School of Biomedical Sciences, CUHK-GIBH CAS Joint Laboratory on Stem Cell and Regenerative Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong, Special Administrative Region of China.
Department of Obstetrics and Gynaecology, The Chinese University of Hong Kong, Shatin, Hong Kong, Special Administrative Region of China.
BMC Biol. 2018 Dec 28;16(1):151. doi: 10.1186/s12915-018-0616-2.
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.
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 CtIP transcripts and produced proteins. Strikingly, the CtIP-disrupted clones raised through another two distinct targeting strategies also produced varied but in-frame aberrant CtIP transcripts. 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.
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.
培养的人类细胞是研究人类基因功能的关键模型，但在二倍体或非整倍体细胞中引入完全功能丧失一直是一个挑战。最近开发的 CRISPR/Cas9 介导的非同源性敲入方法可以高效靶向插入大片段 DNA，为插入破坏选定基因提供了工具。先驱性研究显示出了有前景的结果，但当前的方法仍存在不足，功能结果也未得到很好的检验。利用我们之前研究中建立的无启动子荧光报告系统，在此，我们进一步研究了这种新的插入基因敲除方法的潜力，并检验了其功能结果。
以超二倍体 LO2 细胞为例，我们证明了通过 CRISPR/Cas9 诱导的非同源性 DNA 修复同时敲入双荧光报告基因，可一步生成靶基因在多个等位基因上完全缺失的细胞。通过敲入编码外显子，我们生成了稳定的单细胞克隆，在所有四个等位基因上完全缺失 ULK1 基因，在所有三个等位基因上缺失完整的 FAT10，或在两个等位基因上均缺失完整的 CtIP。我们已经证实，在所获得的细胞克隆中，ULK1 和 FAT10 转录本以及相应蛋白的耗竭。此外，与之前的报道一致，我们观察到 ULK1-/-细胞中的线粒体自噬受损，以及 FAT10-/-克隆中细胞因子诱导的细胞死亡减弱。然而，我们的分析表明，在两个等位基因上均完全缺失 CtIP 基因的单细胞克隆保留了有框异常的 CtIP 转录本，并产生了蛋白。引人注目的是，通过另外两种不同的靶向策略产生的 CtIP 缺失克隆也产生了不同但有框异常的 CtIP 转录本。序列分析表明，多种 DNA 加工和选择性 RNA 剪接参与了这些有框异常的 CtIP 转录本的生成，并且一些罕见事件在 CtIP 缺失细胞克隆中被偏倚性富集。
通过 CRISPR/Cas9 诱导的非同源性双荧光报告基因敲入，随后直接跟踪和细胞分离，可轻松引入多等位基因缺失。通过多种 DNA 和 RNA 加工机制产生部分功能转录本，细胞中存在强大的机制来避免必需基因的功能丧失修饰。
