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CRISPR-dCas9-表觀遺傳調(diào)控系統(tǒng)
表觀遺傳調(diào)節(jié)是往往是通過影響一段染色質(zhì)的結(jié)構(gòu)起作用,比如將染色質(zhì)壓縮成緊密狀態(tài)(異染色質(zhì)),使基因難以轉(zhuǎn)錄,或者使染色質(zhì)打開以促進(jìn)轉(zhuǎn)錄。表觀遺傳調(diào)節(jié)包括DNA甲基化、組蛋白甲基化、組蛋白乙?;?、組蛋白磷酸化等等。
最近,人們將dCas9與表觀遺傳修飾酶融合在一起,形成dCas9表觀遺傳編輯系統(tǒng),該系統(tǒng)成員如表1所示。
Table 1 - The dCas9 Epigenetics Toolbox
Construct | Function | Gene Expression | |
Histone Modifications | dCas9-p300 | Acetylation | Activation |
dCas9-LSD1 | Demethylation | Repression | |
DNA Methylation | dCas9-TET1-CD | Demethylation | Activation |
dCas9-DNMT3A | Methylation | Repression |
dCas9 組蛋白修飾
組蛋白乙?;亲顝?qiáng)大的基因表達(dá)增強(qiáng)子系統(tǒng)之一。dCas9-p300的開發(fā),使得人們能夠直接改變靶標(biāo)基因附近的染色質(zhì)狀態(tài)。在這個(gè)系統(tǒng)中,人類E1A相關(guān)蛋白p300的催化核心與dCas9融合,這是組蛋白乙?;年P(guān)鍵成分(圖4a)。當(dāng)靶向編碼區(qū)或啟動(dòng)子區(qū)時(shí),這個(gè)系統(tǒng)可成功誘導(dǎo)基因高表達(dá)。特別是,當(dāng)靶向啟動(dòng)子或增強(qiáng)子時(shí),基因表達(dá)可增強(qiáng)50至10,000倍(圖4b)。通過轉(zhuǎn)錄組分析評(píng)估得到,基因激活是高度特異性的。這表明該系統(tǒng)可以精確調(diào)控某段染色質(zhì)的變化。
dCas9-p300是一種簡單而獨(dú)特的工具,可用于研究調(diào)控元件和靶基因表達(dá)之間的復(fù)雜關(guān)系。
Figure 4 :The dCas9-p300 system for epigenetic activation. a) dCas9 fused to the core catalytic domain of p300 can acetylate target sites in the genome. Acetylation of the target gene synergizes with the action of transcription factors and RNA Polymerase II, resulting in transcriptional upregulation. Adapted from Figure 1b of Zentner et al. (2015). b) Comparison of relative expression levels of IL1RN when dCas9 by itself vs. dCas9 fused to the p300 catalytic core is targeted to the IL1RN promoter. Relative expression levels determined by qRT-PCR. Adapted from Figure 1c of Hilton et al. (2015).
dCas9-LSD1是dCas9-p300激活系統(tǒng)的互補(bǔ)基因抑制系統(tǒng)。在該系統(tǒng)中,dCas9與賴氨酸特異性組蛋白去甲基化酶1(LSD1)融合(圖5a)。 Kearns等人在穩(wěn)定表達(dá)dCas9-LSD1的小鼠胚胎干細(xì)胞系中,設(shè)計(jì)Oct4基因的遠(yuǎn)端增強(qiáng)子區(qū)域的gRNA,Oct4基因被抑制(12)。然而,當(dāng)dCas9-LSD1針對(duì)Oct4啟動(dòng)子時(shí),沒有觀察到效果。這使得dCas9-LSD1成為研究增強(qiáng)子調(diào)控活性的工具。這與其他系統(tǒng)如dCas9-KRAB(它們是更全面的基因表達(dá)控制器)互補(bǔ)。當(dāng)靶向上游增強(qiáng)子時(shí),dCas9-LSD1也能夠使下游基因表達(dá)沉默(圖5b)(12),而不破壞基因組結(jié)構(gòu)。
由于在增強(qiáng)子區(qū)域內(nèi)發(fā)現(xiàn)了與人類疾病相關(guān)的許多基因組區(qū)域,因此dCas9-LSD1以高度特異的方式功能性地靶向增強(qiáng)子元件的能力使其在定義增強(qiáng)子 - 基因關(guān)系方面具有無價(jià)之寶。當(dāng)與增強(qiáng)子靶向的sgRNA池組合使用時(shí),該系統(tǒng)可以提供高通量的方式來鑒定與基因相關(guān)的所有增強(qiáng)子。
Figure 5 : The dCas9-LSD1 system for epigenetic repression. a) dCas9 fused to LSD1 can demethylate target sites in the genome. Demethylation of the target gene results in transcriptional downregulation. Adapted from Figure 1b of Zentner et al. (2015). b) The relative expression level of the TBX3 gene was assessed via quantitative PCR analysis when the sgRNA of the dCas9-LSD1 system was fused with a distal enhancer vs. a promoter. sgRNAs specific to an unrelated genomic region were used as controls. Adapted from Figure 1e of Kearns et al. (2015).
dCas9-DNA甲基化修飾
哺乳動(dòng)物細(xì)胞中的靶向DNA甲基化主要發(fā)生在CpG二核苷酸序列內(nèi)胞嘧啶的第五個(gè)碳上。細(xì)胞發(fā)育,分化和腫瘤都可以通過DNA甲基化來調(diào)節(jié),高甲基化與癌癥和神經(jīng)系統(tǒng)疾病密切相關(guān)。使DNA甲基化易于調(diào)節(jié)的技術(shù)可以直接探索甲基化狀態(tài)和基因表達(dá)之間的功能關(guān)系,甚至可以開發(fā)治療疾病的療法。
dCas9-Tet1-CD是以這種方式編輯表觀基因組的新技術(shù)之一。該系統(tǒng)由與Tet1(十 - 十一易位甲基胞嘧啶雙加氧酶1)的催化結(jié)構(gòu)域(CD)融合dCas9組成,該酶觸發(fā)DNA去甲基化(圖6a)。伴隨的sgRNA也可以進(jìn)行修飾,使之包含一個(gè)MS2二聚體,每個(gè)二聚體再與兩個(gè)Tet1-CD模塊融合。有研究表明,該系統(tǒng)轉(zhuǎn)染后4天后觀察到基因的激活(圖6b)。在不同的人和小鼠細(xì)胞系中,結(jié)合精確的sgRNA設(shè)計(jì)和控制該系統(tǒng)不同組分的比例,dCas9-Tet1-CD系統(tǒng)及其伴隨的MS2-Tet1-CD系統(tǒng)能夠在特定位置有效去甲基化,且具有很小的脫靶效應(yīng)。
dCas9-Tet1-CD系統(tǒng)地靶向特異性將幫助科學(xué)家輕易地探索特定基因啟動(dòng)子靶點(diǎn)甲基化對(duì)下游基因的影響。最近,dCas9-Tet1-CD系統(tǒng)結(jié)合常規(guī)sgRNA也被用于靶向BRCA1啟動(dòng)子的表觀遺傳修飾,BRCA1是一種腫瘤抑制基因,其過度甲基化沉默與非家族性乳腺癌和卵巢癌相關(guān)。這樣的系統(tǒng)也可以用來恢復(fù)其他腫瘤抑制基因的功能活性。
Figure 6 : The dCas9-TET1-CD system for targeted DNA demethylation. a) dCas9 is fused to the catalytic domain of TET1 (TET1-CD). The accompanying sgRNA is additionally modified to contain two RNA aptamers that recruit MS2 coat proteins, each fused to a TET1-CD. Adapted from Figure 1a of Xu et al. (2016). b) qRT-PCR was used to assess mRNA levels of RANKL gene expression using two sgRNAs designed to target the RANKL promoter region (-800 bp upstream of transcription start site). Adapted from Figure 2c of Xu et al. (2016).
與基于組蛋白的細(xì)胞表型控制不同,DNA甲基化對(duì)基因表達(dá)的作用更穩(wěn)定和長期?;?/span>dCas9的甲基化系統(tǒng)不僅具有跨物種能力,而且對(duì)CpG甲基化不敏感。
dCas9-DNMT3A是先前討論的dCas9-Tet1-CD系統(tǒng)的甲基化對(duì)應(yīng)物。由Vojta等開發(fā),該系統(tǒng)是dCas9(通過靈活的Gly4Ser接頭)與DNMT3A的催化結(jié)構(gòu)域(一種能夠在體內(nèi)甲基化CpG位點(diǎn)的活性DNA甲基轉(zhuǎn)移酶)融合。這個(gè)dCas9-DNMT3A系統(tǒng)在HEK293細(xì)胞中成功地誘導(dǎo)BACH2啟動(dòng)子上游和下游的位點(diǎn)特異性CpG甲基化,最高濃度的甲基化活性(60%)位于PAM序列下游27bp處。當(dāng)針對(duì)IL6ST啟動(dòng)子時(shí),轉(zhuǎn)染后10天后兩種基因的表達(dá)水平均顯著降低。此外,通過dCas9-DNMT3A與sgRNA池結(jié)合同時(shí)靶向多個(gè)基因特異性位點(diǎn)導(dǎo)致甲基化的協(xié)同作用,將甲基化提高2倍,擴(kuò)大該系統(tǒng)的基因沉默能力。
自其發(fā)展以來,dCas9-DMNT3A也被用于多種癌癥相關(guān)的腫瘤抑制基因的啟動(dòng)子的甲基化如(CDKN1A和2A啟動(dòng)子甲基化)。該項(xiàng)研究表明,CDKN1A啟動(dòng)子的甲基化導(dǎo)致CDKN1A的表達(dá)下降和細(xì)胞增殖能力的增加,進(jìn)一步證明了該系統(tǒng)用于功能基因組學(xué)研究。
Figure 7 :The dCas9-DNMT3A system for targeted DNA methylation. a) dCas9 is fused to the catalytic domain of DNMT3A. In vivo, DNMT3A recruits a partner for dimerization along with DNMT3L proteins (shown in dashed red box and ovals, respectively). Adapted from Figure 1a of Vojta et al. (2016). b) RT-qPCR analysis of IL6ST gene expression via dCas9-fused to active DMNT3A. Expression levels induced by dCas9 fused to inactive DMNT3A along with dCas9 accompanied by non-targeting sgRNAs were measured as negative controls. Fold change is relative to mock-transfected cells. Adapted from Figure 4a of Vojta et al. (2016).