Lung and liver editing by lipid nanoparticle delivery of a stable CRISPR-Cas9 RNP

Abstract

AbstractLipid nanoparticle (LNP) delivery of CRISPR ribonucleoproteins (RNPs) has the potential to enable high-efficiencyin vivogenome editing with low toxicity and an easily manufactured technology, if RNP efficacy can be maintained during LNP production. In this study, we engineered a thermostable Cas9 fromGeobacillus stearothermophilus(GeoCas9) using directed evolution to generate iGeoCas9 evolved variants capable of robust genome editing of cells and organs. iGeoCas9s were significantly better at editing cells than wild-type GeoCas9, with genome editing levels >100X greater than those induced by the native GeoCas9 enzyme. Furthermore, iGeoCas9 RNP:LNP complexes edited a variety of cell lines and induced homology-directed repair (HDR) in cells receiving co-delivered single-stranded DNA (ssDNA) templates. Using tissue-selective LNP formulations, we observed genome editing of 35‒56% efficiency in the liver or lungs of mice that received intravenous injections of iGeoCas9 RNP:LNPs. In particular, iGeoCas9 complexed to acid-degradable LNPs edited lung tissuein vivowith an average of 35% efficiency, a significant improvement over editing efficiencies observed previously using viral or non-viral delivery strategies. These results show that thermostable Cas9 RNP:LNP complexes are a powerful alternative to mRNA:LNP delivery vehicles, expanding the therapeutic potential of genome editing.