Lung and liver editing using 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 >100X more genome editing of cells and organs compared to 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 genome editing levels of 16‒37% efficiency 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 edited the disease-causing SFTPC gene in lung tissue with 19% average efficiency, 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.