A comparison of CRISPR/Cas9 and siRNA-mediated ALDH2 gene silencing in human cell lines

Wang, Fei; Guo, Tao; Jiang, Hongmei; Li, Ruobi; Wang, Ting; Zeng, Ni; Dong, Guang‐Hui; Zeng, Xiao‐Wen; Li, Daochuan; Xiao, Yongmei; Hu, Qiansheng; Chen, Wen; Xing, Xiumei; Wang, Qing

doi:10.1007/s00438-018-1420-y

Cited by 19 publications

(12 citation statements)

References 32 publications

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“…Additionally, we observed that siCCM3 treatment destroys the morphology of mitochondria, and the immunofluorescence images of CCM3-KO cells confirmed the dramatic impact of long-term loss of CCM3 on mitochondrial architecture. Although the Western Blotting results of CCM3-related signaling pathway markers in selected cells didn't resemble the siRNA treated cells, the difference in signaling pathways between CRISPR-knockout cells and siRNA-transfected cells has been demonstrated in earlier studies [37,38]. The compromised mitochondrial membrane potential in CRISPR-CCM3 EPCs was also in line with the changes in morphology.…”

Section: Discussionmentioning

confidence: 71%

ATPIF1 maintains normal mitochondrial structure which is impaired by CCM3 deficiency in endothelial cells

et al. 2021

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Background Numerous signaling pathways have been demonstrated experimentally to affect the pathogenesis of cerebral cavernous malformations (CCM), a disease that can be caused by CCM3 deficiency. However, the understanding of the CCM progression is still limited. The objective of the present work was to elucidate the role of CCM3 by RNA-seq screening of CCM3 knockout mice. Results We found that ATPIF1 was decreased in siCCM3-treated Human Umbilical Vein Endothelial Cells (HUVECs), and the overexpression of ATPIF1 attenuated the changes in cell proliferation, adhesion and migration caused by siCCM3. The probable mechanism involved the conserved ATP concentration in mitochondria and the elongated morphology of the organelles. By using the CRISPR-cas9 system, we generated CCM3-KO Endothelial Progenitor Cells (EPCs) and found that the knockout of CCM3 destroyed the morphology of mitochondria, impaired the mitochondrial membrane potential and increased mitophagy. Overexpression of ATPIF1 contributed to the maintenance of normal structure of mitochondria, inhibiting activation of mitophagy and other signaling proteins (e.g., KLF4 and Tie2). The expression of KLF4 returned to normal in CCM3-KO EPCs after 2 days of re-overexpression of CCM3, but not other signaling proteins. Conclusion ATPIF1 maintains the normal structure of mitochondria, inhibiting the activation of mitophagy and other signaling pathway in endothelial cells. Loss of CCM3 leads to the destruction of mitochondria and activation of signaling pathways, which can be regulated by KLF4.

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Section: Discussionmentioning

confidence: 71%

ATPIF1 maintains normal mitochondrial structure which is impaired by CCM3 deficiency in endothelial cells

et al. 2021

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“…2,3 Currently, gene editing approaches using CRISPR-Cas9 technology have greatly facilitated the targeted disruption of genes allowing the generation of cell lines with fully abrogated gene function. 26 However, to date, most knockout cell lines produced by this technology rely on constitutive Cas9 expression that is capable of inducing untoward genotoxic effects. In addition, to achieve biallelic gene disruption, cell cloning or isolation based on selectable markers is required, a possibility restricted mostly to cell lines.…”

Section: Discussionmentioning

confidence: 99%

Efficient CRISPR-Cas9-Mediated Gene Ablation in Human Keratinocytes to Recapitulate Genodermatoses: Modeling of Netherton Syndrome

Gálvez

Chacón-Solano

Bonafont

et al. 2020

Molecular Therapy - Methods & Clinical Development

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Current efforts to find specific genodermatoses treatments and define precise pathogenesis mechanisms require appropriate surrogate models with human cells. Although transgenic and gene knockout mouse models for several of these disorders exist, they often fail to faithfully replicate the clinical and histopathological features of the human skin condition. We have established a highly efficient method for precise deletion of critical gene sequences in primary human keratinocytes, based on CRISPR-Cas9-mediated gene editing. Using this methodology, in the present study we generated a model of Netherton syndrome by disruption of SPINK5. Gene-edited cells showed absence of LEKTI expression and were able to recapitulate a hyperkeratotic phenotype with most of the molecular hallmarks of Netherton syndrome, after grafting to immunodeficient mice and in organotypic cultures. To validate the model as a platform for therapeutic intervention, we tested an ex vivo gene therapy approach using a lentiviral vector expressing SPINK5. Re-expression of SPINK5 in an immortalized clone of SPINK5-knockout keratinocytes was capable of reverting from Netherton syndrome to a normal skin phenotype in vivo and in vitro. Our results demonstrate the feasibility of modeling genodermatoses, such as Netherton syndrome, by efficiently disrupting the causative gene to better understand its pathogenesis and to develop novel therapeutic approaches.

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“…However, correlation analysis between KIAA1377 expression versus clinicopathological variables revealed that there was a significant correlation of KIAA1377 expression with lymph nodes metastases, which leads to the suggestion that KIAA1377 might be involved in the lymph node metastasis of ESCC cells. The slight discrepancy between in vivo and in vitro prompted us to rework the biological roles of KIAA1377 implicated in the proliferation and motility of SCC cells by using the alternative CRISPR‐Cas9 technique (Shalem et al, ), which is distinctively different from the shRNA system in the case of silencing efficiency (Tan and Martin, ; Wang et al, ). In the current observation, knock‐out of KIAA1377 mediated by the CRISPR‐Cas9 system turns out to be able to markedly slow down the growth and motility of KYSE‐150 and HeLa cells, preliminarily defining the oncogenic roles of KIAA1377 in SCC cells.…”

Section: Discussionmentioning

confidence: 99%

Let‐7b‐5p inhibits proliferation and motility in squamous cell carcinoma cells through negative modulation of KIAA1377

Liu

Tan

et al. 2019

Cell Biology International

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KIAA1377 has been found to be linked with lymph node metastasis in esophageal squamous cell carcinoma (SCC) in our previous study; however, the regulation of KIAA1377 remains far from understood. Herein, to understand the regulation of KIAA1377 from the angle of microRNA (miRNA)–messenger RNA (mRNA) modulation in the setting of SCC cells, the basal level of KIAA1377 was determined by quantitative real‐time polymerase chain reaction (qRT‐PCR) and western blot analysis in KYSE‐150 and HeLa cells; biological roles of KIAA1377 contributing in the proliferation, migration, and invasion were evaluated using 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2H‐tetrazolium bromide (MTT), wound‐healing and Transwell assays, respectively, after KIAA1377 was knocked out mediated by the CRISPR‐Cas9 system. Bioinformatic prediction revealed that let‐7b‐5p was a putative miRNA regulating KIAA1377, which was ensuingly validated by the luciferase reporter assay; after which, variation of KIAA1377 expression was further verified by qRT‐PCR and western blot analysis. Moreover, the biological roles of let‐7b‐5p in proliferation, migration, and invasion of KYSE‐150 and HeLa cells were also evaluated. It was exhibited that KIAA1377 was able to promote the proliferation and motility of both KYSE‐150 and HeLa cells, which can be reverted by re‐expression of let‐7b‐5p. The luciferase reporter assay verified that let‐7b‐5p can diametrically target KIAA1377. Collectively, our data demonstrated that let‐7b‐5p can directly but negatively regulate KIAA1377 in SCC cell lines, Ecal109, and HeLa cells.

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A comparison of CRISPR/Cas9 and siRNA-mediated ALDH2 gene silencing in human cell lines

Cited by 19 publications

References 32 publications

ATPIF1 maintains normal mitochondrial structure which is impaired by CCM3 deficiency in endothelial cells

ATPIF1 maintains normal mitochondrial structure which is impaired by CCM3 deficiency in endothelial cells

Efficient CRISPR-Cas9-Mediated Gene Ablation in Human Keratinocytes to Recapitulate Genodermatoses: Modeling of Netherton Syndrome

Let‐7b‐5p inhibits proliferation and motility in squamous cell carcinoma cells through negative modulation of KIAA1377

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