Genome engineering offers significant potential to treat or cure genetic disorders and shows great promise in biomedicine. The CRISPR/Cas9 system, which is an adaptive immune system that is naturally present in the majority of bacteria and archaea, became a powerful genome editing tool holding great prospects for successful gene therapy of various genetic diseases. Although it is known that the Cas9 scissile profile is flexible and might affect DNA repair outcomes, the factors influencing Cas9 cleavage decisions remain largely unknown. Therefore, our lab developed BreakTag, a time-effective and high-throughput methodology for the profiling of Cas9-induced DNA double-strand breaks (DSBs) at nucleotide resolution across the genome. This tool is used to characterize the Cas9 scissile profile and identification of molecular determinants of Cas9 incisions. The findings reveal that the Cas9 scissile profile is non-random but is strongly dependent on the nucleotide sequence of the protospacer and the presence of gRNA-DNA mismatches. Through comparing matched datasets of Cas9 incisions and subsequent repair outcomes, current work from our lab has determined that Cas9-induced staggered breaks are associated with precise, template-driven, and predictable single-nucleotide insertions. This suggests that regulating these cutting patterns could enable the prediction of repair genotypes featuring desirable indels. To pinpoint the responsible factors for mediating single-nucleotide insertions, the goal of this study was to develop and characterise a cell-based fluorescent system in which the anticipated repair of CRISPR-induced DSB towards a single nucleotide insertion restores a functional GFP gene. Our objective is to utilize this system to carry out a genome-wide screening to identify relevant repair factors responsible for these predictable insertions. Our research lays the foundation for utilizing the flexible Cas9 scissile profile for precise and predictable, template-free genome editing, particularly targeting pathogenic single-nucleotide deletions.
University of Patras
