Sumoylation of Cas9 at lysine 848 regulates protein stability and DNA binding

Abstract

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.

Eukaryotic-driven directed evolution of Cas9 nucleases

“…While the effect of chromatin structure on Cas efficiency has been well documented, other factors could influence nuclease activity in eukaryotic cells, such as interactions with host proteins. Post-translational modifications (PTMs) have been shown to inhibit Cas proteins in prokaryotes (32)(33)(34), however it remains unclear to what extent nuclease activity could be modulated by PTMs introduced by host factors in eukaryotes (35). These results suggest that limited insights on Cas interactions with the eukaryotic environment will severely limit the molecular engineering of these tools.…”

“…Overall UltraCjCas9, generated by EPICA, showed an enhanced activity while preserving specificity. (35). These results suggest that limited insights on Cas interactions with the eukaryotic environment will severely limit the molecular engineering of these tools.…”

Umut Şahin: SUMOylation in health and disease

“…Many key proteins are now emerging as prime sumoylation targets, and our core scientific interest is to define how sumoylation regulates their function. For example, we have recently reported in LSA our discovery of Cas9 sumoylation ( 2 ), the first post-translational modification to be ever described on this very important enzyme. Even though the CRISPR/Cas9 system is a popular genome editing platform with many future prospects for therapeutics, surprisingly, little is known about how this system behaves in human cells.…”

Cas9 is mostly orthogonal to human systems of DNA break sensing and repair

“…The cellular factors that might facilitate the enzyme turnover and expose the nascent DSB are poorly known; Cas9 holds on DNA so tightly that it could hardly be displaced even by replication and transcription machinery [21][22][23]. Two ways that have found some experimental support so far involve FACT, a nucleosome disassembly factor that normally operates on H2A/ H2B histones removing them from chromatin, and ubiquitylation or sumoylation followed by proteolysis [24,25]. Even with this, the exact mechanism of Cas9 removal has not been addressed, and many other chromatin remodelling factors could be involved.…”