1系统是由多个蛋白的复合物结合在一起,共同行使功能的,包括I型,III型和IV型;C2系统则由单独Cas蛋白发挥作用,包括II型和V型。II型CRISPR系统主要使用RuvC和HNH内切酶结构域行使功能,V型CRISPR系统则使用单独的RuvC内切酶结构域,包括Cpf1,C2c1和C2c3等。这些系统都是靶向DNA的。VI型CRISPR系统由单独的C2c2效应蛋白组成,这一蛋白没有脱氧核糖核酸酶结构域,但是有两个核酸酶结构域(Higher Eukaryotesand Prokaryotes Nucleotide-binding domain,HEPN)。因此,C2c2是由RNA介导的靶向RNA的CRISPR效应器,并且是一个单分子的内切核糖核酸酶。进一步的报道证明,C2c2是一个由crRNA介导的单链RNA(ssRNA)内切酶。C2c2的靶向切割既取决于靶向序列,又取决与序列的二级结构。在离体实验中,C2c2-crRNA复合体在被靶向RNA激活并开始剪切后,还能够非特异性的剪切其他RNA。这一系统的特性被应用为体外的核酸检测。同时,来源于Leptotrichia shahii的Cas13a,以其更为高效的RNA酶切活性得到了使用。然而目前在真核生物中,对于Cas13a相关应用的研究基本尚未开始,对于CRISPR/Cas13a系统在真核和肿瘤细胞中的应用,必将产生巨大的价值。
发明内容
本发明为了解决CRISPR/Cas13a系统在真核和肿瘤细胞中应用较少的问题,所提出一种crRNA介导的CRISPR/Cas13a基因编辑系统在肿瘤细胞中的应用。
本发明是按照以下技术方案实现的。
crRNA介导的CRISPR/Cas13a基因编辑系统在肿瘤细胞中的应用。
进一步的,所述CRISPR/Cas13a基因编辑系统在肿瘤细胞中通过触发随机剪切效应抑制或杀伤肿瘤细胞。
进一步的,所述肿瘤细胞为胶质瘤细胞系、胶质瘤突变型细胞或人肾癌细胞系。
进一步的,所述胶质瘤细胞系为人U87细胞、人LN229细胞系或小鼠GL261细胞系;所述胶质瘤突变型细胞为U87EGFR VIII细胞;所述人肾癌细胞系为人ACHN细胞系。
进一步的,所述Cas13a基因在肿瘤细胞中的表达载体为质粒表达载体或病毒表达载体。
进一步的,所述质粒表达载体为原核与真核质粒表达载体;所述病毒表达载体为腺病毒载体或慢病毒载体。
进一步的,所述质粒表达载体为pcDNA3.1,病毒表达载体为GV341。
进一步的,在U87-Cas13a-EGFP细胞中crRNA的序列为SEQ ID NO.1。
进一步的,在U87-Cas13a-EGFR VIII细胞中crRNA的序列为SEQ ID NO.2。
本发明获得了如下有益效果。
本发明的Cas13a蛋白在U87胶质瘤细胞系中,可以由单链的crRNA介导到互补的目的RNA上,并进行切割。同时,Cas13a蛋白还能够在真核生物细胞中触发连带剪切的效应,即在开始切割第一条目的RNA之后,Cas13a蛋白对其他遇到的RNA,进行无目的的随机切割,从而起到降低肿瘤细胞指数,抑制小鼠成瘤率和肿瘤大小等抑制和杀伤肿瘤的作用。
附图说明
图1是本发明Cas13a病毒在真核细胞中的表达检测图;
图2是本发明CRISPR-Cas13a系统有效敲低EGFP在U87细胞中的表达图;
图3是本发明EGFP的mRNA表达量图(以GAPDH为内参);
图4是本发明CRISPR-Cas13a系统在U87真核细胞中触发核糖体的随机剪切图;
图5是本发明单细胞测序tSNE图;
图6是本发明不同crRNA处理小鼠胶质瘤模型成像结果图;
图7是本发明CRISPR-Cas13a系统在LN229,GL261和ACHN细胞中存在的非特异剪切效应图。
具体实施方式
下面结合附图及实施例对本发明作进一步描述。
Cas13a基因序列为:ATGAAAGTGACCAAGGTCGACGGCATCAGCCACAAGAAGTACATCGAAGAGGGCAAGCTCGTGAAGTCCACCAGCGAGGAAAACCGGACCAGCGAGAGACTGAGCGAGCTGCTGAGCATCCGGCTGGACATCTACATCAAGAACCCCGACAACGCCTCCGAGGAAGAGAACCGGATCAGAAGAGAGAACCTGAAGAAGTTCTTTAGCAACAAGGTGCTGCACCTGAAGGACAGCGTGCTGTATCTGAAGAACCGGAAAGAAAAGAACGCCGTGCAGGACAAGAACTATAGCGAAGAGGACATCAGCGAGTACGACCTGAAAAACAAGAACAGCTTCTCCGTGCTGAAGAAGATCCTGCTGAACGAGGACGTGAACTCTGAGGAACTGGAAATCTTTCGGAAGGACGTGGAAGCCAAGCTGAACAAGATCAACAGCCTGAAGTACAGCTTCGAAGAGAACAAGGCCAACTACCAGAAGATCAACGAGAACAACGTGGAAAAAGTGGGCGGCAAGAGCAAGCGGAACATCATCTACGACTACTACAGAGAGAGCGCCAAGCGCAACGACTACATCAACAACGTGCAGGAAGCCTTCGACAAGCTGTATAAGAAAGAGGATATCGAGAAACTGTTTTTCCTGATCGAGAACAGCAAGAAGCACGAGAAGTACAAGATCCGCGAGTACTATCACAAGATCATCGGCCGGAAGAACGACAAAGAGAACTTCGCCAAGATTATCTACGAAGAGATCCAGAACGTGAACAACATCAAAGAGCTGATTGAGAAGATCCCCGACATGTCTGAGCTGAAGAAAAGCCAGGTGTTCTACAAGTACTACCTGGACAAAGAGGAACTGAACGACAAGAATATTAAGTACGCCTTCTGCCACTTCGTGGAAATCGAGATGTCCCAGCTGCTGAAAAACTACGTGTACAAGCGGCTGAGCAACATCAGCAACGATAAGATCAAGCGGATCTTCGAGTACCAGAATCTGAAAAAGCTGATCGAAAACAAACTGCTGAACAAGCTGGACACCTACGTGCGGAACTGCGGCAAGTACAACTACTATCTGCAAGTGGGCGAGATCGCCACCTCCGACTTTATCGCCCGGAACCGGCAGAACGAGGCCTTCCTGAGAAACATCATCGGCGTGTCCAGCGTGGCCTACTTCAGCCTGAGGAACATCCTGGAAACCGAGAACGAGAACGGTATCACCGGCCGGATGCGGGGCAAGACCGTGAAGAACAACAAGGGCGAAGAGAAATACGTGTCCGGCGAGGTGGACAAGATCTACAATGAGAACAAGCAGAACGAAGTGAAAGAAAATCTGAAGATGTTCTACAGCTACGACTTCAACATGGACAACAAGAACGAGATCGAGGACTTCTTCGCCAACATCGACGAGGCCATCAGCAGCATCAGACACGGCATCGTGCACTTCAACCTGGAACTGGAAGGCAAGGACATCTTCGCCTTCAAGAATATCGCCCCCAGCGAGATCTCCAAGAAGATGTTTCAGAACGAAATCAACGAAAAGAAGCTGAAGCTGAAAATCTTCAAGCAGCTGAACAGCGCCAACGTGTTCAACTACTACGAGAAGGATGTGATCATCAAGTACCTGAAGAATACCAAGTTCAACTTCGTGAACAAAAACATCCCCTTCGTGCCCAGCTTCACCAAGCTGTACAACAAGATTGAGGACCTGCGGAATACCCTGAAGTTTTTTTGGAGCGTGCCCAAGGACAAAGAAGAGAAGGACGCCCAGATCTACCTGCTGAAGAATATCTACTACGGCGAGTTCCTGAACAAGTTCGTGAAAAACTCCAAGGTGTTCTTTAAGATCACCAATGAAGTGATCAAGATTAACAAGCAGCGGAACCAGAAAACCGGCCACTACAAGTATCAGAAGTTCGAGAACATCGAGAAAACCGTGCCCGTGGAATACCTGGCCATCATCCAGAGCAGAGAGATGATCAACAACCAGGACAAAGAGGAAAAGAATACCTACATCGACTTTATTCAGCAGATTTTCCTGAAGGGCTTCATCGACTACCTGAACAAGAACAATCTGAAGTATATCGAGAGCAACAACAACAATGACAACAACGACATCTTCTCCAAGATCAAGATCAAAAAGGATAACAAAGAGAAGTACGACAAGATCCTGAAGAACTATGAGAAGCACAATCGGAACAAAGAAATCCCTCACGAGATCAATGAGTTCGTGCGCGAGATCAAGCTGGGGAAGATTCTGAAGTACACCGAGAATCTGAACATGTTTTACCTGATCCTGAAGCTGCTGAACCACAAAGAGCTGACCAACCTGAAGGGCAGCCTGGAAAAGTACCAGTCCGCCAACAAAGAAGAAACCTTCAGCGACGAGTTGGAACTGATCAACCTGCTGAACCTGGACAACAACAGAGTGACCGAGGACTTCGAGCTGGAAGCCAACGAGATCGGCAAGTTCCTGGACTTCAACGAAAACAAAATCAAGGACCGGAAAGAGCTGAAAAAGTTCGACACCAACAAGATCTATTTCGACGGCGAGAACATCATCAAGCACCGGGCCTTCTACAATATCAAGAAATACGGCATGCTGAATCTGCTGGAAAAGATCGCCGATAAGGCCAAGTATAAGATCAGCCTGAAAGAACTGAAAGAGTACAGCAACAAGAAGAATGAGATTGAAAAGAACTACACCATGCAGCAGAACCTGCACCGGAAGTACGCCAGACCCAAGAAGGACGAAAAGTTCAACGACGAGGACTACAAAGAGTATGAGAAGGCCATCGGCAACATCCAGAAGTACACCCACCTGAAGAACAAGGTGGAATTCAATGAGCTGAACCTGCTGCAGGGCCTGCTGCTGAAGATCCTGCACCGGCTCGTGGGCTACACCAGCATCTGGGAGCGGGACCTGAGATTCCGGCTGAAGGGCGAGTTTCCCGAGAACCACTACATCGAGGAAATTTTCAATTTCGACAACTCCAAGAATGTGAAGTACAAAAGCGGCCAGATCGTGGAAAAGTATATCAACTTCTACAAAGAACTGTACAAGGACAATGTGGAAAAGCGGAGCATCTACTCCGACAAGAAAGTGAAGAAACTGAAGCAGGAAAAAAAGGACCTGTACATCCGGAACTACATTGCCCACTTCAACTACATCCCCCACGCCGAGATTAGCCTGCTGGAAGTGCTGGAAAACCTGCGGAAGCTGCTGTCCTACGACCGGAAGCTGAAGAACGCCATCATGAAGTCCATCGTGGACATTCTGAAAGAATACGGCTTCGTGGCCACCTTCAAGATCGGCGCTGACAAGAAGATCGAAATCCAGACCCTGGAATCAGAGAAGATCGTGCACCTGAAGAATCTGAAGAAAAAGAAACTGATGACCGACCGGAACAGCGAGGAACTGTGCGAACTCGTGAAAGTCATGTTCGAGTACAAGGCCCTGGAA(Gootenberg JS,Abudayyeh OO,Lee JW,Essletzbichler P,Dy AJ,Joung J,Verdine V,Donghia N,Daringer NM,Freije CA,Myhrvold C,BhattacharyyaRP,Livny J,Regev A,Koonin EV,Hung DT,Sabeti PC,Collins JJ,Zhang F.Nucleicacid detection with CRISPR-Cas13a/C2c2.Science.2017Apr 28;356(6336):438-442.doi:10.1126/science.aam9321.Epub 2017Apr 13)
目的基因Cas13a由上海艾博思生物科技有限公司化学合成。
一、Cas13a在真核系统中的表达
首先将Cas13a蛋白基因克隆到了真核表达的慢病毒中,载体名称:GV341(购自吉凯基因);元件顺序:Ubi-MCS-3FLAG-SV40-puromycin;克隆位点:AgeI/NheI。
具体操作步骤如下:
1.利用限制性内切酶消化获得线性化载体
配制50μl酶切体系。按列表顺序依次加入各种试剂,用移液器轻轻吹打混匀,短暂离心,置于37℃反应3h或过夜。对载体酶切产物进行琼脂糖凝胶电泳,回收目的条带。
试剂 | 体积(μl) |
ddH2O | 41 |
10×CutSmart Buffer2 | 5 |
纯化的质粒DNA(1μg/μL) | 2 |
Age I(10U/μl) | 1 |
NheI(10U/μl) | 1 |
Total | 50 |
2.PCR扩增制备目的基因片段
所用扩增引物在设计时需在其5’端添加同源重组序列使用该引物扩增目的基因片段,扩增产物5’和3’最末端的序列分别与线性化克隆载体两末端序列完全一致。
配制如下反应体系,轻轻吹打混匀,短暂离心,置于PCR仪中进行反应。
反应体系:
试剂 | 体积(μL) |
ddH2O | 32.5 |
5×PS Buffer | 10 |
dNTP Mix(2.5mM each) | 4 |
上游扩增引物(10μM) | 1 |
下游扩增引物(10μM) | 1 |
模板1(10ng/μL) | 1 |
PrimeSTAR HS DNA polymerase | 0.5 |
Total | 50 |
引物序列:
Cas13a-F:
CCAACTTTGTGCCAACCGGTCGCCACCATGAAAGTGACCAAGGTCGACGGCATC
Cas13a-R:
AATGCCAACTCTGAGCTTTTCCAGGGCCTTGTACTCGAACATG
反应条件:
PCR产物3501bp,目的片段测序结果:正确。
3.以线性化载体和目的基因扩增产物配制反应体系,进行重组反应,实现线性化载体和目的基因片段的体外环化
于冰水浴中配制如下反应体系。用移液器轻轻吹打混匀,短暂离心,避克产生气泡。37℃反应30min,随后置于冰水浴中冷却5min后立即转化。
4.重组产物直接进行转化,挑取平板上的单克隆进行PCR鉴定,对阳性克隆进行测序及结果分析
将10μL交换反应产物加入到100μL感受态细胞中,轻弹管壁数下混匀,在冰上放置30min。42℃热激90s,冰水浴孵育2min。加入500μL LB培养基,置于37℃摇床振荡培养1h。取适量菌液均匀涂布在含有相应抗生素的平板上,在恒温培养箱中倒置培养12-16h。
5.将正确克隆菌液扩大培养、抽提,获得高纯度质粒,用于下游病毒包装
将测序正确的菌液转接于10ml含相应抗生素的LB液体培养基中,37℃培养过夜,用天根无内毒素质粒小提中量试剂盒进行质粒抽提,抽提合格的质粒进入下游扩增。
详细操作步骤如下:
(1)收集过夜培养的菌液于标记好的5ml离心管,12000rpm,离心2min收菌;
(2)弃上清,加入250μl细胞重悬液,充分振荡,使菌块悬浮均匀;
(3)加入250μl细胞裂解液,再加入10μl蛋白酶K,上下颠倒5-6次,轻轻混匀;静置1-2min,致使菌体裂解澄清;
(4)加入350μl中和液,上下颠倒混匀,使蛋白完全析出,冰浴静置5min;
(5)10000rpm离心10min,弃蛋白,收集上清于另一干净无菌的1.5ml EP管;
(6)12000rpm离心5min,同时准备标记好的回收柱,将上清转秱至回收柱中,12000rpm离心1min,弃下层废液;
(7)加入600μl预先配置好的漂洗液,12000rpm离心1min,弃下层废液,重复一次,12000rpm空离2min,进一步除去残留的漂洗液;
(8)在超净台中将回收柱转秱至新的1.5ml EP管中,静置10-20min,自然晾干;
(9)往回收柱中加入95μl Nuclease-Free Water,静置2min,12000rpm离心2min,收集样品做好编号,电泳、测定浓度,进行质检。
6.质粒转染与慢病毒收获
(1)转染前24h,用胰蛋白酶消化对数生长期的293T细胞,以含10%血清的培养基调整细胞密度约5x 106细胞/15ml,重新接种于10cm细胞培养皿,37℃、5%CO2培养箱内培养。24h待细胞密度达70%~80%时即可用于转染;
(2)转染前2h更换为无血清培养基;
(3)向一灭菌离心管中加入所制备的各DNA溶液(GV载体质粒20μg、pHelper 1.0载体质粒15μg、pHelper 2.0载体质粒10μg),利用lipo3000转染试剂混合均匀,调整总体积为1ml,在室温下温育5min;
(4)混合液缓慢滴加至293T细胞的培养液中,混匀,于37℃、5%CO2细胞培养箱中培养;
注:加入过程一定要均匀,尽可能地不要将细胞吹起。
(5)培养6h后弃去含有转染混和物的培养基,加入10ml的PBS液清洗一次,轻柔晃动培养皿以洗涤残余的转染混和物后倒弃;
(6)缓慢加入含10%血清的细胞培养基20ml,于37℃、含5%CO2培养箱内继续培养48-72h。
7.慢病毒浓缩与纯化
(1)根据细胞状态,收集转染后48h(转染即可为0h计起)的293T细胞上清液;
(2)于4℃,4000g离心10min,除去细胞碎片;
(3)以0.45μm滤器过滤上清液于40ml超速离心管中;
(4)分别配平样品,将带有病毒上清液的超速离心管逐一放入至Beckman超速离心机内,设置离心参数为25000rpm,离心时间为2h,离心温度控制在4℃;
(5)离心结束后,弃去上清,尽量去除残留在管壁上的液体,加入病毒保存液(可用PBS或细胞培养基替代),轻轻反复吹打重悬;
注:该步骤病毒回收会存在一定程度损失,尽可能地避免病毒长时间暴露在室温中。
(6)经充分溶解后,高速离心10000rpm,离心5min后,取上清按要求分装;
(7)准备样品待检测。
8.Cas13a病毒在真核细胞中的表达检测
目的细胞:293T(购自ATCC)
培养基:DMEM培养基(含10%胎牛血清)
抗体信息:
一抗:FLAG,Sigma,F1804,1:3000
二抗:Mouse,santa-cruz,sc-2005,1:4000
Western Blot实验过程:
细胞准备完毕,弃去培养基,用预冷的4℃PBS清洗细胞3次,弃净PBS后将培养皿置于冰上。RIPA细胞裂解液与PMSF以100:1混合,冰上预冷。10cm大皿加200μl裂解液,将细胞刮下,将细胞和裂解液转移至1.5ml EP管,冰上裂解30min。于4℃下12000rpm离心15min。将上清移至另一1.5m lEP管。
BCA蛋白浓度检测:按照BCA试剂盒说明制作标准曲线。BCA和CuCl2以50:1混合均匀,每个副孔加入混合好的BCA溶液200μl。收集的蛋白用PBS稀释至1/10,每个副孔加入20μl。蛋白和BCA混合均匀和置于37℃恒温箱30min,酶标仪在562nm处检测吸光度。利用标准曲线计算蛋白浓度。
将剩下的蛋白上清加入蛋白上样缓冲液,100℃煮沸10min,-20℃保存。
聚丙烯酰胺凝胶电泳:配置10%的聚丙烯酰胺凝胶配方如下:
每孔加入20-40μg蛋白,80V电泳40min,后改为150V电泳至上样缓冲液至胶底,终止电泳,将蛋白转移至PVDF膜上,100V转膜约60min。5%BSA封闭PVDF膜1h,一抗封闭过夜(抗Flag抗体(CST,1:1000稀释)。第二天将PVDF膜在常温下复温1h,弃去抗体,PBST洗3遍,每次10min。使用相应抗鼠或是抗兔结合辣根过氧化氢酶的二抗常温下孵育1h,再次使用PBST洗3遍,每次10min。使用G:BOX F3凝胶成像系统(英国Syngene)免疫成像。
结果如图1所示,1#:WB标准品—SURVIVIN-3FLAG-GFP(分子大小:48KD);2#:对照组为293T细胞;3#:为目的基因质粒转染293T后样品。
经Western Blot检测,可以观察到129KD附近处有特征条带,其大小和目的基因融合蛋白相吻合。结果说明:该病毒用FLAG抗体检测到目的条带,过表达成功。
二、CRISPR-Cas13a系统在胶质瘤U87细胞中能够触发随机剪切
通过慢病毒过表达的方式构建U87-Cas13a过表达细胞。将纯化所得Cas13a慢病毒(滴度2E+8/TU/ml)滴加入U87细胞中,计量为1ul/ml。48h后利用嘌呤霉素进行筛选,工作浓度为2ug/ml。筛选一周后,得到U87-Cas13a过表达细胞。EGFP-PEST是一种可以快速降解的EGFP蛋白,非常便于检测细胞内RNA水平的变化。将EGFP-PEST的荧光病毒(购自吉凯基因)转染入U87-Cas13a细胞中,得到了U87-Cas13a-EGFP细胞。转染方法同Cas13a慢病毒转染过程。
针对EGFP设计crRNA序列
crRNA-EGFP:CCACCCUGACCUACGGCGUGCAGUGCUUC
将crRNA-EGFP利用lipo3000(购自英潍捷基)转染入U87细胞,crRNA的转染剂量为300ng/ml,转染方法按照lipo3000说明书进行。分别在0h,2h,4h,8h时间点固定细胞,并在共聚焦显微镜下观察细胞EGFP荧光水平的变化。免疫荧光的结果显示,CRISPR-Cas13a系统能够有效的在U87细胞中减弱EGFP的表达(如图2所示)。
进一步的,在不同时间点提取转染crRNA-EGFP的U87-Cas13a-EGFP细胞的RNA,利用实时定量PCR的方法进行检测(Real Time PCR),并根据GAPDH进行qRT-PCR的结果显示,2h和4h时间点内,EGFP的mRNA表达量相较对照组有明显的下降,而8h时间点,EGFP的表达量反而开始上升(如图3所示)。
查看Real Time PCR的原始数据发现从4h开始,在RNA初始反转录量一致的情况下,GAPDH循环数逐渐升高,表明GAPDH失去了持家基因的功能(见表1)。
表1.Real Time PCR实验中EGFP和GAPDH的ct值列表
由此推断,随着时间的增加,GAPDH可能存在由于CRISPR-Cas13a系统造成的降解情况。为了验证此推断,对两个不相关基因进行检测,L3MBTL1和HOTAIR在不同时间点的ct值变化。结果发现,在4h和8h,这两个基因的ct值都出现了上升,与GAPDH的变化吻合(见表2)。
表2:Real Time实验中,L3MBTL1和HOTAIR基因的循环数
为了进一步验证CRISPR-Cas13a系统在U87细胞中的随机剪切效应,在0h,0.5h,1h,2h,4h和8h这几个时间点,提取crRNA-EGFP转染的U87-Cas13a-EGFP细胞总RNA,并利用变性凝胶电泳对总RNA进行了检测,结果如图4所示,在4h和8h时间点上,核糖体28S和18S亚基均出现降解。这一直接的证据,说明了随机剪切效应在真核细胞中的存在,也为随机剪切效应的应用奠定了可能。
三、CRISPR-Cas13a系统能够触发EGFRvIII特异性表达细胞的凋亡
EGFR的过表达和下游信号通路的激活,常见于多种癌症。EGFR的III型突变(EGFRvIII),则是胶质瘤中特异的突变形式。EGFRvIII是野生型EGFR缺失2~7号外显子后,1号与8号外显子直接连接的突变体。EGFRvIII能够持续激活,过表达EGFRvIII的胶质瘤细胞,恶性程度很高。本发明希望应用CRISPR-Cas13a系统清除EGFRvIII阳性的胶质瘤细胞。为此,针对EGFRvIII特异性的1号、8号外显子连接点,设计了5条crRNA。
crRNA-EGFRvIII-1(crRNA1):CUGGAGGAAAAGAAAGGUAAUUAUGUGGU
crRNA-EGFRvIII-2(crRNA2):GGAGGAAAAGAAAGGUAAUUAUGUGGUGA
crRNA-EGFRvIII-3(crRNA3):GAGGAAAAGAAAGGUAAUUAUGUGGUGAC
crRNA-EGFRvIII-4(crRNA4):AAGAAAGGUAAUUAUGUGGUGACAGAUCA
crRNA-EGFRvIII-5(crRNA5):AGAAAGGUAAUUAUGUGGUGACAGAUCAC
将EGFRvIII的病毒(购自吉凯基因)转染进入U87-Cas13a细胞中,得到了U87-Cas13a-EGFRvIII细胞。利用lipo3000,分别将crRNA1~5转染入细胞中,并在4h和8h提取RNA,读取Real Time PCR的ct值。结果显示,crRNA2的处理,能够提高EGFRvIII和GAPDH的循环数(如表3)。
表3.Real Time实验中,EGFRvIII和GAPDH基因的循环数
为了进一步验证CRISPR-Cas13a系统在胶质瘤中的随机剪切效应,将U87-Cas13a-EGFRvIII细胞分成三组,一组转染crRNA2,一组转染crRNA5,对照组单加转染试剂lipo3000。处理4h后,收集细胞,进行单细胞测序。在上机细胞数目相同的情况下,对照组建库细胞6763个,crRNA5组建库细胞6240个,而crRNA2组只收集到2504个细胞,且这些能够建库的细胞,内含RNA的数目也远小于对照组和crRNA5组(如图5)。
进一步的,用lipo3000,crRNA2和crRNA5预处理U87-Cas13a-EGFRvIII细胞4h,并将50万细胞颅内原位注射入小鼠脑内,建立小鼠胶质瘤模型。小动物成像的结果显示,crRNA2处理组有效的抑制了小鼠的成瘤率和肿瘤大小。实验结果表明(如图6所示),CRISPR-Cas13系统能够在真核肿瘤系统中触发随机剪切效应,并起到抑制和杀伤肿瘤的作用。
本发明在其他肿瘤细胞中进一步验证了CRISPR/Cas13a的随机剪切效应,分别为LN229细胞系(人胶质瘤细胞)、GL261(小鼠胶质瘤细胞系)和ACHN(人肾癌细胞系)。在这三种细胞中过表达了Cas13a和EGFRvIII,方法与U87中的步骤一致。通过Real Time PCR的方法,验证了这几种细胞在转染crRNA2后,EGFRvIII,GAPDH和Cas13a的表达变化。以0h的表达量作为标准,发现无论目的基因EGFRvIII,还是非特异性的基因GAPDH和Cas13a,表达量在4h和8h都显著下降,说明在LN229,GL261和ACHN细胞中也存在非特异剪切效应(图7)。
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Claims (9)
1.crRNA介导的CRISPR/Cas13a基因编辑系统在肿瘤细胞中的应用。
2.根据权利要求1所述的crRNA介导的CRISPR/Cas13a基因编辑系统在肿瘤细胞中的应用,其特征在于:所述CRISPR/Cas13a基因编辑系统在肿瘤细胞中通过触发随机剪切效应抑制或杀伤肿瘤细胞。
3.根据权利要求1所述的crRNA介导的CRISPR/Cas13a基因编辑系统在肿瘤细胞中的应用,其特征在于:所述肿瘤细胞为胶质瘤细胞系、胶质瘤突变型细胞或人肾癌细胞系。
4.根据权利要求3所述的crRNA介导的CRISPR/Cas13a基因编辑系统在肿瘤细胞中的应用,其特征在于:所述胶质瘤细胞系为人U87细胞、人LN229细胞系或小鼠GL261细胞系;所述胶质瘤突变型细胞为U87EGFR VIII细胞;所述人肾癌细胞系为人ACHN细胞系。
5.根据权利要求1所述的crRNA介导的CRISPR/Cas13a基因编辑系统在肿瘤细胞中的应用,其特征在于:所述Cas13a基因在肿瘤细胞中的表达载体为质粒表达载体或病毒表达载体。
6.根据权利要求5所述的crRNA介导的CRISPR/Cas13a基因编辑系统在肿瘤细胞中的应用,其特征在于:所述质粒表达载体为原核与真核质粒表达载体;所述病毒表达载体为腺病毒载体或慢病毒载体。
7.根据权利要求5所述的crRNA介导的CRISPR/Cas13a基因编辑系统在肿瘤细胞中的应用,其特征在于:所述质粒表达载体为pcDNA3.1,病毒表达载体为GV341。
8.根据权利要求4所述的crRNA介导的CRISPR/Cas13a基因编辑系统在肿瘤细胞中的应用,其特征在于:在U87-Cas13a-EGFP细胞中crRNA的序列为SEQ ID NO.1。
9.根据权利要求4所述的crRNA介导的CRISPR/Cas13a基因编辑系统在肿瘤细胞中的应用,其特征在于:在U87-Cas13a-EGFR VIII细胞中crRNA的序列为SEQ ID NO.2。
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