紀(jì)慈數(shù),趙志剛,賀 穎*

(1.廈門(mén)大學(xué)實(shí)驗(yàn)動(dòng)物中心,福建廈門(mén)361102;2.廣州軍區(qū)武漢總醫(yī)院,湖北武漢430000)

·研究簡(jiǎn)報(bào)·

利用CRISPR/Cas9技術(shù)制備Mindin基因敲除小鼠

紀(jì)慈數(shù)1,趙志剛2,賀 穎1*

(1.廈門(mén)大學(xué)實(shí)驗(yàn)動(dòng)物中心,福建廈門(mén)361102;2.廣州軍區(qū)武漢總醫(yī)院,湖北武漢430000)

針對(duì)Mindin基因編碼序列設(shè)計(jì)3條單鏈向?qū)NA(sgRNA),制備相應(yīng)質(zhì)粒,將表達(dá)sgRNA和Cas9蛋白的質(zhì)粒共同電轉(zhuǎn)入小鼠成纖維細(xì)胞L929中,通過(guò)藥物篩選獲得細(xì)胞庫(kù).經(jīng)單克隆測(cè)序檢測(cè)細(xì)胞庫(kù)中基因組的突變效率,選擇效率最佳的sgRNA.體外轉(zhuǎn)錄獲取sg RNA和Cas9 m RNA,進(jìn)行小鼠受精卵的顯微注射后提取小鼠基因組DNA測(cè)序鑒定.產(chǎn)生的40只小鼠中17只發(fā)生不同程度的堿基插入或缺失,其中23號(hào)小鼠缺失10個(gè)堿基造成移碼突變,Mindin基因表達(dá)被破壞,成功構(gòu)建出Mindin基因敲除小鼠,為深入研究Mindin基因的生物學(xué)功能奠定了基礎(chǔ).

CRISPR/Cas9;基因敲除;Mindin

CRISPR/Cas9是近年來(lái)從古細(xì)菌免疫系統(tǒng)中發(fā)展起來(lái)的一種新型高效的基因組核酸內(nèi)切酶編輯技術(shù),因其操作簡(jiǎn)單、成本低、效率高、限制條件少等優(yōu)點(diǎn)而備受關(guān)注,是目前生命醫(yī)學(xué)領(lǐng)域的研究熱點(diǎn)[1-3].

Mindin是一種高度保守的分泌性細(xì)胞外基質(zhì)蛋白,通過(guò)介導(dǎo)細(xì)胞與基質(zhì)之間的相互作用對(duì)細(xì)胞的黏附、分化、遷移和增殖等產(chǎn)生重要影響[4].之前的研究大都集中在Mindin對(duì)免疫調(diào)節(jié)的重要作用[7]上,對(duì)Mindin在腫瘤生成和發(fā)展中的作用研究較少.已有研究表明Mindin在多種人類腫瘤中異常表達(dá)[6-7].增強(qiáng)Mindin的表達(dá)會(huì)引起腸癌細(xì)胞遷移、侵襲和增殖能力的提升[18],提示Mindin在腸癌病理過(guò)程中發(fā)揮重要的抗癌作用.本研究擬利用CRISPR/Cas9技術(shù)顯微注射C56BL/6J小鼠受精卵制備Mindin基因敲除小鼠,為深入研究Mindin基因的生物學(xué)功能奠定基礎(chǔ),為腸癌的研究、預(yù)防與治療提供有力的模型支持.

1 材料和方法

1.1材料

細(xì)胞株:L929細(xì)胞株(小鼠成纖維細(xì)胞株)由廈門(mén)大學(xué)李勤喜教授實(shí)驗(yàn)室饋贈(zèng),用含10%(體積分?jǐn)?shù))胎牛血清(FBS,Gibco公司)的DMEM培養(yǎng)基(Gibco公司)于5%(體積分?jǐn)?shù))CO2恒溫濕熱培養(yǎng)箱中培養(yǎng).

質(zhì)粒:p UC57-sgRNA質(zhì)粒載體(Addgene 51132)以及pST1374-Cas9-NLS表達(dá)質(zhì)粒由上?？萍即髮W(xué)黃行許教授實(shí)驗(yàn)室饋贈(zèng);p BKS質(zhì)粒由廈門(mén)大學(xué)林圣彩教授實(shí)驗(yàn)室饋贈(zèng).

感受態(tài)細(xì)胞:大腸桿菌(Escherichia coli)DH5α感受態(tài)細(xì)胞購(gòu)自Genentech公司.

1.2主要試劑與儀器

PCR儀(BioRad公司),凝膠成像系統(tǒng)(Genentech公司),電轉(zhuǎn)儀(BioRad公司),顯微注射儀(NIKON&NARISHIGE GROUP).

PCR擴(kuò)增及質(zhì)粒構(gòu)建:BsaⅠ限制性內(nèi)切酶(NEB,R0535S),SolutionⅠ(Ta KaRa,6022Q),Primerstar(Premix)(Ta KaRa,DR040A).

體外轉(zhuǎn)錄:MEGAshortscriptTMT7 Kit(Life Technology,AM1354),m MESSAGE m MACHINE? T7 ULTRA Kit(Life Techonology,AM1345), MEGAclearTMKit(Ambion,AM1908),A-PlusTMPoly(A)Polymerase Tailing Kit(CellScript,C-PAP5104H).體外轉(zhuǎn)錄依據(jù)試劑盒使用說(shuō)明書(shū)進(jìn)行.

單鏈向?qū)NA(sgRNA)寡聚核苷酸鏈及單克隆引物由廈門(mén)安特奧生物有限公司合成;質(zhì)粒測(cè)序及單克隆菌液測(cè)序由廈門(mén)閩博生物有限公司完成.

1.3實(shí)驗(yàn)方法

1)質(zhì)粒構(gòu)建:針對(duì)Mindin基因中前兩段蛋白編碼序列,利用麻省理工大學(xué)張鋒教授實(shí)驗(yàn)室提供的在線網(wǎng)站(http:∥crispr.mit.edu/)分析可能的sg RNA位點(diǎn)(PAM序列為NGG),從中挑選3個(gè)高評(píng)分的sgRNA位點(diǎn).構(gòu)建相應(yīng)sgRNA表達(dá)質(zhì)粒,通過(guò)測(cè)序檢驗(yàn)所構(gòu)建質(zhì)粒的正確性.

2)質(zhì)粒的電轉(zhuǎn)及敲除效率檢測(cè):將測(cè)序正確的sgRNA質(zhì)粒連同Cas9質(zhì)粒共同電轉(zhuǎn)入小鼠成纖維細(xì)胞L929中,加入殺稻瘟菌素(blasticidin)篩選獲得細(xì)胞庫(kù);提取細(xì)胞庫(kù)基因組,用相應(yīng)單克隆引物PCR構(gòu)建單克隆進(jìn)行測(cè)序,獲得細(xì)胞庫(kù)敲除效率.

3)體外轉(zhuǎn)錄及sgRNA和Cas9 m RNA的顯微注射:利用合成的帶T7啟動(dòng)子序列的引物通過(guò)PCR從相應(yīng)質(zhì)粒上擴(kuò)增sgRNA和Cas9序列,回收PCR產(chǎn)物作為體外轉(zhuǎn)錄的模板;利用T7體外轉(zhuǎn)錄試劑盒體外轉(zhuǎn)錄sgRNA和Cas9 mRNA,Cas9 mRNA轉(zhuǎn)錄完后在3′端加入poly(A)尾以增加m RNA的穩(wěn)定性;最后利用試劑盒回收sgRNA和Cas9 m RNA,并稀釋至終質(zhì)量濃度分別為50和100 ng/μL進(jìn)行受精卵顯微注射(胞質(zhì)注射).

4)小鼠基因組的提取及突變基因的檢測(cè):受精卵顯微注射后,將其移回至假孕母鼠的輸卵管內(nèi).待所生小鼠(F0代)斷奶后切取尾部組織(約3 mm),酚-三氯甲烷抽提法提取基因組DNA,PCR獲得相應(yīng)靶基因片段并進(jìn)行測(cè)序,篩選突變的小鼠.

5)F0代小鼠的傳代及F1代小鼠的檢測(cè):將獲得的F0代突變小鼠與野生型小鼠配對(duì),獲得F1代小鼠,提取腸上皮細(xì)胞進(jìn)行蛋白質(zhì)免疫印跡,檢測(cè)Mindin蛋白的表達(dá).

2 結(jié)果和分析

2.1sgRNA的設(shè)計(jì)和相應(yīng)質(zhì)粒的構(gòu)建

利用http:∥crispr.mit.edu/網(wǎng)站針對(duì)Mindin基因編碼序列設(shè)計(jì)挑選了3個(gè)sg RNA作用靶點(diǎn)(表1).加入通用連接序列合成相應(yīng)寡聚核苷酸鏈,退火后連接入BsaⅠ切割的p UC57-sgRNA載體,完成sg RNA質(zhì)粒載體的構(gòu)建,并通過(guò)測(cè)序驗(yàn)證.

表1　針對(duì)小鼠Mindin基因編碼序列的sg RNA位點(diǎn)信息Tab.1 Information of sg RNAs targeting coding sequence in mammalian Mindin gene

2.2sgRNA敲除效率的檢測(cè)

為了確定所選sgRNA介導(dǎo)Mindin靶序列的編輯效率,將上述3條sg RNAs的表達(dá)質(zhì)粒分別與表達(dá)Cas9蛋白的質(zhì)粒共同電轉(zhuǎn)入小鼠成纖維細(xì)胞L929中,以空白載體作為對(duì)照,實(shí)驗(yàn)組和對(duì)照組轉(zhuǎn)染24 h后加入殺稻瘟菌素處理,待對(duì)照組細(xì)胞全部死亡后,將實(shí)驗(yàn)組剩下的細(xì)胞繼續(xù)培養(yǎng)至24孔板中.利用酚-三氯甲烷抽提法提取細(xì)胞基因組進(jìn)行PCR,所得DNA片段回收后連入pBKS質(zhì)粒載體中,挑選單克隆菌落進(jìn)行測(cè)序分析,統(tǒng)計(jì)各sgRNA的敲除效率.結(jié)果表明,所有單克隆中Mindin的靶位點(diǎn)序列相較于野生型Mindin序列都發(fā)生了改變,并且突變位點(diǎn)都位于sgRNA后NGG序列附近(如圖1所示),說(shuō)明這3條sgRNAs都可以介導(dǎo)Cas9蛋白高效切割Mindin基因靶序列.

2.3利用CRISPR/Cas9制備Mindin基因敲除小鼠

選擇sg RNA3進(jìn)行后續(xù)Mindin基因敲除小鼠的制備,利用帶T7啟動(dòng)子的特異性引物PCR獲得相應(yīng)sg RNA和Cas9編碼序列.回收的PCR產(chǎn)物利用T7 RNA聚合酶進(jìn)行體外轉(zhuǎn)錄,回收RNA后按sg RNA 50 ng/μL,Cas9 mRNA 100 ng/μL的終質(zhì)量濃度混合,通過(guò)胞質(zhì)注射200個(gè)小鼠受精卵,最終成活160個(gè)二細(xì)胞胚胎,移植6只假孕小鼠,有5只小鼠生育,最后共獲得40只F0代小鼠.

小鼠出生21 d后,切取尾部組織提取基因組DNA,用單克隆引物(表2)進(jìn)行PCR.將擴(kuò)增產(chǎn)物構(gòu)建至p BKS載體中,次日挑選單克隆進(jìn)行測(cè)序分析.結(jié)果如圖2所示:12號(hào)小鼠的Mindin編碼序列缺少了3 bp,但不影響其正常編碼,為缺失突變體;23號(hào)小鼠的Mindin基因缺少了10 bp,形成移碼突變,可能導(dǎo)致Mindin基因無(wú)法正常表達(dá),實(shí)現(xiàn)了Mindin基因的敲除.

圖1　sgRNA1～3細(xì)胞庫(kù)的單克隆測(cè)序檢測(cè)結(jié)果Fig.1 Single clone sequencing results of sgRNA1-3 cell pools

表2　sg RNA3單克隆測(cè)序引物Tab.2 sg RNA3 single clone sequencing primers

為了驗(yàn)證所產(chǎn)生的基因組突變能否穩(wěn)定傳代并盡量消除可能的脫靶效應(yīng),將23號(hào)小鼠與野生型小鼠進(jìn)行交配,產(chǎn)生的F1代雜合小鼠進(jìn)行基因型鑒定,測(cè)序結(jié)果如圖2中23號(hào)小鼠的序列,說(shuō)明Mindin基因的缺失能穩(wěn)定遺傳;同時(shí)提取了F1代雜合小鼠的腸上皮組織,對(duì)Mindin蛋白的表達(dá)進(jìn)行蛋白質(zhì)免疫印跡檢測(cè),發(fā)現(xiàn)F1代雜合小鼠腸組織中Mindin的表達(dá)有明顯降低(圖3),表明通過(guò)CRISPR/Cas9系統(tǒng)成功制備了Mindin基因敲除小鼠.

3 討 論

基因組編輯是研究基因功能以及進(jìn)行基因治療的重要手段,人工核酸酶技術(shù)的出現(xiàn)為基因組編輯提供了強(qiáng)有力的工具.但早期的鋅指核酸酶技術(shù)(zinc finger nuclease,ZFN)因?yàn)閷＠拗萍颁\指蛋白自身識(shí)別序列限制,應(yīng)用范圍不廣泛[9].后期發(fā)展起來(lái)的類轉(zhuǎn)錄激活因子效應(yīng)核酸酶(transcription activator-like effector nuclease,TALEN)技術(shù)克服了ZFN編輯技術(shù)中的識(shí)別序列限制,但其制備過(guò)程耗時(shí)而繁冗,限制了其廣泛應(yīng)用[10].CRISPR/Cas9作為新一代基因組編輯技術(shù),其對(duì)靶位點(diǎn)的識(shí)別依賴于sg RNA 5′端約20 bp序列與靶位點(diǎn)DNA序列間的互補(bǔ)配對(duì),針對(duì)不同靶位點(diǎn)序列的sgRNA僅需替換識(shí)別位點(diǎn)處20 bp序列即可,且其切割效率明顯高于ZFN和TALEN,應(yīng)用簡(jiǎn)單易行,具有顯著的優(yōu)勢(shì)[11].

真核生物基因組DNA具有復(fù)雜高級(jí)結(jié)構(gòu),尋找高效的sg RNA位點(diǎn)是CRISPR/Cas9有效工作的重要保證.為了獲得高效的sg RNA,在本研究中針對(duì)腸癌抑制基因Mindin的編碼序列,利用已有網(wǎng)站設(shè)計(jì)并構(gòu)建了針對(duì)Mindin編碼序列的3條sgRNAs,并將其導(dǎo)入小鼠成纖維細(xì)胞L929中,檢測(cè)sg RNA的效率.結(jié)果表明,細(xì)胞庫(kù)的所有單克隆都發(fā)生了突變,并且突變位點(diǎn)都位于NGG序列附近,證明了這3條sgRNAs都可以介導(dǎo)Cas9蛋白高效切割Mindin基因靶序列.利用體外轉(zhuǎn)錄RNA和受精卵顯微注射技術(shù)成功制備了可以穩(wěn)定遺傳的Mindin基因突變小鼠,其F1代雜合小鼠腸上皮細(xì)胞中Mindin蛋白的表達(dá)量顯著下調(diào),從而為深入研究Mindin在結(jié)直腸癌發(fā)生過(guò)程中的作用提供了可用的動(dòng)物模型.

值得注意的是,40只F0代中總共獲得了17只突變小鼠,突變率為42.5%,這與已有報(bào)道的超過(guò)70%的效率[12-13]仍有差距.之前的研究指出,體外轉(zhuǎn)錄RNA的質(zhì)量是影響CRISPR制備基因敲除動(dòng)物的關(guān)鍵因素[14-15].在本研究系統(tǒng)中,突變率低的可能原因在于顯微注射過(guò)程中RNA的降解,即由于所用顯微注射儀在操作過(guò)程中會(huì)發(fā)生受精卵培養(yǎng)基回流至注射針管導(dǎo)致RNA的降解,從而使效率降低;此外,Cas9 m RNA體外加poly(A)的效率也是一個(gè)可能的影響因素,poly(A)會(huì)影響CRISPR蛋白的翻譯效率[16],如果體外加入poly(A)的效率偏低,也會(huì)導(dǎo)致效率偏低.因此,今后將從RNA質(zhì)量方面進(jìn)一步優(yōu)化該系統(tǒng).

圖2　F0代突變小鼠中突變?nèi)旧w的單克隆測(cè)序結(jié)果Fig.2 Single clone sequencing results of mutant chromosome in F0 mutant mouse

圖3　F1代雜合小鼠腸上皮中Mindin表達(dá)量顯著下調(diào)Fig.3 Down regulation of Mindin expression in colon epithelial cells in F1 heterozygote mice

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Application of CRISPR/Cas9 Technology for Establishing Mindin Gene Knockout Mice

JI Cishu1,ZHAO Zhigang2,HE Ying1*

(1.Laboratory Animal Center,Xiamen University,Xiamen 361102,China; 2.Wuhan General Hospital,Guangzhou Military,Wuhan 430000,China)

To establish Mindin knockout mice using CRISPR/Cas9 gene targeting technology,three sg RNA targets of Mindin were designed according to Mindin genome sequence.Vectors expressing sgRNA and Cas9 were constructed separately,and co-transfected into L929 cells by with electroporation method.Transfected cells were selected with blasticitin and the mutation rate was determined using DNA sequencing with proper primers.The sgRNA which had resulted in the highest mutation rate in L929 cells was transcribed in vitro,along with Cas9.Then sgRNA and Cas9 m RNAs were microinjected into mice zygotes and 40 mice were born after the microinjection.Genotypes of the mice were determined by PCR with genomic DNA from the new born mice tail and 17 neonatal mice were found carrying mutations in Mindin gene by subsequent sequencing.Gene sequencing results showed that all mutant mice had different levels of nucleotide insertion or deletion,with the maximum number of 10 base deletion found in mouse No.23,leading to the reading frame shift and Mindin gene silence.Taking together,we have successfully established a mouse model with Mindin gene knockout,which lays a solid foundation for related future research.

CRISPR/Cas9;gene knockout;Mindin

Q 789

A

0438-0479(2016)06-0922-05

10.6043/j.issn.0438-0479.201604028

2016-04-15 錄用日期:2016-06-22

福建省自然科學(xué)基金重點(diǎn)項(xiàng)目(2013Y01010490)

hey@xmu.edu.cn

紀(jì)慈數(shù),趙志剛,賀穎.利用CRISPR/Cas9技術(shù)制備Mindin基因敲除小鼠[J].廈門(mén)大學(xué)學(xué)報(bào)(自然科學(xué)版),2016,55 (6):922-926.

JI C S,ZHAO Z G,HE Y.Application of CRISPR/Cas9 technology for establishing Mindin gene knockout mice[J]. Journal of Xiamen University(Natural Science),2016,55(6):922-926.(in Chinese)