Identification of the major sumoylation site on crispr CAS9 protein

Abstract

CRISPR/Cas9 system is a type of adaptive immune system, which operates in prokaryotic organisms as an antiviral defense. Ever since its adaptation to eukaryotic systems as a gene editing tool, CRISPR has been one of the most popular genetic engineering technologies. Its diverse applications in molecular biology and medicine such as gene therapy, drug design, epigenome regulation, etc. have made this technology worth exploring further and further. In order to set up a more specific and efficient gene targeting system, it is essential to learn how the CRISPR pathway is regulated, in particular the Cas9 enzyme (CRISPR-associated protein 9), which is the key player of this system. In the lights of strongly supported data provided by a current lab member, it is revealed that Cas9 is covalently modified by SUMO-1 and SUMO-2 proteins. This was the first ever demonstration for a post-translational modification to which Cas9 is subjected. In the scope of this study, we intended to identify the major sumoylation site of Cas9 protein and generate a Cas9 mutant that is defective in sumoylation. For this purpose, a series of site-directed mutagenesis experiments were conducted. As a result, 10 single mutants for the 10 sumoylation motifs found on Cas9 protein were generated, as well as an 11th mutant in which all 10 sumoylation consensus motifs were destroyed altogether.