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

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

doi:10.1038/s41587-024-02437-3

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

Nat Biotechnol. 2025 Sep;43(9):1445-1457. doi: 10.1038/s41587-024-02437-3. Epub 2024 Oct 16.

Authors

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.

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.

MeSH terms

Animals
CRISPR-Associated Protein 9* / genetics
CRISPR-Associated Protein 9* / metabolism
CRISPR-Cas Systems* / genetics
Gene Editing* / methods
Humans
Lipids / chemistry
Liposomes
Liver* / metabolism
Lung* / metabolism
Mice
Nanoparticles* / chemistry
Ribonucleoproteins* / genetics
Ribonucleoproteins* / metabolism

Substances

Ribonucleoproteins
CRISPR-Associated Protein 9
Lipid Nanoparticles
Lipids
Liposomes

Abstract

MeSH terms

Substances

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