The effect of guide RNA thermal denaturation on the quality of Cas9 ribonucleoprotein-loaded lipid nanoparticle formulations

Abstract

Gene-editing technology for the treatment of genetic diseases uses a system that delivers clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 as ribonucleoproteins (RNPs) in a faster and more transiently effective manner than other delivery methods that involve gene expression. Lipid nanoparticles (LNPs) are an integral part of technology that is used to deliver Cas9 RNPs, and in recent years the in vivo delivery of RNPs has been achieved. While single-guide RNA (sgRNA) forms complex higher-order structures, which are known to result in the formation of heterogeneous RNPs, the impact on RNP-loaded LNP formulations has been overlooked. The results of this study show that the heterogeneity of sgRNA significantly affects the internal structure, physical properties, and knockout activity of RNP-loaded LNP formulations through changes in molecular weight distribution and RNP charge.