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Generation of Brown Fat-Specific Knockout Mice Using a Combined Cre-LoxP, CRISPR-Cas9, and Adeno-Associated Virus Single-Guide RNA System
Published on: March 24, 2023
Establishing a dual knock-out cell line by lentivirus based combined CRISPR/Cas9 and Loxp/Cre system
Ya Li1, Weifeng Zhang1, Junli Zhao1
1Laboratory of Gene Therapy, Department of Biochemistry, College of Life Sciences, Shaanxi Normal University, 199 South Chang'an Road, Xi'an, 710062, Shaanxi, People's Republic of China.
Abstract:
The clustered regulatory interspersed short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system has been widely used for gene knock-out. Lentiviral vectors have been commonly used as a delivery method for this system, however, prolonged Cas9/sgRNA expression due to lentiviral integration can lead to accumulating off-target mutations. To solve this issue in engineering a gene knock-out cell line, this study established a novel system, which was composed of two lentiviral vectors. One lentiviral vector carried simultaneously sgRNAs and CRISPR/Cas9 expression cassettes targeting single or multiple gene(s); the other lentiviral vector carried Cre that could remove excess sgRNAs and Cas9 expression cassettes in the genome after gene targeting was achieved. To prove the principle, two candidate genes, extracellular matrix protein 1 (ECM1) and progranulin (PGRN), both highly expressed in MDA-MB-231 cells, were selected for testing the novel system. A dual knock-out of ECM1 and PGRN was successfully achieved in MDA-MB-231 cell line, with the sgRNAs and Cas9 expression cassettes being removed by Cre. This system should have great potential in applications for multiple genes knock-out in vitro.
Insights
This study introduces a novel CRISPR/Cas9 gene editing system using two lentiviral vectors to achieve efficient multiple gene knock-out. The system removes Cas9 and sgRNA expression cassettes post-editing, minimizing off-target mutations.
Area of Science:
- Molecular Biology
- Gene Editing Technologies
- Cancer Research
Background:
- The clustered regulatory interspersed short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system is a powerful tool for gene knock-out.
- Lentiviral vector delivery of CRISPR/Cas9 can cause persistent expression leading to off-target mutations.
Purpose of the Study:
- To develop a novel CRISPR/Cas9 delivery system to overcome limitations of prolonged expression and off-target mutations.
- To establish an efficient method for engineering multiple gene knock-out cell lines.
Main Methods:
- A two-lentiviral vector system was designed: one vector for CRISPR/Cas9 and sgRNAs, the other for Cre recombinase.
- Cre recombinase was used to excise the Cas9 and sgRNA expression cassettes after gene targeting.
- Extracellular matrix protein 1 (ECM1) and progranulin (PGRN) were targeted in MDA-MB-231 cells.
Main Results:
- Successful dual knock-out of ECM1 and PGRN was achieved in MDA-MB-231 cells.
- The Cas9 and sgRNA expression cassettes were efficiently removed by Cre-mediated excision.
- The novel system demonstrated effective gene targeting and minimized potential for off-target effects.
Conclusions:
- The developed two-lentiviral vector system provides a robust method for multiple gene knock-out.
- This system effectively removes CRISPR/Cas9 components, reducing off-target mutation risks.
- The technology holds significant promise for in vitro applications requiring precise multi-gene editing.
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