| CRISPR/Cas9介導的非同源DNA敲入在多拷貝基因敲除中的應用 | BMC Biology |
論文標題：Homology-independent multiallelic disruption via CRISPR/Cas9-based knock-in yields distinct functional outcomes in human cells
期刊：BMC Biology
作者：Chenzi Zhang†, Xiangjun He†, Yvonne K. Kwok, Feng Wang, Junyi Xue, Hui Zhao, Kin Wah Suen, Chi Chiu Wang, Jianwei Ren, George G. Chen, Paul B. S. Lai, Jiangchao Li, Yin Xia, Andrew M. Chan, Wai-Yee Chan and Bo Feng
發表時間：2018/12/28
數字識別碼：10.1186/s12915-018-0616-2
原文連結：https://bmcbiol.biomedcentral.com/articles/10.1186/s12915-018-0616-2?utm_source=other&utm_medium=other&utm_content=null&utm_campaign=BSCN_2_DD_BMCBio_Art_Scinet
微信連結：https://mp.weixin.qq.com/s/_G1shHW7FxO7um8QDwSHZQ
生物醫學研究中，體外培養的各種細胞系是研究人類基因功能的重要模型。但是，這些細胞系在篩選建立和適應培養條件的過程中經常積累以至攜帶了大量的基因組異常，許多基因均呈現為三個拷貝甚至更多拷貝（圖1）。因此，在研究過程中，對體外細胞系中多拷貝基因進行基因編輯並完全敲除，經常存在一定難度。
圖1. LO2 細胞的雙色螢光原位雜交 (Fluorescence in situ hybridization, FISH) 分析結果
原文 Figure. S1, Zhang et al. BMC Biology, 2018
之前的研究發現，由CRISPR/Cas9誘導的、經非同源末端連接路徑（non-homology end joining, NHEJ pathway）介導的DNA修復，可以用於大片段DNA（或基因）在基因組中的定點敲入。 該方法的敲入效率遠遠高於傳統的、由同源依賴性DNA修復路徑（homology-dependent repair, HDR pathway）介導的敲入（He et al., Nucleic Acids Research 2016; Suzuki et al., Nature 2016; Zhou et al., FEBS Letters 2016）。因此，該方法或可為多拷貝基因敲除提供了一個很好的工具。
最近，香港中文大學生物醫學學院的馮波博士課題組（張辰子等）在BMC Biology發表的最新研究工作，以LO2人類細胞係為模型，運用CRISPR/Cas9- NHEJ介導的方法， 針對目的基因同時插入了兩個不攜帶啟動子的螢光報告基因（ires-GFP/ires-Tdomato）。這些報告基因只有在以預先設計的方式插入目的基因後，才可通過目的基因啟動子來表達報告螢光蛋白。隨後，通過流式細胞螢光分選技術（Fluorescence activated cell sorting, FACS）對樣品進行分析，可以檢測並分選獲得雙陽性螢光細胞，以此，可一步達到同時阻斷兩個或者多個拷貝基因表達的目的。運用這個方法，他們對LO2中的候選基因ULK1（4拷貝），FAT10（3拷貝）進行基因敲除實驗及檢測（圖2）。
圖2. 經CRIPSR/Cas9- NHEJ介導，在ULK1和FAT10基因中同時插入雙螢光報告基因進行目的基因敲除
原文Figure. 2A；Zhang et al. BMC Biology, 2018
在單克隆細胞系的分析中，基因組PCR驗證了在ULK1和FAT10基因的剪切位點均插入了相應螢光報告基因；mRNA、蛋白水平及功能性分析均驗證了ULK1和FAT10蛋白在單克隆細胞系中的缺失（以ULK1單克隆細胞為例，圖3）。而且，總體實驗結果證明，該方法確實可以大幅提高獲得攜帶目的基因敲除的單克隆細胞系的效率（原文Figure. 2D）。
圖3. 對ULK1敲除單克隆細胞系的分析
原文Figure. 2E~G；Zhang et al. BMC Biology, 2018
與此同時，該研究還運用同一方法對CTIP基因（2拷貝；圖1）外顯子進行了雙螢光報告基因的插入，並且意外地發現，在所獲得的單克隆細胞系中，雖然基因組水平的PCR分析上已明確地檢測到了CTIP雙等位基因中均插入了螢光報告基因，但在mRNA和蛋白水平仍然可以檢測到CTIP轉錄和蛋白的表達。
進一步對這些克隆細胞系中CTIP mRNA進行測序的結果顯示，這些mRNA轉錄本確實來自報告基因插入後的基因組，而且攜帶了各種不同的剪切印記。但是意外的是，所有這些異常CTIP mRNA轉錄本均都保留了原有CTIP基因的閱讀框架（in-frame aberrant transcripts），（圖4）。
圖4. CTIP敲除單克隆細胞系 (RT-PCR)測序分析結果
原文Figure. 3E, F；Zhang et al. BMC Biology, 2018
結論和意義
本研究利用CRISPR/Cas9誘導的NHEJ修複方式插入雙螢光報告基因從而實現了體外細胞系中多拷貝基因的敲除。該方法可利用雙螢光流式分選來對多拷貝基因敲除細胞進行富集，簡化和提高了多拷貝基因敲除的效率。
另外，本研究通過對CTIP的基因編輯，發現細胞中所存在的多種DNA修復及RNA剪切機制會產生各種不同的隨機產物，這使得在生存壓力下，細胞將有機會富集具有功能的基因產物，使得對細胞生存起關鍵作用的必要基因免於被完全「敲除」。
這項研究進一步加深了對基因編輯過程中細胞反應的理解，同時，也對今後CRISPR/Cas9基因編輯的應用提供了重要的指導意義。
摘要：
Background
Cultured human cells are pivotal models to study human gene functions, but introducing complete loss of function in diploid or aneuploid cells has been a challenge. The recently developed CRISPR/Cas9-mediated homology-independent knock-in approach permits targeted insertion of large DNA at high efficiency, providing a tool for insertional disruption of a selected gene. Pioneer studies have showed promising results, but the current methodology is still suboptimal and functional outcomes have not been well examined. Taking advantage of the promoterless fluorescence reporter systems established in our previous study, here, we further investigated potentials of this new insertional gene disruption approach and examined its functional outcomes.
Results
Exemplified by using hyperploid LO2 cells, we demonstrated that simultaneous knock-in of dual fluorescence reporters through CRISPR/Cas9-induced homology-independent DNA repair permitted one-step generation of cells carrying complete disruption of target genes at multiple alleles. Through knocking-in at coding exons, we generated stable single-cell clones carrying complete disruption of ULK1 gene at all four alleles, lacking intact FAT10 in all three alleles, or devoid of intact CtIP at both alleles. We have confirmed the depletion of ULK1 and FAT10 transcripts as well as corresponding proteins in the obtained cell clones. Moreover, consistent with previous reports, we observed impaired mitophagy in ULK1−/− cells and attenuated cytokine-induced cell death in FAT10−/− clones. However, our analysis showed that single-cell clones carrying complete disruption of CtIP gene at both alleles preserved in-frame aberrant CtIPtranscripts and produced proteins. Strikingly, the CtIP-disrupted clones raised through another two distinct targeting strategies also produced varied but in-frame aberrant CtIPtranscripts. Sequencing analysis suggested that diverse DNA processing and alternative RNA splicing were involved in generating these in-frame aberrant CtIP transcripts, and some infrequent events were biasedly enriched among the CtIP-disrupted cell clones.
Conclusion
Multiallelic gene disruption could be readily introduced through CRISPR/Cas9-induced homology-independent knock-in of dual fluorescence reporters followed by direct tracing and cell isolation. Robust cellular mechanisms exist to spare essential genes from loss-of-function modifications, by generating partially functional transcripts through diverse DNA and RNA processing mechanisms.
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期刊介紹:
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（來源：科學網）
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