利用CRISPR/Cas9技術(shù)制備Mindin基因敲除小鼠

紀(jì)慈數(shù),趙志剛,賀 穎*

(1.廈門大學(xué)實驗動物中心,福建廈門361102;2.廣州軍區(qū)武漢總醫(yī)院,湖北武漢430000)

·研究簡報·

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.廈門大學(xué)實驗動物中心,福建廈門361102;2.廣州軍區(qū)武漢總醫(yī)院,湖北武漢430000)

針對Mindin基因編碼序列設(shè)計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,通過藥物篩選獲得細(xì)胞庫.經(jīng)單克隆測序檢測細(xì)胞庫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號小鼠缺失10個堿基造成移碼突變,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是近年來從古細(xì)菌免疫系統(tǒng)中發(fā)展起來的一種新型高效的基因組核酸內(nèi)切酶編輯技術(shù),因其操作簡單、成本低、效率高、限制條件少等優(yōu)點而備受關(guān)注,是目前生命醫(yī)學(xué)領(lǐng)域的研究熱點[1-3].

Mindin是一種高度保守的分泌性細(xì)胞外基質(zhì)蛋白,通過介導(dǎo)細(xì)胞與基質(zhì)之間的相互作用對細(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對免疫調(diào)節(jié)的重要作用[7]上,對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)由廈門大學(xué)李勤喜教授實驗室饋贈,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)黃行許教授實驗室饋贈;p BKS質(zhì)粒由廈門大學(xué)林圣彩教授實驗室饋贈.

感受態(tài)細(xì)胞:大腸桿菌(Escherichia coli)DH5α感受態(tài)細(xì)胞購自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)試劑盒使用說明書進(jìn)行.

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

1.3實驗方法

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

2)質(zhì)粒的電轉(zhuǎn)及敲除效率檢測:將測序正確的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;提取細(xì)胞庫基因組,用相應(yīng)單克隆引物PCR構(gòu)建單克隆進(jìn)行測序,獲得細(xì)胞庫敲除效率.

3)體外轉(zhuǎn)錄及sgRNA和Cas9 m RNA的顯微注射:利用合成的帶T7啟動子序列的引物通過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: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)行測序,篩選突變的小鼠.

5)F0代小鼠的傳代及F1代小鼠的檢測:將獲得的F0代突變小鼠與野生型小鼠配對,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Mindin蛋白的表達(dá).

2 結(jié)果和分析

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

利用http:∥crispr.mit.edu/網(wǎng)站針對Mindin基因編碼序列設(shè)計挑選了3個sg RNA作用靶點(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,并通過測序驗證.

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

2.2sgRNA敲除效率的檢測

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,以空白載體作為對照,實驗組和對照組轉(zhuǎn)染24 h后加入殺稻瘟菌素處理,待對照組細(xì)胞全部死亡后,將實驗組剩下的細(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)行測序分析,統(tǒng)計各sgRNA的敲除效率.結(jié)果表明,所有單克隆中Mindin的靶位點序列相較于野生型Mindin序列都發(fā)生了改變,并且突變位點都位于sgRNA后NGG序列附近(如圖1所示),說明這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,通過胞質(zhì)注射200個小鼠受精卵,最終成活160個二細(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)行測序分析.結(jié)果如圖2所示:12號小鼠的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基因無法正常表達(dá),實現(xiàn)了Mindin基因的敲除.

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

表2　sg RNA3單克隆測序引物Tab.2 sg RNA3 single clone sequencing primers

為了驗證所產(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,測序結(jié)果如圖2中23號小鼠的序列,說明Mindin基因的缺失能穩(wěn)定遺傳;同時提取了F1代雜合小鼠的腸上皮組織,對Mindin蛋白的表達(dá)進(jìn)行蛋白質(zhì)免疫印跡檢測,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)因為專利限制及鋅指蛋白自身識別序列限制,應(yīng)用范圍不廣泛[9].后期發(fā)展起來的類轉(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)中的識別序列限制,但其制備過程耗時而繁冗,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),其對靶位點的識別依賴于sg RNA 5′端約20 bp序列與靶位點DNA序列間的互補(bǔ)配對,針對不同靶位點序列的sgRNA僅需替換識別位點處20 bp序列即可,且其切割效率明顯高于ZFN和TALEN,應(yīng)用簡單易行,具有顯著的優(yōu)勢[11].

真核生物基因組DNA具有復(fù)雜高級結(jié)構(gòu),尋找高效的sg RNA位點是CRISPR/Cas9有效工作的重要保證.為了獲得高效的sg RNA,在本研究中針對腸癌抑制基因Mindin的編碼序列,利用已有網(wǎng)站設(shè)計并構(gòu)建了針對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sg RNA的效率.結(jié)果表明,細(xì)胞庫的所有單克隆都發(fā)生了突變,并且突變位點都位于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)生過程中的作用提供了可用的動物模型.

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%,這與已有報道的超過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程中RNA的降解,即由于所用顯微注射儀在操作過程中會發(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)的效率也是一個可能的影響因素,poly(A)會影響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

[1] BARRANGOU R,FREMAUX C,DEVEAU H,et al. CRISPR provides acquired resistance against viruses in prokaryotes[J].Science,2007,315(5819):1709-1712.

[2] WANG H,FU Y,REYON D,et al.Efficient genome editing in zebrafish using a CRISPR-Cas system[J].Nat Biotechnol,2013,31(3):227-229.

[3] MALI P,ESVELT K M,CHURCH G M.Cas9 as a versatile tool for engineering biology[J].Nat Methods,2013, 10(10):957-963.

[4] LI Z,GARANTZIOTIS S,JIA W,et al.The extracellular matrix protein Mindin regulates trafficking of murine eosinophils into the airspace[J].J Leukoc Biol,2009,85(1): 124-131.

[5] JIA,W.The extracellular matrix protein Mindin serves as an integrin ligand and is critical for inflammatory cell recruitment[J].Blood,2005,106(12):3854-3859.

[6] LUO,J H,REN B,KERYANOV S,et al.Transcriptomic and genomic analysis of human hepatocellular carcinomas and hepatoblastomas[J].Hepatology,2006,44(4): 1012-1024.

[7] MANDA R,KOHNO T,MATSUNO Y,et al. Identification of genes SPON2 and C20orf2 differentially expressed between cancerous and noncancerous lung cells by mRNA differential display[J].Genomics,1999,61(1): 5-14.

[8] BA G,LIAN Y M,REN J L.Mindin is upregulated during colitis and may activate NF-kappaB in a TLR-9 mediated manner[J].World J Gas,2010,16(9):1070-1075.

[9] GEURTS A M,COST G J,FREYVERT Y,et al. Knockout rats via embryo microinjection of zinc-finger nuclease[J].Science,2009,325(5939):433.

[10] CERMAK T,DOYLE E L,CHRISTIAN M,et al.Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting[J]. Nucleic Acids Res,2011,39(12):82-84.

[11] CONG L,RAN F A,COX D,et al.Multiplex genome engineering using CRISPR/Cas systems[J].Science,2013, 339(6121):819-823.

[12] WANG H,YANG H,SHIVALILA C S,et al.One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering[J].Cell, 2013,153(4):910-918.

[13] SHEN B,ZHANG J,WU H,et al.Generation of genemodified mice via Cas9/RNA-mediated gene targeting [J].Cell Res,2013,23(5):720-723.

[14] YANG H,WANG H,JAENISCH R,et al.Generating genetically modified mice using CRISPR/Cas-mediated genome engineering[J].Nat Protoc,2014,9(8): 1956-1968.

[15] QIN W,KUTNY P M,MASER R S,et al.Generating mouse models using CRISPR-Cas9-mediated genome editing[J].Curr Protoc Mouse Biol,2016,6(1):39-66.

[16] CHU V T,WEBER T,GRAF R,et al.Efficient generation of Rosa26 knock-in mice using CRISPR/Cas9 in C57BL/6 zygotes[J].BMC Biotechnol,2016,16:4.

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)基金重點項目(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].廈門大學(xué)學(xué)報(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)