A Targeted and Tuneable DNA Damage Tool Using CRISPR/Cas9

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.

A strain of Vibrio alginolyticus isolated from Azumapecten farreri and its pathogenic mechanism using CRISPRI-Cas9 technology

“…CRISPR/Cas system has been applied in basic research of bioengineering due to its high accuracy and short operation time (Wege et al, 2021;Zhu et al, 2020;Wright et al, 2016;Barrangou and Doudna., 2016), which can be designed to induce DSB at any genomic site (Gaj et al, 2013;Martin et al, 2012;Maeder et al, 2008;Miller et al, 2010;Claudio et al, 2011). Ioannis Emmanouilidis's research shows that the average transfection rate is up to 60% when electroporation is performed using recombinant Cas9 instead of traditional transfection (Emmanouilidis et al, 2021). In our present study, the electroporation transformation method improved the transformation efficiency and reduced the probability of false positives.…”

High-Throughput Imaging of CRISPR- and Recombinant Adeno-Associated Virus–Induced DNA Damage Response in Human Hematopoietic Stem and Progenitor Cells

“…While CRISPR-Cas9-induced DDR with gRNAs that have multiple on-target sites has been shown previously, 16 a much more challenging task that is relevant for deploying CRISPR in the clinic is comparing the DDR effects of gRNAs with only a single on-target site characterized by low levels of DSBs. Since CRISPR-Cas9 is known to elicit a DDR, 46 which is thought to be at least partially due to off-target activity, it is critical to identify a gRNA with the most specific editing possible.…”

“…Repair-center clusters manifest as foci under the microscope, 12 , 13 facilitating quantification of DNA damage caused by gamma irradiation, zinc-finger nucleases (ZFNs), 14 , 15 and extremely promiscuous CRISPR gRNAs. 16 …”

Plant chromatin on the move: an overview of chromatin mobility during transcription and DNA repair

“…The most comprehensive studies of chromatin mobility to date have been those associated with DNA damage. DNA (Emmanouilidis et al, 2021;Wang et al, 2019) or genotoxic agents such as zeocin or bleomycin, or c radiation. In different model species, it has been shown that, subsequent to DSBs, chromatin becomes generally more mobile (Dion et al, 2012;Dion et al, 2013;Hauer et al, 2017;Herbert et al, 2017;Lawrimore et al, 2017;Min e-Hattab et al, 2017;Min e-Hattab & Rothstein, 2012;Strecker et al, 2016).…”