Development of a novel translocation reporter system
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Abstract
Genome integrity is vital for the propagation of genetic information. However, genomes are under the constant exposure to DNA damaging factors. One of the most deleterious types of DNA damage are DSBs. Illegitimate repair of broken chromosomes may result in the formation of chromosome rearrangements, including chromosome translocations. Their occurrence may lead to the deregulation of gene expression or activation of oncogenes that can be causal in a variety of cancers. The formation of chromosome translocations is a multistep process that involves the chromosome breakage, chromosome-end synapsis and misrepair to form fusions. However, still little is known about the factors that contribute to those individual steps. Due to their rare occurrence, probing and quantifying chromosome translocations is difficult. Current tools, such as FISH and PCR are laborious and not sensitive enough for such low-frequency events. The major focus of the field has been to understand the repair mechanisms and studying the characteristics of individual factors. The purpose of this study was to develop the system that would enable the identification of novel factors involved in the formation of chromosome translocations through performing a genome-wide screening. Created translocation reporter assay is based on the CRISPR/Cas9 technology for inducing DSBs, which is a new golden standard for creating translocation relevant cancer models. In addition, the custom-made selection cassette was integrated into the genome of the haploid cell line. It contains the antibiotic resistance gene, which expression is activated only upon translocation. Therefore, the main advantage of the system is that it enables the selection and enrichment of cells positive for translocations. Our results show that the system is suitable for the generation of different types of chromosome rearrangements, such as deletions, inversions and translocations. It is also compatible with PCR based methods, which allows quantification of frequencies of occurring events. We anticipate our assay to be a starting point for more sophisticated studies on the processes behind the translocation biogenesis.