Biological Procedures Online, Volume (20), No (21), Year (2018-11) , Pages (1-12)

Title : ( CRISPR/Cas9 Knockout Strategies to Ablate CCAT1 lncRNA Gene in Cancer Cells )

Authors: khadije zare , Milad Shademan , Mohammad Mahdi Ghahramani Seno , Hesam Dehghani ,

Citation: BibTeX | EndNote


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.


CRISPR/Cas9 Knockout Cancer cell Long noncoding RNA CCAT1
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author = {Zare, Khadije and Shademan, Milad and Ghahramani Seno, Mohammad Mahdi and Dehghani, Hesam},
title = {CRISPR/Cas9 Knockout Strategies to Ablate CCAT1 lncRNA Gene in Cancer Cells},
journal = {Biological Procedures Online},
year = {2018},
volume = {20},
number = {21},
month = {November},
issn = {1480-9222},
pages = {1--12},
numpages = {11},
keywords = {CRISPR/Cas9 Knockout Cancer cell Long noncoding RNA CCAT1},


%0 Journal Article
%T CRISPR/Cas9 Knockout Strategies to Ablate CCAT1 lncRNA Gene in Cancer Cells
%A Zare, Khadije
%A Shademan, Milad
%A Ghahramani Seno, Mohammad Mahdi
%A Dehghani, Hesam
%J Biological Procedures Online
%@ 1480-9222
%D 2018