PT  - JOURNAL ARTICLE
AU  - Tunahan Ergünay
AU  - Özgecan Ayhan
AU  - Arda B Celen
AU  - Panagiota Georgiadou
AU  - Emre Pekbilir
AU  - Yusuf T Abaci
AU  - Duygu Yesildag
AU  - Mandy Rettel
AU  - Ulduz Sobhiafshar
AU  - Anna Ogmen
AU  - NC Tolga Emre
AU  - Umut Sahin
TI  - Sumoylation of Cas9 at lysine 848 regulates protein stability and DNA binding
AID  - 10.26508/lsa.202101078
DP  - 2022 Apr 01
TA  - Life Science Alliance
PG  - e202101078
VI  - 5
IP  - 4
4099  - https://www.life-science-alliance.org/content/5/4/e202101078.short
4100  - https://www.life-science-alliance.org/content/5/4/e202101078.full
SO  - Life Sci. Alliance2022 Apr 01; 5
AB  - CRISPR/Cas9 is a popular genome editing technology. Although widely used, little is known about how this prokaryotic system behaves in humans. An unwanted consequence of eukaryotic Cas9 expression is off-target DNA binding leading to mutagenesis. Safer clinical implementation of CRISPR/Cas9 necessitates a finer understanding of the regulatory mechanisms governing Cas9 behavior in humans. Here, we report our discovery of Cas9 sumoylation and ubiquitylation, the first post-translational modifications to be described on this enzyme. We found that the major SUMO2/3 conjugation site on Cas9 is K848, a key positively charged residue in the HNH nuclease domain that is known to interact with target DNA and contribute to off-target DNA binding. Our results suggest that Cas9 ubiquitylation leads to decreased stability via proteasomal degradation. Preventing Cas9 sumoylation through conversion of K848 into arginine or pharmacologic inhibition of cellular sumoylation enhances the enzyme’s turnover and diminishes guide RNA-directed DNA binding efficacy, suggesting that sumoylation at this site regulates Cas9 stability and DNA binding. More research is needed to fully understand the implications of these modifications for Cas9 specificity.