本发明属于基因编辑技术领域,具体为一种用于iii-a型crispr-csm系统中rna编辑的组合物及应用。
背景技术:
crispr-cas系统是一种细菌和古菌的免疫防御系统,通过识别和切割外源dna或rna,干扰基因的表达从而达到抵抗病毒的入侵。因crispr-cas系统具有dna和rna靶向切割作用,crispr-cas系统现已被开发成一种高效的基因编辑工具,是继锌指核酸内切酶和类转录激活因子效应物核酸酶后的第三代基因组和基因定点编辑技术,可用于用于动、植物及微生物的基因功能解析、新种质创制、核酸检测和疾病诊断及治疗等基础研究及应用中。尽管ii型crispr-cas9基因组编辑技术在多种细胞以及动植物模型中实现靶向编辑dna,但是尚未有以iii型crispr-csm系统建立的基因编辑技术的应用,因此,急需建立和发展iii型crispr-csm系统介导的rna编辑技术体系。
技术实现要素:
有鉴于此,本发明的目的之一在于提供一种用于iii-a型crispr-csm系统中rna编辑的组合物,本发明的目的之二在于提供所述组合物在细胞中进行rna编辑的方法,本发明的目的之三在于提供一种用于iii-a型crispr-csm系统中rna编辑的试剂盒,本发明目的之四在于提供一种药物制剂,本发明目的之五在于提供一种所述药物制剂在制备抗病毒治疗药物中的应用。
为达到上述目的,本发明提供如下技术方案:
1、一种用于iii-a型crispr-csm系统中rna编辑的组合物,所述组合物包括csm复合物和靶向定位目标rna序列的向导rna,所述csm复合物由cas10、csm2、csm3、csm4和csm5组成,核苷酸序列为seqidno:1-seqidno:5,氨基酸序列为seqidno:6-seqidno:10。
作为优选的技术方案之一,所述目标rna为sars冠状病毒、流感病毒、人类冠状病毒的rna基因组及转录本。
作为优选的技术方案之一,所述人类冠状病毒为hku1-cov、229e-cov、nl63-cov。
作为优选的技术方案之一,所述rna编辑为在细胞中或体外进行编辑。
作为优选的技术方案之一,所述细胞为哺乳动物细胞、植物细胞、细菌细胞或酵母细胞。
2、利用所述组合物在细胞中进行rna编辑的方法,具体步骤为:
(1)构建含有csm复合物核苷酸序列的质粒,表达并纯化出csm复合物蛋白;
(2)根据目标rna序列,设计并合成向导rna;
(3)将csm复合物蛋白与向导rna共同转染至细胞中。
作为优选的技术方案之一,所述目标rna序列为新型冠状病毒sars-cov-2的rdrp基因序列,所述向导rna核苷酸序列为seqidno:14或seqidno:15。
作为优选的技术方案之一,所述目标rna序列为流感病毒基因,所述向导rna核苷酸序列为seqidno:16或seqidno:17。
3、一种用于iii-a型crispr-csm系统中rna编辑的试剂盒,所述试剂盒包括所述csm复合物蛋白和向导rna。
4、一种药物制剂,所述药物制剂含有所述组合物和一种或多种药学上可以接受的载体或辅料。
5、所述药物制剂在制备抗病毒治疗药物中的应用。
本发明的有益效果在于:
本发明所提供的iii-a型crispr-csm系统中rna编辑的组合物(csm-grna)中csm复合物蛋白核苷酸序列由cas10、csm2、csm3、csm4和csm5的核酸序列组成。csm-grna组合物能够靶向特异识别与向导rna互补的rna,进而特异切割rna分子;通过lipofectaminetmcrisprmaxtm转染试剂介导的组合物转染细胞,有助于实现对靶向目标rna的高效编辑,为基因改造提供有效的rna编辑技术体系;此外,本发明所提供得组合物还可以用于制备抗病毒治疗的药物,具有良好的应用前景和医药用途。
附图说明
图1为iii-a型crispr-csm系统中rna编辑技术体系及其rna编辑组合物的活性验证,a为iii-a型crispr-csm系统中rna编辑技术体系的示意图;b为哺乳细胞hek293t中rna编辑组合物的活性验证原理示意图;c为gaussia荧光素酶定量检测rna编辑组合物的活性效率结果。
图2为用于iii-a型crispr-csm系统中rna编辑的组合物靶向编辑新型冠状病毒sars-cov-2的rdrp基因结果图,a为靶向编辑新型冠状病毒sars-cov-2的rdrp基因的示意图;b为qpcr结果图;c为westernblot结果图;d为流式细胞检测hek293t细胞中rdrp表达水平结果图。
图3为用于iii-a型crispr-csm系统中rna编辑的组合物靶向编辑流感病毒结果,a为靶向编辑流感病毒示意图;b为qrt-pcr检测iav病毒rna表达水平结果;c为iav病毒滴度评估病毒感染的效率。
具体实施方式
以下通过特定的具体实例说明本发明的实施方式,本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用,本说明书中的各项细节也可以基于不同观点与应用,在没有背离本发明的精神下进行各种修饰或改变。
实施例1
用于iii-a型crispr-csm系统中rna编辑的组合物
iii型crispr-csm系统由cas1-cas2-cas6-cas10-csm2-csm3-csm4-csm5-csm6-csm6组成(图1中a)。在iii型crispr-cas中,cas10-csm2-csm3-csm4-csm5能够靶向特异识别与grna互补的rna,进而特异切割rna分子;同时,能够激活csm6的rna降解酶,引起非特异性的rna降解,导致细胞死亡。由于csm6的非特异性的切割rna,限制了开发iii型crispr系统作为真正的应用工具。为了避免该缺陷,设计了一种用于iii-a型crispr-csm系统介导rna编辑的组合物,由csm复合物和向导rna(guiderna,grna)组成,所述csm复合物由cas10、csm2、csm3、csm4和csm5组成,核苷酸序列为seqidno:1-seqidno:5,氨基酸序列为seqidno:6-seqidno:10。构建csm复合物基因的重组质粒pcas/csm/his-csm2质粒,cas/csm/his-csm2质粒由geneuniversal公司合成。
csm复合物的表达及纯化:将构建的pcas/csm/his-csm2质粒转化e.colibl21感受肽细胞,获得重组质粒菌,扩大培养后用7000rpm离心10分钟收集,用pbs清洗3次,液氮反复冻融3次后超声波破碎30min,16000rpm离心30分钟收集上清,上清用0.45μm滤膜抽滤;将抽滤后的上清上柱到用pbs平衡过的ni亲和柱中,分别用30mlpbs溶液和20mm咪唑的pbs快速冲洗柱子以洗去未结合的蛋白,分别用10ml含100mm、500mm和1m咪唑的pbs缓慢冲洗柱子并收集洗脱液,每2ml收集一管,将收集到的洗脱液进行sds-page电泳检测,以丢弃含有杂蛋白的洗脱液,用pbs溶液进行透析36h,每8h换透析液一次,最后进行sds-page电泳检测,成功分离纯化csm复合物蛋白;并用bca蛋白浓度测定试剂盒测定其浓度。
根据目标gaussia基因的rna序列(ncbi登录号mf882921.1):设计grna,seqidno:11作空白对照,检测原理如图1中b所示。
not-targetinggrna序列(seqidno:11):
5’-acggaaacggacacgcgaacuuguggccguuuacgucgc-3’;
gaussiagrna1序列(seqidno:12):
5’-acggaaacgcagaucagggcaaacagaacuuugacuccca-3’;
gaussiagrna2序列(seqidno:13):
5’-acggaaacguucucggugggcuuggccucggccacagcga-3’;
参照lipofectaminecrisprmaxtmtransfectionreagent(thermofisherscienctific)转染试剂说明书,将3000ngiii-a型csm复合物蛋白和1200nggrna(grna1/grna2/空白对照)组成的转染体系,转染到hek293t细胞;通过lipofectamine3000(thermofisherscienctific)转染试剂将50ngptk-gaussialuc和pcmv-redfireflyluc质粒转染到hek293k细胞。按piercetmgaussia-fireflyluciferasedualassaykit操作说明,检测luciferase含量。
结果如图1中c所示,在哺乳细胞中用于iii-a型crispr-csm系统介导rna编辑的组合物可以实现对报告质粒中gaussia的转录rna进行切割,其rna的切割效率分别为65.8%(grna1)和69.0%(grna2),表明此组合物可以用于开展基因敲降,抑制基因表达,从而实现rna编辑。
实施例2
用于iii-a型crispr-csm系统中rna编辑的组合物靶向编辑新型冠状病毒sars-cov-2的rdrp基因
rna病毒的基因组复制过程完成不涉及dna形式,因此需要自身编码的依赖rna的rna聚合酶(rna-dependentrnapolymerase,rdrp)来主导完成。根据rdrp基因(ncbi登录号:43740578)设计grna,以实施例1中seqidno:11作空白对照,检测原理如图2中a所示,
rdrpgrna3序列(seqidno:14):
5’-acggaaacacgugauauaugugguaccauguucaccgucua-3’;
rdrpgrna4序列(seqidno:15):
5’-acggaaacuaugucugaucccaauauuuaaaauaacgguc-3’
参照lipofectaminecrisprmaxtmtransfectionreagent(thermofisherscienctific)转染试剂说明书,将3000ngiii型csm复合物和1200nggrnas(grna3/grna4/空白对照)组成的转染体系,转染至hek293t细胞;用sars-cov2-rdrp-gfplentivirus感染hek293k细胞(sars-cov-2-rdrp-gfp由斯坦福大学leis.qi惠赠)。24h后通过qpcr、westernblot和流式细胞检测rdrp-gfp的表达水平。
结果如图2中b-d所示,用于iii-a型crispr-csm系统介导rna编辑的组合物可以敲低rdrp基因的表达。
实施例3
用于iii-a型crispr-csm系统中rna编辑的组合物靶向编辑流感病毒
根据流感病毒(influenzaavirus,iav)核衣壳基因(ncbi登录号:mh006711.1)设计grna,以实施例1中seqidno:11作空白对照,检测原理如图3中a所示,
iavgrna5序列(seqidno:16):
5’-acggaaacguaaugaaggaucuuauuucuucggagacaau-3’;
iavgrna6序列(seqidno:17):
5’-acggaaacggucgguugcucacaaguccugccugccugcc-3’;
按照lipofectaminecrisprmaxtmtransfectionreagent(thermofisherscienctific)转染试剂说明书,将3000ngiii型csm复合物和1200nggrnas(grna5/grna6/空白对照)组成的转染体系转染至hek293t细胞,感染iav病毒(moi=0.01和0.5,multiplicityofinfection),24h后检测iav病毒的rna以及病毒滴度。
结果如图3中b、c所示,在hek293t细胞中用于iii-a型crispr-csm系统介导rna编辑的组合物可有效抑制iav感染,并有助于抵抗或者消除rna病毒的感染,可应用于靶向防治新型冠状病毒,以及人类多种常见或致病性的rna病毒,并应用于预防和治疗未来可能新出现的流行性病毒。
最后说明的是,以上实施例仅用以。说明本发明的技术方案而非限制,尽管参照较佳实施例对本发明进行了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本技术方案的宗旨和范围,其均应涵盖在本发明的权利要求范围当中。
序列表
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tccatctcccctctgaccaagatcgagttcgagatcacaacaacaaccgacgaggccggc720
agactgattgaggagctgggcaagagggcccaggccttttacaaggactacaaggccttc780
ttcctgtccgagtttcccgacgacaagatccaggccaatctgcagtaccccatctacctg840
ggcgccggcagcggcgcttggacaaagaccctgttcaagcaggccgacggcatcctgcag900
agaagatacagcaggatgaagaccaagatggtgaagaagggcgtgctgaagctgacaaag960
gcccctctgaagatcgtgaagatccctagcggcaatcacagcctgatcaagaatcacgag1020
agcttctacgagatgggcaaggccaactttatgatcaaggagatcgacaag1071
<210>6
<211>758
<212>prt
<213>人工序列(artificialsequence)
<400>6
metlyslysglulysileaspleuphetyrglyalaleuleuhisasp
151015
ileglylysvalileglnargalathrglygluarglyslyshisala
202530
leuvalglyalaasptrppheaspgluilealaaspasnglnvalile
354045
seraspglnileargtyrhismetalaasntyrglnserasplysleu
505560
glyasnasphisleualatyrilethrtyrilealaaspasnileala
65707580
serglyvalaspargargglnserasnglugluseraspgluaspala
859095
seralalysiletrpaspthrtyrthrasnglnalaaspilepheasn
100105110
valpheglyalaglnthrasplysargtyrphelysprothrvalleu
115120125
asnleulysserlysproasnphealaseralathrtyrgluprophe
130135140
serlysglyasptyralaalailealathrargilelysasngluleu
145150155160
alaglupheglupheasnglnalaglnileaspserleuleuasnleu
165170175
pheglualaileleuserphevalproserserthrasnserlysglu
180185190
ilealaaspileserleualagluhisserargleuthralaalaphe
195200205
alaleualailetyrasptyrleugluasplysglyarghisasntyr
210215220
lysgluaspleuphethrlysalaseralaphetyrgluglugluala
225230235240
pheleuleualaserpheaspleuserglyileglnasppheiletyr
245250255
asnilealathrserglyalaalalysglnleulysalaargserleu
260265270
tyrleuaspphemetserglutyrilealaaspserleuleuasplys
275280285
leuglyleuasnargalaasnleuleutyrvalglyglyglyhisala
290295300
tyrphevalleualaasnthrglulysthrvalgluthrleuvalgln
305310315320
pheglulysasppheasnglnpheleuleualaasnpheglnthrarg
325330335
leutyrvalalapheglytrpglyserphealaalalysaspilemet
340345350
sergluleuasnserproglusertyrargglniletyrglnlysala
355360365
serargmetileserglulyslysileserargtyrasptyrargthr
370375380
leumetleuleuasnargglyglylyssersergluargglucysglu
385390395400
ilecyshisservalgluasnleuvalsertyrhisaspglnlysval
405410415
cysaspilecysargglyleutyrglnpheserlysgluilealahis
420425430
asphispheileilethrgluasngluglyleuproileglyproasn
435440445
alacysleulysglyvalalapheglulysleuserglngluserphe
450455460
serargvaltyrvallysasnasptyrlysalaglythrilelysala
465470475480
thrhisvalphevalglyasptyrglncysaspgluilehislystyr
485490495
alaalaleuserlysasngluaspglyleuglyilelysargleuala
500505510
valvalargleuaspvalaspaspleuglyalaalaphemetalagly
515520525
pheserargglnglyasnglyglntyrserthrleuserargserala
530535540
thrpheserargsermetserleuphephelysvaltyrileasngln
545550555560
phealaserasplyslysleuserileiletyralaglyglyaspasp
565570575
valphealaileglysertrpglnaspileilealaphethrvalglu
580585590
leuargglnasnpheilelystrpthrasnglylysleuthrleuser
595600605
alaglyileglyleuphealaasplysthrproileserleumetala
610615620
hisglnthrglygluleugluglualaalalysglyasnglulysasp
625630635640
serileserleupheserserasptyrthrphelyspheaspargphe
645650655
ilethrasnvaltyraspasplysleugluglnileargtyrphephe
660665670
asnhisglnaspgluargglylysasnpheiletyrlysleuileglu
675680685
leuleuargasntyrglusergluglulysmetasnvalalaargleu
690695700
alatyrtyrleuthrargleuglugluleuthrasplysaspgluarg
705710715720
asplysphelysglnphelyslysleuphephelystrptyrthrasn
725730735
asngluserasparglysglualagluleualaleuleuleutyrval
740745750
tyrgluilearglysasp
755
<210>7
<211>121
<212>prt
<213>人工序列(artificialsequence)
<400>7
metalaileleuthraspgluasntyrvalasplysalagluargala
151015
ileserleuleuglulysaspasnlysglyasntyrleuleuthrthr
202530
serglnilearglysleuleuserleucysserserleutyrasparg
354045
serlysgluarglyspheaspgluleuileasnaspvalsertyrleu
505560
argvalglnphevaltyrglnalaglyarggluilealavallysasp
65707580
leuileglulysalaglnileleuglualaleulysgluilelysasp
859095
arggluthrleuglnargphecysargtyrmetglualaleuvalala
100105110
tyrphelysphetyrglyglylysasp
115120
<210>8
<211>220
<212>prt
<213>人工序列(artificialsequence)
<400>8
metthrphealalysilelyspheseralaglnileargleugluthr
151015
glyleuhisileglyglyseraspalaphealaalaileglyalaile
202530
aspserprovalilelysaspproilethrasnileproileilepro
354045
glyserserleulysglylysmetargthrleuleualalysvaltyr
505560
asnglulysvalalaglulysproseraspaspseraspileleuser
65707580
argleupheglyasnserlysasplysargphelysmetglyargleu
859095
ilepheargaspalapheleuserasnalaaspgluleuaspserleu
100105110
glyvalargsertyrthrgluvallysphegluasnthrileasparg
115120125
ilethralaglualaasnproargglnilegluargalaileargasn
130135140
serthrpheaspphegluleuiletyrgluilethraspgluasnglu
145150155160
asnglnvalglugluaspphelysvalileargaspglyleulysleu
165170175
leugluleuasptyrleuglyglyserglyserargglytyrglylys
180185190
valalapheglulysleulysalathrthrvalpheglyasntyrasp
195200205
vallysthrleuasngluleuleuthralagluval
210215220
<210>9
<211>299
<212>prt
<213>人工序列(artificialsequence)
<400>9
metthrtyrlysleutyrilemetthrpheglnasnalahisphegly
151015
serglythrleuaspserserlysleuthrpheseralaaspargile
202530
pheseralaleuvalleugluserleulysmetglylysleuaspala
354045
pheleualaglualaasnglnasplysphethrleuthraspalaphe
505560
propheglnpheglypropheleuprolysproileglytyrprolys
65707580
hisaspglnileaspglnservalaspvallysgluvalargarggln
859095
alalysleuserlyslysleuglnpheleualaleugluasnvalasp
100105110
asptyrleuasnglygluleuphegluasnglugluhisalavalile
115120125
aspthrvalthrlysasnglnprohislysaspglyasnleutyrgln
130135140
valalathrthrargpheserasnaspthrserleutyrvalileala
145150155160
asngluseraspleuleuasngluleumetserserleuglntyrser
165170175
glyleuglyglylysargserserglypheglyargphegluleuasp
180185190
ileglnasnileproleugluleuseraspargleuthrlysasnhis
195200205
serasplysvalmetserleuthrthralaleuprovalaspalaasp
210215220
leugluglualametgluaspglyhistyrleuleuthrlysserser
225230235240
glyphealapheserhisalathrasngluasntyrarglysglnasp
245250255
leutyrlysphealaserglyserthrpheserlysthrpheglugly
260265270
glnilevalaspvalargproleuasppheprohisalavalleuasn
275280285
tyralalysproleuphephelysleugluval
290295
<210>10
<211>357
<212>prt
<213>人工序列(artificialsequence)
<400>10
metlysasnasptyrargthrphelysleuserleuleuthrleuala
151015
proilehisileglyasnglyglulystyrthrserargglupheile
202530
tyrgluasnlyslysphetyrpheproaspmetglylysphetyrasn
354045
lysmetvalglulysargleualaglulyspheglualapheleuile
505560
glnthrargproasnalaargasnasnargleuileserpheleuasn
65707580
aspasnargilealagluargserpheglyglytyrserileserglu
859095
thrglyleugluseraspargasnproasnseralaglyalaileasn
100105110
gluvalasnlyspheileargaspalapheglyasnprotyrilepro
115120125
glyserserleulysglyalaileargthrileleumetasnthrthr
130135140
prolystrpasnasngluasnalavalasnasppheglyargphepro
145150155160
lysgluasnlysasnleuileprotrpglyprolyslysglylysglu
165170175
tyraspaspleupheasnalaileargvalseraspserlysprophe
180185190
aspasnlysargleuileleuvalglnlystrpasptyrseralalys
195200205
thrasnlysalalysproleuproleutyrarggluserileserpro
210215220
leuthrlysilegluphegluilethrthrthrthraspglualagly
225230235240
argleuileglugluleuglylysargalaglnalaphetyrlysasp
245250255
tyrlysalaphepheleuserglupheproaspasplysileglnala
260265270
asnleuglntyrproiletyrleuglyalaglyserglyalatrpthr
275280285
lysthrleuphelysglnalaaspglyileleuglnargargtyrser
290295300
argmetlysthrlysmetvallyslysglyvalleulysleuthrlys
305310315320
alaproleulysilevallysileproserglyasnhisserleuile
325330335
lysasnhisgluserphetyrglumetglylysalaasnphemetile
340345350
lysgluileasplys
355
<210>11
<211>39
<212>rna
<213>人工序列(artificialsequence)
<400>11
acggaaacggacacgcgaacuuguggccguuuacgucgc39
<210>12
<211>40
<212>rna
<213>人工序列(artificialsequence)
<400>12
acggaaacgcagaucagggcaaacagaacuuugacuccca40
<210>13
<211>40
<212>rna
<213>人工序列(artificialsequence)
<400>13
acggaaacguucucggugggcuuggccucggccacagcga40
<210>14
<211>41
<212>rna
<213>人工序列(artificialsequence)
<400>14
acggaaacacgugauauaugugguaccauguucaccgucua41
<210>15
<211>40
<212>rna
<213>人工序列(artificialsequence)
<400>15
acggaaacuaugucugaucccaauauuuaaaauaacgguc40
<210>16
<211>40
<212>rna
<213>人工序列(artificialsequence)
<400>16
acggaaacguaaugaaggaucuuauuucuucggagacaau40
<210>17
<211>40
<212>rna
<213>人工序列(artificialsequence)
<400>17
acggaaacggucgguugcucacaaguccugccugccugcc40