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Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells
Published on: June 16, 2017
A Targeted and Tuneable DNA Damage Tool Using CRISPR/Cas9
Ioannis Emmanouilidis1, Natalia Fili2, Alexander W Cook2
1School of Biosciences, University of Kent, Canterbury CT2 7NJ, UK.
Abstract:
Mammalian cells are constantly subjected to a variety of DNA damaging events that lead to the activation of DNA repair pathways. Understanding the molecular mechanisms of the DNA damage response allows the development of therapeutics which target elements of these pathways. Double-strand breaks (DSB) are particularly deleterious to cell viability and genome stability. Typically, DSB repair is studied using DNA damaging agents such as ionising irradiation or genotoxic drugs. These induce random lesions at non-predictive genome sites, where damage dosage is difficult to control. Such interventions are unsuitable for studying how different DNA damage recognition and repair pathways are invoked at specific DSB sites in relation to the local chromatin state. The RNA-guided Cas9 (CRISPR-associated protein 9) endonuclease enzyme is a powerful tool to mediate targeted genome alterations. Cas9-based genomic intervention is attained through DSB formation in the genomic area of interest. Here, we have harnessed the power to induce DSBs at defined quantities and locations across the human genome, using custom-designed promiscuous guide RNAs, based on in silico predictions. This was achieved using electroporation of recombinant Cas9-guide complex, which provides a generic, low-cost and rapid methodology for inducing controlled DNA damage in cell culture models.
Insights
This study introduces a novel method using Cas9 (CRISPR-associated protein 9) to create precise DNA double-strand breaks (DSBs) in mammalian cells. This controlled DNA damage induction allows for better study of DNA repair pathways at specific genomic locations.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- Mammalian cells possess DNA repair pathways to counteract constant DNA damage.
- Double-strand breaks (DSBs) are critical DNA lesions impacting cell viability and genome stability.
- Current methods for inducing DSBs (e.g., irradiation, drugs) lack precision in location and dosage.
Purpose of the Study:
- To develop a method for inducing targeted and controlled DNA double-strand breaks (DSBs) at specific genomic locations.
- To enable the study of DNA damage response and repair mechanisms in relation to local chromatin states.
- To provide a versatile tool for investigating genome stability and repair pathway activation.
Main Methods:
- Utilized the RNA-guided Cas9 (CRISPR-associated protein 9) endonuclease.
- Designed custom promiscuous guide RNAs based on in silico predictions.
- Employed electroporation of recombinant Cas9-guide complexes to induce DSBs in human cell lines.
Main Results:
- Successfully induced DSBs at defined quantities and specific locations across the human genome.
- Demonstrated a generic, low-cost, and rapid methodology for controlled DNA damage induction.
- Established a new model system for studying DNA repair at targeted genomic sites.
Conclusions:
- The Cas9-mediated induction of targeted DSBs offers a significant advancement over traditional methods.
- This technique provides unprecedented control for investigating DNA damage response and repair in mammalian cells.
- The methodology is adaptable for various research applications in genome stability and therapeutic development.
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