Dataset Information

CRISPR/Cas9 Knockout Strategies to Ablate CCAT1 lncRNA Gene in Cancer Cells.

ABSTRACT: Background:With the increasing discovery of long noncoding RNAs (lncRNAs), the application of functional techniques that could have very specific, efficient, and robust effects and readouts is necessary. Here, we have applied and analyzed three gene knockout (KO) strategies to ablate the CCAT1 gene in different colorectal adenocarcinoma cell lines. We refer to these strategies as "CRISPR excision", "CRISPR HDR", and "CRISPR du-HITI". Results:In order to obstruct the transcription of lncRNA or to alter its structure, in these strategies either a significant segment of the gene is removed, or a transcription termination signal is inserted in the target gene. We use RT-qPCR, RNA-seq, MTT, and colony formation assay to confirm the functional effects of CCAT1 gene ablation in knockout colorectal adenocarcinoma cell lines. We applied three different CRISPR/Cas9 mediated knockout strategies to abolish the transcription of CCAT1 lncRNA. CCAT1 knockout cells displayed dysregulation of genes involved in several biological processes, and a significant reduction for anchorage-independent growth. The du-HITI strategy introduced in this study removes a gene segment and inserts a reporter and a transcription termination signal in each of the two target alleles. The preparation of donor vector for this strategy is much easier than that in "CRISPR HDR", and the selection of cells in this strategy is also much more practical than that in "CRISPR excision". In addition, use of this technique in the first attempt of transfection, generates single cell knockouts for both alleles. Conclusions:The strategies applied and introduced in this study can be used for the generation of CCAT1 knockout cell lines and in principle can be applied to the deletion of other lncRNAs for the study of their function.

SUBMITTER: Zare K

PROVIDER: S-EPMC6211572 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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CRISPR/Cas9 Knockout Strategies to Ablate <i>CCAT1</i> lncRNA Gene in Cancer Cells.

Zare Khadijeh K Shademan Milad M Ghahramani Seno Mohammad M MM Dehghani Hesam H

Biological procedures online 20181101

<h4>Background</h4>With the increasing discovery of long noncoding RNAs (lncRNAs), the application of functional techniques that could have very specific, efficient, and robust effects and readouts is necessary. Here, we have applied and analyzed three gene knockout (KO) strategies to ablate the <i>CCAT1</i> gene in different colorectal adenocarcinoma cell lines. We refer to these strategies as "CRISPR excision", "CRISPR HDR", and "CRISPR du-HITI".<h4>Results</h4>In order to obstruct the transcr ...[more]

PMID: 30410426

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Project description:BackgroundThe growing detection of long noncoding RNAs (lncRNAs) required the application of functional approaches in order to provide absolutely precise, conducive, and reliable processed information along with effective consequences. We utilized genetic knockout (KO) techniques to ablate the Long Intergenic Noncoding RNA 00,511 gene in several humans who suffered from breast cancer cells and at the end we analyzed and examined the results.ResultsThe predictive relevance of LINC00511 expression pattern was measured by using a pooled hazard ratio (HR) with a 95% confidence interval (CI). The link among LINC00511 expression profiles and cancer metastasis was measured by using a pooled odds ratio (OR) with a 95% confidence interval. This meta- analysis was composed of fifteen studies which contained a total of 1040 tumor patients. We used three distinct CRISPR/Cas9-mediated knockdown techniques to prevent the LINC00511 lncRNA from being transcribed. RT-PCR was used to measure lncRNA and RNA expression. We used CCK-8, colony formation tests, and the invasion transwell test to measure cell proliferation and invasion. The stemness was measured by using a sphere-formation test. To validate molecular attachment, luciferase reporter assays were performed. The functional impacts of LINC00511 gene deletion in knockdown breast cancer cell lines were confirmed by using RT-qPCR, MTT, and a colony formation test. This meta-analysis was composed of 15 trials which contained a total of 1040 malignant tumors. Greater LINC00511 expression was ascribed to a lower overall survival (HR = 1.93, 95% CI 1.49-2.49, < P 0.001) and to an increased proportion of lymph node metastasis (OR = 3.07, 95% CI 2.23-4.23, P < 0.001) in the meta-analysis. It was found that the role of LINC00511 was overexpressed in breast cancer samples, and this overexpression was ascribed to a poor prognosis. The gain and loss-of-function tests demonstrated findings such as LINC00511 increased breast cancer cell proliferation, sphere-forming ability, and tumor growth. Additionally, the transcription factor E2F1 binds to the Nanog gene's promoter site to induce transcription. P57, P21, Prkca, MDM4, Map2k6, and FADD gene expression in the treatment group (LINC00511 deletion) was significantly higher than in the control group (P < 0.01). In addition, knockout cells had lower expression of BCL2 and surviving genes than control cells P < 0.001). In each of the two target alleles, the du-HITI approach introduced a reporter and a transcription termination signal. This strategy's donor vector preparation was significantly easier than "CRISPR HDR," and cell selection was likewise much easier than "CRISPR excision." Furthermore, when this approach was used in the initial transfection attempt, single-cell knockouts for both alleles were generated.ConclusionsThe methods employed and described in this work could be extended to the production of LINC00511 knockout cell lines and, in theory, to the deletion of other lncRNAs to study their function.

Project description:The editing efficiency primarily hinders the utility of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology in poultry. For a better understanding of the factors that influence the efficiency of gene knockout mediated by CRISPR/Cas9 in chicken DF1 cells, the single or dual single guide RNA (sgRNA) targeted exon regions of genes (taking anti-Müllerian hormone, TGF-beta receptor type-2 and Peroxisome proliferator-activated receptor gamma as examples) were designed. The sgRNA-CRISPR/Cas9 vectors with corresponding reporter vectors were transfected into DF1 cells. T7 endonuclease 1 (T7E1) and amplicon sequencing assay were compared for evaluating genome editing efficiency and the indel profiles were analyzed based on the data of amplicon sequencing. Meanwhile, to evaluate the precision of Cas9 cleavage, we also analyzed the homology of small insertion with the nucleotides of upstream and downstream of cleave sties. The surrogate reporter systems showed strong enrichment function, and the indel percentages were increased after puromycin selection. The indel ratios of T7E1 assay were lower than amplicon sequencing assay, which indicated T7E1 isn't fit to be used as the sole evaluation criterion for the targeting efficiency of CRISPR/Cas9. Based on the amplicon sequencing analysis, the editing efficiency showed noticeable differences among cells treated with different sgRNAs. However, the variety of indel efficiencies was not related to the GC content of sgRNA or chromosome types of targeted genes. The results showed that the dual sgRNA might not raise the indel ratios compared with individual sgRNA, but they could increase the ratios of the fragment deletions. The present study suggested that the surrogate reporter was an effective method to promote the editing efficiencies of CRISPR/Cas9 in chicken cells. The dual sgRNA could increase the fragment deletions, and the sensitivity of amplicon sequencing to detect cleavage was higher than the T7 endonuclease 1 assay. These results are essential to improve the application of CRISPR/Cas9 technology in chicken cells.

Dataset Information

CRISPR/Cas9 Knockout Strategies to Ablate CCAT1 lncRNA Gene in Cancer Cells.

Publications

CRISPR/Cas9 Knockout Strategies to Ablate <i>CCAT1</i> lncRNA Gene in Cancer Cells.

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