Efficient genome editing with Cas9–sgRNA in vivo has required the use of viral delivery systems, which have limitations for clinical applications. Translational efforts to develop other RNA therapeutics have shown that judicious chemical modification of RNAs can improve therapeutic efficacy by reducing susceptibility to nuclease degradation. Guided by the structure of the Cas9–sgRNA complex, we identify regions of sgRNA that can be modified while maintaining or enhancing genome-editing activity, and we develop an optimal set of chemical modifications for in vivo applications. Using lipid nanoparticle formulations of these enhanced sgRNAs (e-sgRNA) and mRNA encoding Cas9, we show that a single intravenous injection into mice induces >80% editing of Pcsk9 in the liver. Serum Pcsk9 is reduced to undetectable levels, and cholesterol levels are significantly lowered about 35% to 40% in animals. This strategy may enable non-viral, Cas9-based genome editing in the liver in clinical settings.
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We thank T. Jacks, P. Sharp, T. Tammela, Z. Weng, G. Gao, E. Sontheimer and A. Vegas for discussions and for sharing reagents, Y. Li and A. Park for technical assistance, and K. Cormier for histology. This work is supported by grants from the National Institutes of Health (NIH), 5R00CA169512, DP2HL137167 and P01HL131471 (to W.X.). V.K. acknowledges support from the Russian Scientific Fund, Grant number 14-34-00017. H.Y. is supported by Skoltech Center and 5-U54-CA151884-04 (NIH). This work is supported in part by Cancer Center Support (core) grant P30-CA14051 from the NIH. We thank the Swanson Biotechnology Center for technical support.
H.Y., D.G.A. and R.L. have applied for patents related to this study. D.G.A. is a scientific co-founder of CRISPR Therapeutics.
Integrated supplementary information
HEK293 cells stably expressing both EF1α promoter-GFP and EFs promoter-spCas9 were incubated with a GFP targeting sgRNA with various chemical modifications or without modification (unmodified, as Native Strand). a) FACS analysis was performed after 6-7 days. Indels at GFP locus in total DNA from HEK293 cells were determined by b) T7EI assay and c) TIDE analysis. n = 4.
a) HEK293 cells expressing both GFP and spCas9 were incubated with various doses of unmodified, 5'&3' modified and e-sgRNA targeting GFP. TIDE analysis was performed to determine indels at GFP locus. b) HEK293 cells expressing GFP were incubated with Cas9 mRNA and e-sgRNA, or Cas9 protein/e-sgRNA Ribonucleoproteins (RNP). TIDE analysis was performed to determine indels at GFP locus. n = 4.
Supplementary Figure 3 In vivo delivery of chemical modified sgRNAs and Cas9 mRNA induced knockout of targeted gene.
C57BL/6 mice were i.v. injected with two e-sgRNAs targeting Pcsk9 (PCSK9-1 & 2) and Cas9 mRNA encapsulated in lipid nanoparticles (Fig. 4). PCR was performed using two pairs of primers. The size of uncut and deletion bands (red pairs) is indicated. Both primer pairs show similar level of deletion band. Gel images were cropped. See full length gels with molecular weight standard in Supplementary information
Supplementary Figure 4 Determine the genome editing efficiency in hepatocytes and hepatic nonparenchymal cells.
C57BL/6 female mice were injected with lipid nanoparticles encapsulated with two e-sgRNAs targeting Pcsk9 and Cas9 mRNA. Hepatocytes enriched population and hepatic nonparenchymal cells (NPC) were isolated from the liver. Indels at Pcsk9 locus in total DNA from the liver, hepatocytes enriched population and NPC were measured by T7EI assay. Red arrow indicates PCR fragment with deletion, and blue arrow indicates cleaved fragment. n = 3. Gel images were cropped. See full length gels with molecular weight standard in Supplementary information
C57BL/6 female mice were injected with lipid nanoparticles encapsulated with two unmodified, 5'&3'sgRNAs or e-sgRNAs targeting Pcsk9 and Cas9 mRNA. Indels at Pcsk9 locus in total DNA from the liver were measured by T7EI assay. Red arrow indicates PCR fragment with deletion, and blue arrow indicates cleaved fragment. n = 4 for native sgRNA and 5'&3' sgRNA groups, n=3 for e-sgRNA group. Gel images were cropped. See full length gels with molecular weight standard in Supplementary information
Supplementary Figure 6 Determine the activities of unmodified sgRNA targeting Fah in FAHmut/mut mice.
a) The unmodified sgRNA targeting Fumarylacetoacetate hydrolase (Fah) was formulated into LNP (nano.sgRNA) and co-injected with Cas9 mRNA encapsulated into LNP (nano.Cas9). FAHmut/mut mice were kept on NTBC water and euthanized 7 days after treatment to estimate indels rate. b) Indels of total DNA from liver by Illumina sequencing. n = 3.
Mice were injected with lipid nanoparticles encapsulated with two e-sgRNAs targeting Pcsk9 and Cas9 mRNA. a) H&E staining of liver 5 days after injection. b) Body weight before and 10 days after injections. c-e) Serum biomarkers c) 5 days, d) 1 day and e) 18 days after injection. f) H&E staining of liver 1 days and 18 days after injection. g) Serum cytokines and chemokines 1 day after injection. n = 4. Scale bars are 200μm.
Supplementary Figure 8 Determine the activity of genome editing in lung and spleen of mice after CRISPR treatment.
C57BL/6 or FAHmut/mut mice were injected with lipid nanoparticles encapsulated with e-sgRNAs targeting Pcsk9, or Fah (for FAHmut/mut mice) or ROSA26 and Cas9 mRNA. Indels at a) Pcsk9, b) Fah and c) ROSA26 loci in total DNA from the lung and spleen were measured by T7EI assay. n = 3. Gel images were cropped. See full length gels with molecular weight standard in Supplementary information
Supplementary Figure 9 Determine the indels at the Fah sgRNA's off-target site obtained from GUIDE-seq.
FAHmut/mut mice were injected with lipid nanoparticles encapsulated with e-sgRNA targeting Fah and Cas9 mRNA. Indels at off-target site 1 (OT1) concluded from GUIDE-seq were measured by T7EI assay. n = 4. Gel images were cropped. See full length gels with molecular weight standard in Supplementary information
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Yin, H., Song, CQ., Suresh, S. et al. Structure-guided chemical modification of guide RNA enables potent non-viral in vivo genome editing. Nat Biotechnol 35, 1179–1187 (2017). https://doi.org/10.1038/nbt.4005
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