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.
Highlights
Our approach utilises electroporation of recombinant Cas9 instead of traditional transfection or engineering stable expression within cell line models. This reduces the perturbation to the cell line, removes the time delay to express Cas9 and enables multiple cells lines to be readily studied
We first established the electroporation efficiency with nuclease-deficient Cas9 to avoid any complications arising from non-specific nuclease activity
We observed that 40% of cells contained dCas9 (Figure 1B)
Summary
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. One of the most critical processes for living organisms is to maintain genome integrity using DNA damage surveillance and repair mechanisms. These mechanisms prevent cells from progressing through cell division, which would propagate the defective genome to daughter cells [1]. If lesions are not repaired, mutations can accumulate, leading to cell senescence, ageing and the onset of disease, such as cancer
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