Abstract
BackgroundWith 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”.ResultsIn 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.ConclusionsThe 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.
Highlights
With the increasing discovery of long noncoding RNAs, the application of functional techniques that could have very specific, efficient, and robust effects and readouts is necessary
* Correspondence: dehghani@um.ac.ir 1Department of Basic Sciences, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Azadi Square, Mashhad, Iran 2Division of Biotechnology, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Azadi Square, Mashhad, Iran Full list of author information is available at the end of the article phenomena such as genomic imprinting, dosage compensation, pluripotency, and differentiation commitment [4]. These findings have proven Long noncoding RNA (lncRNA) to be important molecules with significant functions
cancer associated transcript 1 (CCAT1) transcription is highly upregulated in the pre-malignant adenomatous polyps and malignant colorectal carcinoma [8]
Summary
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. 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”. Long non-coding RNAs (lncRNAs) play important roles in the regulation of transcription and post-transcriptional processes of coding and non-coding RNAs. Different mechanisms of function have been reported for lncRNAs including guiding chromatin modifiers to specific genomic loci, sequestering transcription factors, allosteric modulation of transcriptional regulatory proteins, alteration of nuclear domains, modulation of translation, modulation of mRNA stability, and working as competing endogenous RNAs [1,2,3]. The third category includes programmable nucleases including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR/Cas (CRISPR-associated protein-9 nuclease)
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