Kinetic Analyses of SaCas9[D10E] in vitro
Abstract
CRISPR is a well-known adaptive defense mechanism that gained attention through its ability to be easily reprogrammable. Over time, the native CRISPR-Cas system evolved to tolerate mismatches, broadening its cleavage preferences; however, this development poses concerns with off-target cleavage in gene editing. Here, we introduced a D10E mutation into SaCas9 to potentially reduce off-target cleavage. To characterize SaCas9[D10E] and its tolerance for mutations, we designed 21 different substrates that each contained a single transversion in a therapeutically relevant EMX1-1gene. Through a series of in vitro cleavage assays, SaCas9[WT] and SaCas9[D10E] were compared across these substrates. A kinetic analysis of SaCas9[D10E] revealed trends in initial cleavage rates as PAM proximal mutations exhibited reduced cleavage activity and PAM distal mutations displayed enhanced activity. Furthermore, the ability of SaCas9[D10E] was highlighted through a competition assay that displayed discrimination between single nucleotide differences.