Authors: Kai Chen, Hesong Han, Sheng Zhao, Bryant Xu, Boyan Yin, Atip Lawanprasert, Marena Trinidad, Benjamin W. Burgstone, Niren Murthy, and Jennifer A. Doudna
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.
Fig. iGeoCas9 RNP–LNPs efficiently edit the liver and lungs of mice. (A) Schematic diagram of the experimental design used to evaluate iGeoCas9 RNP–LNP-mediated editing in Ai9 mice. (B) Schematic presentation of LNP preparation procedures. (C) The modified FX12 LNP formulation (FX12m, with lipid compositions indicated in the table) primarily edits the liver tissue with 37% efficiency. In vivo genome-editing levels in different tissues and different cell types in the liver were quantified by tdTom(+) signals using flow cytometry. (D) The modified FC8 LNP formulation (FC8m, with lipid compositions indicated in the table) primarily edits the lung tissue with 16% efficiency. In vivo genome editing levels in different tissues and different cell types in the lungs were quantified by tdTom(+) signals using flow cytometry. For c and d, n = 5 for each group; data are presented as mean values with individual data points and the s.d.; IVT sgRNA, tdTom-g3(23), was used. (E, F) Nuclear staining with DAPI (blue) and imaging of tdTomato (red) in the edited and nonedited liver (E) or lung (F) tissues. Editing signals were observed with the tissues from experimental mice (n = 5). RFP, red fluorescent protein. (G) sgRNA target designs for PCSK9 and SFTPC gene editing with iGeoCas9 in the liver and lungs, respectively. (H, I) In vivo PCSK9 and SFTPC gene-editing levels (indels) in the liver and lung tissues using FX12m and FC8m LNP formulations, respectively, as quantified by NGS (n = 5 for each group); data are presented as mean values with individual data points and the s.d.; PBS-only injections are included as negative controls and the indels in the liver (FX12m) or in the lungs (FC8m) are shown as the blank editing levels. iGeoCas9 used in this figure is 2NLS-iGeoCas9(C1)-2NLS. Neg ctrl, negative control.
Selected Figure
Keywords: lipid nanoparticles; CRISPR-Cas9; iGeoCas9; gene therapy; Flex-M
Nat Biotechnol 2024