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Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells
Published on: June 16, 2017
CRISPR/Cas9 Knockout Strategies to Ablate CCAT1 lncRNA Gene in Cancer Cells
Khadijeh Zare1, Milad Shademan1, Mohammad M Ghahramani Seno1,2
11Department of Basic Sciences, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Azadi Square, Mashhad, Iran.
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.
Insights
Researchers developed and compared three CRISPR/Cas9 gene knockout methods to study the CCAT1 long noncoding RNA (lncRNA) in colorectal cancer cells. The CRISPR du-HITI method proved most efficient for creating CCAT1 knockout cell lines.
Area of Science:
- Molecular Biology
- Genetics
- Cancer Research
Background:
- Increasing discovery of long noncoding RNAs (lncRNAs) necessitates efficient functional analysis techniques.
- Accurate gene knockout (KO) strategies are crucial for studying lncRNA function in diseases like colorectal adenocarcinoma.
Purpose of the Study:
- To apply and analyze three CRISPR/Cas9-mediated gene knockout strategies for ablating the CCAT1 lncRNA.
- To evaluate the efficiency and practicality of CRISPR excision, CRISPR HDR, and CRISPR du-HITI methods for lncRNA KO.
Main Methods:
- Utilized three distinct CRISPR/Cas9 strategies: CRISPR excision, CRISPR HDR, and CRISPR du-HITI to knockout the CCAT1 gene.
- Employed RT-qPCR, RNA-seq, MTT, and colony formation assays to assess the functional consequences of CCAT1 gene ablation.
- Introduced the CRISPR du-HITI strategy, involving gene segment removal and reporter/termination signal insertion.
Main Results:
- CCAT1 knockout cells exhibited dysregulation of genes involved in critical biological processes.
- Significant reduction in anchorage-independent growth was observed in CCAT1 knockout cells.
- The CRISPR du-HITI strategy demonstrated easier donor vector preparation and more practical cell selection compared to other methods, achieving biallelic single-cell knockouts efficiently.
Conclusions:
- The evaluated strategies are effective for generating CCAT1 knockout cell lines.
- These methods can be broadly applied for the functional study of other lncRNAs through gene deletion.
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