Physicochemical and Functional Characterization of Differential CRISPR-Cas9 Ribonucleoprotein Complexes

Abstract

DNA cleavage assay using anion-exchange chromatography. Using these methods, we characterized the formation and stability of Cas9 RNP complexes associated with Cas9 and sgRNA characteristics as well as their functional activities. Multi-angle light scattering characterization showed different types and levels of aggregates in different source sgRNA materials, which contribute to form different Cas9 RNP complexes. The aggregations irreversibly dissociated at high temperatures. When the Cas9 RNP complexes derived from non-heated and heated sgRNAs were characterized, the data showed that specific RNP peaks were impacted. The Cas9 RNP complexes derived from the heated sgRNA retained their biological function and cleaved the double-strand target DNA at a higher rate. This work provides new tools to characterize the Cas9 RNP complex formation, stability, and functional activity and provides insights into sgRNA properties and handling procedures to better control the Cas9 RNP complex formation.