Mean expression levels of RNA in cells receiving WT-Cas9 and Mutant Cas9 along with gRNA targeting MIAT locus.

In this study, we report pre-existing human CD8+ T cell immunity to Streptococcus pyogenes Cas9 in the majority of healthy individuals screened. In a proof-of-principle study, we demonstrate that Cas9 protein can be modified to eliminate immunodominant epitopes through targeted mutation while preserving its function and specificity. The data presented here show the functionality of this Mutant Cas9 compared to WT-Cas9 when targeting an endogenous locus. Mammalian cells (Hek293FT) received either WT-Cas9 or a mutant Cas9 along with activator domains and gRNA targeting endogenous MIAT locus. The goal of the experiment was comparing activation capacity of the mutant Cas9 with the WT-Cas9. Activation levels were assessed 3 days post transfection using RNA-seq and qRT-PCR.