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

Lipid nanoparticle (LNP) delivery of CRISPR ribonucleoproteins (RNPs) could enable high-efficiency, low-toxicity, and scalable in vivo genome editing if efficacious RNP:LNP complexes can be reliably produced. Here, we engineered a thermostable Cas9 from Geobacillus stearothermophilus (GeoCas9) to generate iGeoCas9 variants capable of genome editing of cells and organs with several orders of magnitude higher efficiency than the native GeoCas9 enzyme. Furthermore, iGeoCas9 RNP:LNP complexes edited a variety of cell types and induced homology-directed repair (HDR) in cells receiving co-delivered single-stranded DNA (ssDNA) templates. Using tissue-selective LNP formulations, we observed robust genome editing in the liver and lungs of model reporter mice that received single intravenous injections of iGeoCas9 RNP:LNPs. In addition, iGeoCas9 RNP complexed to biodegradable LNPs effectively edited the disease-causing SFTPC gene in lung tissue, representing a major improvement over genome editing levels 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 and expand the therapeutic potential of genome editing.