166. Biophysical Characterization and Direct Delivery of S. Pyogenes Cas9 Ribonucleoprotein Complexes

Abstract

Several groups have demonstrated efficient genome editing in various mammalian cells by cationic lipid mediated delivery of purified Cas9 protein complexed with in-vitro translated or chemically synthesized guide-RNA (gRNA). Such “direct delivery” of the Cas9 ribonucleoprotein (RNP) complex allows for efficient gene-editing while minimizing off-target activity owing to the rapid turnover of the Cas9 protein in cells. Efficiency of gene-editing mediated by RNP delivery varies by locus, depends on the length of guide-RNA and on the amount and ratio of Cas9 protein and gRNA delivered.The Cas9 complex with gRNA has been well characterized structurally and biophysically revealing a large contact area and a high affinity. Thermal melt curves are a useful property to detect the binding and stability of complexes. We have used the large increase in the melting temperature from apo-Cas9 to the Cas9 complexed with gRNA to characterize the affinity of Cas9 for gRNA. Multiple gRNAs with differing lengths were complexed with Cas9 at differing stoichiometries and the tightness of the interaction was measured using thermal shift. These biophysically characterized complexes were then transfected into 293T cells and the efficiency of indel generated was measured. We have found that subtle differences in the gRNA length and base composition affect the binding and formation of RNP complex. Correlating binding affinity with efficiency of genome editing informs the design of an optimal composition of RNPs for cationic lipid mediated direct delivery.