Lung and liver editing by lipid nanoparticle delivery of a stable CRISPR–Cas9 ribonucleoprotein
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.
Authors
- Atip Lawanprasert (ORCID: https://orcid.org/0009-0001-3163-8803)
- Boyan Yin (ORCID: https://orcid.org/0000-0002-9023-6265)
- Marena Trinidad (ORCID: https://orcid.org/0000-0001-7839-4642)
- Jennifer A. Doudna (ORCID: https://orcid.org/0000-0001-9161-999X)
- Niren Murthy (ORCID: https://orcid.org/0000-0002-7815-7337)
- Hesong Han (ORCID: https://orcid.org/0000-0001-5545-5238)
- Benjamin W. Burgstone (ORCID: https://orcid.org/0009-0000-8256-3867)
- Kai Chen (ORCID: https://orcid.org/0000-0002-3325-3536)
- Bryant Xu
- Sheng Zhao (ORCID: https://orcid.org/0009-0002-9890-048X)
Institutions
- QB3 (US)
- Gladstone Institutes (US)
- Howard Hughes Medical Institute (US)
- Lawrence Berkeley National Laboratory (US)
- Innovative Genomics Institute (US)
- University of California, Berkeley (US)
Publication Details
- Journal
- Nature Biotechnology
- Published
- 2024-10-16
- DOI
- https://doi.org/10.1038/s41587-024-02437-3
- Citations
- 93
- Primary Topic
- CRISPR and Genetic Engineering
- Type
- article
- Field-Weighted Citation Impact
- 21.75
Funders
- National Science Foundation
- Howard Hughes Medical Institute
- U.S. Department of Energy
- Cystic Fibrosis Foundation
- California Institute for Regenerative Medicine
- Life Sciences Research Foundation
- Innovative Genomics Institute
- Emerson Collective
- Additional Ventures
- National Institutes of Health
- National Heart, Lung, and Blood Institute
- National Institute of Mental Health
- National Institute of Allergy and Infectious Diseases
- National Institute of Diabetes and Digestive and Kidney Diseases
- National Institute of Neurological Disorders and Stroke
- Lawrence Livermore National Laboratory