Systemic nanoparticle delivery of CRISPR-Cas9 ribonucleoproteins for effective tissue specific genome editing

Abstract

CRISPR-Cas9 has emerged as a powerful technology that relies on Cas9/sgRNA ribonucleoprotein complexes (RNPs) to target and edit DNA. However, many therapeutic targets cannot currently be accessed due to the lack of carriers that can deliver RNPs systemically. Here, we report a generalizable methodology that allows engineering of modified lipid nanoparticles to efficiently deliver RNPs into cells and edit tissues including muscle, brain, liver, and lungs. Intravenous injection facilitated tissue-specific, multiplexed editing of six genes in mouse lungs. High carrier potency was leveraged to create organ-specific cancer models in livers and lungs of mice though facile knockout of multiple genes. The developed carriers were also able to deliver RNPs to restore dystrophin expression in DMD mice and significantly decrease serum PCSK9 level in C57BL/6 mice. Application of this generalizable strategy will facilitate broad nanoparticle development for a variety of disease targets amenable to protein delivery and precise gene correction approaches.

Fig. 1. A modular approach was developed to enable systemic nanoparticle delivery of CRISPR-Cas9 RNPs for tissue-specific genome editing.

a Addition of a permanently cationic supplemental component (e.g., DOTAP) into traditional LNP formulations enabled encapsulation and protection of Cas9/sgRNA complexes using neutral buffers during nanoparticle formation. Precise tuning of the DOTAP percentage mediated tissue-specific gene editing. b Size distribution of Cas9/sgLuc RNPs prepared in PBS buffer (pH 7.4) and citrate buffer (pH 4.0). The size increase is likely due to denaturization. c Size distribution of 5A2-DOT-10 encapsulating Cas9/sgLuc RNPs prepared in PBS and citrate buffer. 5A2-DOT-10 prepared without RNPs was used as control. d Size distribution of Cas9/sgRNA RNPs with Cas9/sgLuc molar ratio of 1/1, 1/3, and 1/5. e Size distribution of 5A2-DOT-10 encapsulating Cas9/sgLuc with molar ratio of 1/1, 1/3, and 1/5. f Zeta potential of Cas9/sgRNA RNPs showing decreasing charge. Data are presented as mean ± s.e.m. (n = 3 biologically independent samples). g No significant difference of zeta potential was observed for 5A2-DOT-10 encapsulating Cas9/sgLuc with different molar ratios. Data are presented as mean ± s.e.m. (n = 3 biologically independent samples). h Time-dependent cellular uptake of 5A2-DOT-10 LNPs encapsulating EGFP-fused Cas9/sgRNAs showing cytoplasmic release and gradual entry into the nucleus (n = 3 biologically independent samples). Scale bar: 10 μm. Red arrows show distribution of EGFP-fused Cas9/sgRNAs inside cells. i Inhibition of 5A2-DOT-10 LNP uptake was studied using specific endocytosis inhibitors. Amiloride (AMI): inhibitor of macropinocytosis; chlorpromazine (CMZ): inhibitor of clathrin-mediated endocytosis; Genistein (GEN): inhibitor of caveolae-mediated endocytosis; Methyl-β-cyclodextrin (MβCD): lipid rafts-mediated endocytosis; 4 degree: energy-mediated endocytosis. Data are presented as mean ± s.e.m. (n = 3 biologically independent samples). Source data are in the Source Data file.

Fig. 2. Gene editing occurs quickly and effectively in vitro.

a T7EI cleavage assay of DNA isolated from HeLa-Luc cells treated with various formulations. Highly effective gene editing was mediated by 5A2-DOT-10 delivering Cas9/sgLuc RNPs (1/3 and 1/5). Red arrows indicate cleavage bands. Indels (%) at Luc locus was quantified by Sanger sequencing and TIDE analysis. This experiment was repeated three times independently with similar results. b Fluorescence microscopy images of HeLa-GFP cells after treatment with various formulations (n = 3 biologically independent samples). Scale bar = 100 μm. 5A2-DOT-10 Cas9/sgGFP treatment significantly decreased GFP fluorescence. c Flow cytometry analysis of HeLa-GFP cells after treatment with various formulations. The peak of GFP-positive cells shifted completely to the left only for the 5A2-DOT-10 Cas9/sgGFP group, indicating almost all GFP-positive cells went dark. d Time-dependent GFP fluorescence intensity of HeLa-GFP cells after various treatments (mean ± s.e.m., n = 3 biologically independent samples). Permanent GFP fluorescence loss was observed with 5A2-DOT-10 Cas9/sgGFP treatment, which was supported by Sanger sequencing and TIDE analysis. e 5A2-DOT-10 Cas9/sgGFP LNPs were stored at 4 °C for 2 months. The nanoparticle diameter and PDI was monitored over time (mean ± s.e.m., n = 4 biologically independent samples). f Periodic treatment of HeLa-GFP cells with stored LNPs showed that no activity was lost, indicating long-term LNP and RNP stability (mean ± s.e.m., n = 3 biologically independent samples). g Mean fluorescence intensity (%) of HeLa-GFP cells after treatment with Cas9/sgGFP alone, 5A2-DOT-10, C12-200-DOT-10, MC3-DOT-10, C12-200 LNPs, MC3 LNPs, and Cas9/sgGFP-loaded RNAiMAX (mean ± s.e.m., n = 3 biologically independent samples). The GFP fluorescence significantly decreased after treated with all three DOTAP-modified formulations. TIDE analysis of Sanger sequencing data further confirmed the highest gene editing efficiency was with 5A2-DOT-10 LNPs. h Mean fluorescence intensity (%) of Hela-GFP cells after treatment with 5A2-DOT-10 formulated with Cas9/sgGFP in different buffers. Neutral buffer was required for RNP encapsulation and delivery (mean ± s.e.m., n = 3 biologically independent samples). One-way ANOVA followed by Dunnett’s multiple comparison test was used to determine the significance in g and h. (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). Source data are in the Source Data file.

Fig. 3. Highly efficient multiplexed genome editing was achieved in vivo.

a Schematic illustration shows how delivery of Cas9/sgTOM RNPs activates Td-Tom expression in Td-Tomato transgenic mice. 5A2-DOT-X LNPs were injected into Td-Tom mice locally (via intra-muscle or intra-brain injections) and systemically (via IV injection through tail vein). In vivo imaging of Td-Tom mice after intra-muscle (1 mg kg⁻¹ sgTOM) (b) or intra-brain (0.15 mg kg⁻¹ sgTOM) (d) injection of 5A2-DOT-10 Cas9/sgTOM showed bright red fluorescence in the leg muscle or brain tissue (respectively). Successful CRISPR-Cas gene editing was further confirmed by confocal imaging of c muscle and e brain tissue sections. Scale bar: 20 μm. 5A2-DOT-10 enabled higher gene editing efficiency than positive control RNAiMAX, which has previously been used for local RNP injections. f In vivo imaging of Td-Tom mice after intravenous (IV) injection of 5A2-DOT-X Cas9/sgTOM LNPs with different molar percentages of DOTAP. Td-Tom fluorescence, as a downstream readout of DNA editing, showed that low DOTAP percentages facilitated liver editing while high DOTAP percentages facilitated lung editing (1.5 mg kg⁻¹ sgTOM, IV). g Successful CRISPR-Cas gene editing was further confirmed by confocal imaging. Scale bar: 20 μm. h The T7EI cleavage assay was performed on DNA isolated from liver and lung tissues after systemic IV treatment with 5A2-DOT-5, 5A2-DOT-10, 5A2-DOT-50, and 5A2-DOT-60 encapsulating Cas9/sgPTEN. Red arrows indicate cleavage bands generated. Indels (%) was calculated using next generation sequencing (NGS) of DNA isolated from harvested tissues. i 5A2-DOT-50 LNPs containing pooled sgRNAs for six targets (sgTOM, sgP53, sgPTEN, sgEml4, sgALK, and sgRB1) (5A2-DOT-50-Pool) were administered to td-Tom mice IV at total RNA dose of 2 mg kg⁻¹ (0.33 mg kg⁻¹ each sgRNA). Gene editing at the TOM locus was confirmed by in vivo imaging and j editing of the other five loci was confirmed using the T7EI assay on lung tissues. Indels percentages were measured using Sanger sequencing and TIDE analysis. Red arrows indicate cleavage bands generated. Data of c, e, g, h, and j were repeated three times independently with similar results.

Fig. 4. 5A2-DOT-X LNPs simplify generation of complex mouse models.

a To create an in situ liver-specific cancer model, 5A2-DOT-5 LNPs encapsulating Cas9/sgP53/sgPTEN/sgRB1 RNPs were injected into adult C57BL/6 mice weekly (three injections, 2.5 mg kg⁻¹ total sgRNA, IV, n = 4). After 12, 15, and 20 weeks, mice were sacrificed and livers were collected to analyze tumor generation. b T7EI cleavage results from genomic DNA extracted from livers confirmed gene editing occurred at all three loci. Red arrows indicate cleavage bands. Indel percentages shown under gel images were measured by Sanger sequencing and TIDE analysis. c Representative photograph of a mouse liver containing tumors excised 20 weeks after injection. d H&E and Ki67 staining further confirmed progressive tumor formation. Higher tumor proliferation biomarker Ki67 expression was detected in tumor lesions. Scale bar = 100 μm. e To create an in situ lung-specific cancer model, 5A2-DOT-50 LNPs encapsulating Cas9/sgEml4/sgAlk RNPs were injected into adult C57BL/6 mice once (2 mg kg⁻¹) or twice (1.5 mg kg⁻¹ weekly for 2 weeks) (IV, n = 5). After 10, 16, and 24 weeks, mice were sacrificed and lungs were collected to analyze tumor generation. f T7EI cleavage results from genomic DNA extracted from lungs confirmed gene editing occurred at loci of Eml4 and Alk. Red arrows indicate cleavage bands. Indel percentages shown under gel images were measured by Sanger sequencing and TIDE analysis. g PCR amplicons of Eml4-Alk rearrangements were also detected in all lungs treated with 5A2-DOT-50 LNPs. h Eml4-Alk rearrangements were further confirmed by sub-cloning and DNA sequencing. i H&E and Ki67 staining further confirmed progressive tumor formation. Higher tumor proliferation biomarker Ki67 expression was detected in lung tumor lesions. Scale bar = 100 μm. Data of b, d, f, g, and i were repeated three times independently with similar results.

Fig. 5. 5A2-DOT-X LNPs achieve CRISPR-Cas-based gene editing in therapeutic models.

a To restore dystrophin expression, 5A2-DOT-10 LNPs encapsulating Cas9/sgDMD RNPs were injected into TA muscles of DMD exon 44 deletion mice weekly (three injections, 1 mg kg⁻¹ sgDMD, n = 3). Three weeks after the last injection, TA muscles were collected to detect expression of dystrophin protein. b Immunofluorescence images indicated that 5A2-DOT-10 LNPs treatment successfully corrected dystrophin gene and restored the expression of dystrophin proteins in TA muscles. 5A2-DOT-10 LNPs nanoparticle only treatment was used as negative control (NC). Scale bar = 100 μm. Data was repeated two times independently with similar results. c Western blot analysis further confirmed the expression of dystrophin protein in the 5A2-DOT-10 LNPs encapsulating Cas9/sgDMD RNPs treatment group. 4.2% of dystrophin protein was restored. NC: 5A2-DOT-10 LNPs nanoparticle only; WT wild-type group. d To knockout the PCSK9 gene in mouse liver, 5A2-DOT-5 LNPs encapsulating Cas9/sgPCSK9 RNPs were administered to adult C57BL/6 mice via tail vein injection weekly (three injections, 2.5 mg kg⁻¹ sgPCSK9, IV, n = 3). One week after the last injection, mouse serum and livers were collected for analyses. e The relative PCSK9 level in the serum was significantly decreased in 5A2-DOT-5 LNPs encapsulating Cas9/sgPCSK9 RNPs treatment group, detected using PCSK9 Elisa Kit. Data are presented as mean ± s.e.m. (n = 3 biologically independent animals). One-way ANOVA followed by Dunnett’s multiple comparison test was used to determine the significance of data. (*P < 0.05). f T7EI cleavage results from genomic DNA extracted from mice livers confirmed gene editing occurred at the PCSK9 locus. Red arrows indicate cleavage bands. Indel percentages shown under the gel image were measured by Sanger sequencing and TIDE analysis. Source data are in the Source Data file.