Lung and liver editing by lipid nanoparticle delivery of a stable CRISPR–Cas9 ribonucleoprotein

Chen, Kai; Han, Hesong; Zhao, Sheng; Xu, Bryant; Yin, Boyan; Lawanprasert, Atip; Trinidad, Marena; Burgstone, Benjamin W.; Murthy, Niren; Doudna, Jennifer A.

doi:10.1038/s41587-024-02437-3

Title: Lung and liver editing by lipid nanoparticle delivery of a stable CRISPR–Cas9 ribonucleoprotein

Journal Article · 16 October 2024 · Nature Biotechnology

DOI: https://doi.org/10.1038/s41587-024-02437-3 · OSTI ID:2491400

^[1]; Han, Hesong ^[1];

^[1];

^[1]; Yin, Boyan ^[1];

^[1]; Trinidad, Marena ^[1];

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^[2]

University of California, Berkeley, CA (United States)
University of California, Berkeley, CA (United States); Gladstone Institutes, San Francisco, CA (United States); San Francisco State Univ., CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Lipid nanoparticle (LNP) delivery of clustered regularly interspaced short palindromic repeat (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 engineer a thermostable Cas9 from Geobacillus stearothermophilus (GeoCas9) to generate iGeoCas9 variants capable of >100× more genome editing of cells and organs compared with the native GeoCas9 enzyme. Furthermore, iGeoCas9 RNP–LNP complexes edit a variety of cell types and induce homology-directed repair in cells receiving codelivered single-stranded DNA templates. Using tissue-selective LNP formulations, we observe genome-editing levels of 16-37% in the liver and lungs of reporter mice that receive single intravenous injections of iGeoCas9 RNP–LNPs. In addition, iGeoCas9 RNPs complexed to biodegradable LNPs edit 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 nonviral delivery strategies. These results show that thermostable Cas9 RNP–LNP complexes can expand the therapeutic potential of genome editing.

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Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER). Biological Systems Science (BSS); National Science Foundation (NSF); National Institutes of Health (NIH); California Institute for Regenerative Medicine (CIRM)

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 2491400

Journal Information:: Nature Biotechnology, Journal Name: Nature Biotechnology; ISSN 1087-0156

Publisher:: Springer NatureCopyright Statement

Country of Publication:: United States

Language:: English

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