. 2024 Oct 16:10.1038/s41587-024-02437-3.

doi: 10.1038/s41587-024-02437-3. Online ahead of print.

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

Kai Chen^#^{1

2}, Hesong Han^#^{2

3}, Sheng Zhao^{2

3}, Bryant Xu^{1

2}, Boyan Yin^{2

3}, Atip Lawanprasert^{2

3}, Marena Trinidad^{1

2

4}, Benjamin W Burgstone^{2

3}, Niren Murthy^{5

6}, Jennifer A Doudna^{7

8

9

10

11

12

13

14}

Affiliations

¹ Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, CA, USA.
² Innovative Genomics Institute, University of California Berkeley, Berkeley, CA, USA.
³ Department of Bioengineering, University of California Berkeley, Berkeley, CA, USA.
⁴ Howard Hughes Medical Institute, University of California Berkeley, Berkeley, CA, USA.
⁵ Innovative Genomics Institute, University of California Berkeley, Berkeley, CA, USA. nmurthy@berkeley.edu.
⁶ Department of Bioengineering, University of California Berkeley, Berkeley, CA, USA. nmurthy@berkeley.edu.
⁷ Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, CA, USA. doudna@berkeley.edu.
⁸ Innovative Genomics Institute, University of California Berkeley, Berkeley, CA, USA. doudna@berkeley.edu.
⁹ Howard Hughes Medical Institute, University of California Berkeley, Berkeley, CA, USA. doudna@berkeley.edu.
¹⁰ Gladstone Institutes, San Francisco, CA, USA. doudna@berkeley.edu.
¹¹ Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA, USA. doudna@berkeley.edu.
¹² Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA. doudna@berkeley.edu.
¹³ Department of Chemistry, University of California Berkeley, Berkeley, CA, USA. doudna@berkeley.edu.
¹⁴ California Institute for Quantitative Biosciences, University of California Berkeley, Berkeley, CA, USA. doudna@berkeley.edu.

^# Contributed equally.

PMID: 39415058
PMCID: PMC12000389 (available on 2026-04-16)
DOI: 10.1038/s41587-024-02437-3

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

Kai Chen et al. Nat Biotechnol. 2024.

. 2024 Oct 16:10.1038/s41587-024-02437-3.

doi: 10.1038/s41587-024-02437-3. Online ahead of print.

Authors

Affiliations

¹ Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, CA, USA.
² Innovative Genomics Institute, University of California Berkeley, Berkeley, CA, USA.
³ Department of Bioengineering, University of California Berkeley, Berkeley, CA, USA.
⁴ Howard Hughes Medical Institute, University of California Berkeley, Berkeley, CA, USA.
⁵ Innovative Genomics Institute, University of California Berkeley, Berkeley, CA, USA. nmurthy@berkeley.edu.
⁶ Department of Bioengineering, University of California Berkeley, Berkeley, CA, USA. nmurthy@berkeley.edu.
⁷ Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, CA, USA. doudna@berkeley.edu.
⁸ Innovative Genomics Institute, University of California Berkeley, Berkeley, CA, USA. doudna@berkeley.edu.
⁹ Howard Hughes Medical Institute, University of California Berkeley, Berkeley, CA, USA. doudna@berkeley.edu.
¹⁰ Gladstone Institutes, San Francisco, CA, USA. doudna@berkeley.edu.
¹¹ Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA, USA. doudna@berkeley.edu.
¹² Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA. doudna@berkeley.edu.
¹³ Department of Chemistry, University of California Berkeley, Berkeley, CA, USA. doudna@berkeley.edu.
¹⁴ California Institute for Quantitative Biosciences, University of California Berkeley, Berkeley, CA, USA. doudna@berkeley.edu.

^# Contributed equally.

PMID: 39415058
PMCID: PMC12000389 (available on 2026-04-16)
DOI: 10.1038/s41587-024-02437-3

Abstract

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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Conflict of interest statement

Competing interests

The Regents of the University of California have patents issued and pending for CRISPR technologies on which K.C., H.H., S.Z., N.M. and J.A.D. are inventors. J.A.D. is a cofounder of Azalea Therapeutics, Caribou Biosciences, Editas Medicine, Evercrisp, Scribe Therapeutics, Intellia Therapeutics and Mammoth Biosciences. J.A.D. is a scientific advisory board member at Evercrisp, Caribou Biosciences, Intellia Therapeutics, Scribe Therapeutics, Mammoth Biosciences, The Column Group and Inari. J.A.D. is also an advisor for Aditum Bio. J.A.D. is chief science advisor to Sixth Street, is a director at Altos, Johnson & Johnson and Tempus and has research projects sponsored by Apple Tree Partners. N.M. and H.H. are founders of Opus Biosciences. The other authors declare no competing interests.

Update of

Lung and liver editing by lipid nanoparticle delivery of a stable CRISPR-Cas9 RNP.
Chen K, Han H, Zhao S, Xu B, Yin B, Trinidad M, Burgstone BW, Murthy N, Doudna JA. Chen K, et al. bioRxiv [Preprint]. 2023 Nov 15:2023.11.15.566339. doi: 10.1101/2023.11.15.566339. bioRxiv. 2023. Update in: Nat Biotechnol. 2024 Oct 16. doi: 10.1038/s41587-024-02437-3. PMID: 38014175 Free PMC article. Updated. Preprint.

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

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

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