Abstract:
The CRISPR/Cas9 system offers a simple method for genome engineering by utilizing the ability of the bacterial Cas9 enzyme to cleave any desired genomic region under the guidance of a complementary RNA molecule. Due to its minimalism and versatility, the CRISPR/Cas9 system is increasingly being used as a gene editing platform in higher organisms, with the current applications encompassing diverse fields such as disease therapy, biotechnology and agriculture. Despite such widespread use of this prokaryotic protein, there is a lack of knowledge regarding its behavior and regulation in eukaryotic systems where the majority of these applications are implemented. In this study, we aim to elucidate the mechanisms of Cas9 regulation in eukaryotic systems with the specific goal of investigating the potential post-translational modifications (PTMs) on this protein. As part of this, we show that Cas9 gets ubiquitylated, promoting its proteasomal degradation. In addition, we present a cell culture-based experimental setup where we aim to discover that the Streptococcus pyogenes Cas9 protein undergoes SUMO modification following the engulfment of these bacteria by human immune cells. We expect that these results will lead to a better understanding of the eukaryotic posttranslational regulation of Cas9. Our hope is that uncovering the functional implications of these PTMs will contribute to the development of safer therapies and improve the current CRISPR-based applications, while paving the way for new ones. Moreover, we hope that a potential discovery of in vivo Cas9 sumoylation may create a new frontier for future research focusing on host-pathogen interactions.
