Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo

Journal name:
Nature Biotechnology
Volume:
34,
Pages:
328–333
Year published:
DOI:
doi:10.1038/nbt.3471
Received
Accepted
Published online

The combination of Cas9, guide RNA and repair template DNA can induce precise gene editing and the correction of genetic diseases in adult mammals. However, clinical implementation of this technology requires safe and effective delivery of all of these components into the nuclei of the target tissue. Here, we combine lipid nanoparticle–mediated delivery of Cas9 mRNA with adeno-associated viruses encoding a sgRNA and a repair template to induce repair of a disease gene in adult animals. We applied our delivery strategy to a mouse model of human hereditary tyrosinemia and show that the treatment generated fumarylacetoacetate hydrolase (Fah)-positive hepatocytes by correcting the causative Fah-splicing mutation. Treatment rescued disease symptoms such as weight loss and liver damage. The efficiency of correction was >6% of hepatocytes after a single application, suggesting potential utility of Cas9-based therapeutic genome editing for a range of diseases.

At a glance

Figures

  1. In vitro delivery of Cas9 mRNA mediates efficient genome editing in cells.
    Figure 1: In vitro delivery of Cas9 mRNA mediates efficient genome editing in cells.

    (a) C12-200 lipid nanoparticle delivery of Cas9 mRNA into cells. 293T cells stably expressing both EF1a promoter-GFP and U6 promoter-GFP targeting sgRNA (sgGFP) were incubated with Cas9 mRNA nanoparticles (nano.Cas9). Cas9-mediated frameshift NHEJ events will result in GFP-negative cells. Red arrowhead indicates the Cas9 cutting site. (b) FACS analysis shows that Cas9 mRNA generates GFP-negative cells. Gate R2 indicates 80% GFP-negative cells after nano.Cas9 treatment (n = 3). (c) GFP locus was deep sequenced in nano.Cas9 treated cells (n = 4). Shown are representative indels. (d) Distribution of indels. (e) Indel phase shows that most indels cause a frameshift. For example, 3N + 1 include 1-, 4- and 7-bp indels, 3N + 2 include 2-, 5- and 8-bp indels, and 3N include 3-, 6- and 9-bp indels. (f,g) Transient Cas9 expression by mRNA delivery can reduce off-target genome editing for a VEGFA sgRNA. 293T cells were co-transfected with Cas9 mRNA and pLKO.sgVEGFA (mRNA). 293T cells infected with lentiviral Cas9 were transfected with pLKO.sgVEGFA alone to represent long-term Cas9 expression (lenti). On-target (TS2) (f) and off-target (OT2-2) (g) indel rate was measured by surveyor assay at 2 d. Arrows denote indel bands. *, nonspecific bands. (h) Relative off-target/on-target ratio. The ratio in lenti.Cas9 was set as 1. *P < 0.01 (n = 3). Error bars, mean ± s.d.

  2. In vivo delivery of Cas9 mRNA and AAV-HDR template cures type I tyrosinemia mice.
    Figure 2: In vivo delivery of Cas9 mRNA and AAV-HDR template cures type I tyrosinemia mice.

    (a) Design of AAV-HDR template and experiments. G->A point mutation at the last nucleotide of exon 8 in Fahmut/mut homozygous mice leads to exon skipping of exon 8. A dual function AAV vector harbors U6-sgRNA and a HDR template (1.7 kb) with the “G” nucleotide to repair the “A” mutation. The “TGG” PAM was modified to “TCC” to prevent self-cleavage. Dashed lines denote homologous recombination. ITR stands for inverted terminal repeat. Black arrows indicate PCR primers for deep sequencing analysis. Fahmut/mut mice were injected with AAV-HDR and nano.Cas9 at indicated time points. Mice were kept off NTBC water at D0. Body weight normalized to pre-injection was monitored over time. (b) Delivery of AAV-HDR and nano.Cas9 fully rescues weight loss upon NTBC withdrawal (n = 3 mice). Error bars, mean ± s.e.m. (c) Liver damage markers (aspartate aminotransferase (AST), alanine aminotransferase (ALT), and bilirubin) were measured in serum *P < 0.01 (n = 3 mice) using one-way ANOVA. Error bars, mean ± s.e.m. (d) Fah+ cells after 30 d off NTBC. Scale bar, 100 μm.

  3. In vivo delivery of Cas9 mRNA and AAV corrects Fah mutation.
    Figure 3: In vivo delivery of Cas9 mRNA and AAV corrects Fah mutation.

    (a) Fahmut/mut mice were kept on NTBC water and euthanized 7 d after nano. Cas9 treatment to estimate initial repair rate. (b) Fah immunohistochemistry (IHC). Scale bars are 200 μm for upper and lower panels, respectively. The lower panel of AAV-HDR + nano.Cas9 is a high-magnification view (box with black dashed line). (c) Fah+ positive cells were counted to determine the percentage. (d) Quantitative RT-PCR measurement of wild-type expression of Fah mRNA. (e) Sequence of repaired Fah mRNA in treated mice. QRT-PCR band of exon 8 to exon 9 spliced mRNA in (d) was sequenced. The corrected G nucleotide is circled. (f) Indels and (g) “G-CC” recombination pattern in total DNA from liver by Illumina sequencing. *P < 0.01 (n = 4 mice) using one-way ANOVA. Error bars, mean ± s.e.m.

  4. Cas9 mRNA nanoparticles characterization.
    Supplementary Fig. 1: Cas9 mRNA nanoparticles characterization.

    (a) nano.Cas9 formulation scheme. Cas9 mRNA was mixed with C12-200, DOPE, Cholesterol, C14PEG2000 and arachidonic acid in a microfluidic chamber. (b) nano.Cas9 structure is characterized by cryo-TEM. Scale bar indicates 100nm. (c) Average diameter of nano.Cas9 was measured by dynamic light scattering. The size of nano.Cas9 (d) and the polydispersity index (PDI) (e) were measured 0, 7, 11 or 18 days after formulation and storage at 4˚C.

  5. The expression of proteins in mouse liver after mRNA nanoparticles treatment.
    Supplementary Fig. 2: The expression of proteins in mouse liver after mRNA nanoparticles treatment.

    (a) C57bl/6 mice were i.v. injected with nanoparticles encapsulated with β-gal (b and c) or Cas9 mRNA (nano.Cas9, d and e), and livers taken. (b) The expression of β-gal protein was measured in liver lysate at 14 hours after injection. (c) The activity of β-gal in liver sections was determined by salmon-gal assay. Scale bar indicates 200 µm. (d) The expression of Cas9 protein was measured in liver lysate 14 hours after injection. 50μg negative control samples mixed with 10, 1 or 0.1ng Cas9 protein served as positive controls. β-actin served as a loading control in (b) and (d). (e) The Cas9 mRNA level in liver was determined by qRT-PCR at 4, 14, and 24 hours after injection (n=3 mice).

  6. Cas9 mRNA nanoparticles are well tolerated.
    Supplementary Fig. 3: Cas9 mRNA nanoparticles are well tolerated.

    C57/Bl6 mice were treated with 2mg/kg nano.Cas9, and histology (a), the levels of liver damage markers (b) and plasma cytokines (c) were determined after 24 hours. Scale bar indicates 50μM. (n=4 mice).

  7. The time course of sgRNA expression in mouse liver.
    Supplementary Fig. 4: The time course of sgRNA expression in mouse liver.

    Mice were injected with AAV-HDR and livers taken at 0, 3, 7 and 14 days after injection. qRT-PCR was performed to determine sgRNA expression in liver. The expression levels were normalized to Day 3 (n = 4 mice).

  8. A PCR approach proves substitution of the correct sequence.
    Supplementary Fig. 5: A PCR approach proves substitution of the correct sequence.

    (a) Design of the PCR primers. Blue arrow indicates the reverse PCR primer, which is outside the repair template. The sequence of the forward primer is presented, and “G” and “CC” in the corrected sequence are highlighted. (b) Genomic DNA of the liver tissue was extracted, and PCR was performed using the primers in (a). The predicted size of PCR product is 1.02kb. A representative sample from each group is shown (n = 3 mice). (c) The PCR product from (b) was cloned to a TA cloning vector and Sanger sequenced. The corrected “G” and “CC” are highlighted.

  9. Viral delivery of Cas9 does not increase HDR rate compared to mRNA delivery.
    Supplementary Fig. 6: Viral delivery of Cas9 does not increase HDR rate compared to mRNA delivery.

    (a) Design of AAV-HDR template. Four “G” point mutations resulting in stabilization of β-Catenin are highlighted. Ad.Cas9 is an adenovirus expressing Cas9. (b) β-Catenin IHC. AAV-HDR-Ctnnb1 alone serves as a control. Arrows denote β-Catenin positive hepatocytes. (c-d) The Ctnnb1 locus in the liver total DNA of Ad.Cas9+ AAV-HDR-Ctnnb1 treated mice (n=2) were deep sequenced to measure indels. (e) β-Catenin positive hepatocytes were counted to determine the percentage of HDR. P < 0.01 (n = 3 mice). mRNA delivery of Cas9 yields higher rate of HDR for Fah (>6%).

  10. Cas9 mRNA delivery has minimal off-target effects at assayed sites in vivo.
    Supplementary Fig. 7: Cas9 mRNA delivery has minimal off-target effects at assayed sites in vivo.

    (a) Top ranking off-target sites (OT1, OT3 and OT4) for sgFah and the predicted score (Hsu et al, 2013). Mismatch bases are in red. Score for the wildtype sgFah.2 targets the mutant Fah which has one mismatch with wildtype Fah (wt Fah). (b) Indel frequency is low and is comparable between control mouse and nano-Cas9+ AAV-HDR mouse. OT1, OT3 and OT4 regions were PCR amplified from mouse liver genomic DNA and analyzed by deep sequencing. (c) Surveyor assay did not detect indels at OT1, OT3 and OT4. Predicted size of uncut and cut bands are indicated.

  11. Indel rate measured by deep sequencing for GUIDE-Seq off-target sites.
    Supplementary Fig. 8: Indel rate measured by deep sequencing for GUIDE-Seq off-target sites.

    OT1 is the strongest off-target sites identified by GUIDE-Seq. GOT1-11 are additional genomic sites that displayed GUIDE-Seq oligonucleotide insertions. (a) Mouse Hepa1-6 liver cells transfected with pX330.sgFah.2. #1 and #2 are replicates. (b) Mouse livers treated with nano.Cas9 and AAV-HDR (treated) or control treated (control). Fah is the on-target site. See Table S9 for details.

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References

  1. Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819823 (2013).
  2. Doudna, J.A. & Charpentier, E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 346, 1258096 (2014).
  3. Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823826 (2013).
  4. Mali, P., Esvelt, K.M. & Church, G.M. Cas9 as a versatile tool for engineering biology. Nat. Methods 10, 957963 (2013).
  5. Sander, J.D. & Joung, J.K. CRISPR-Cas systems for editing, regulating and targeting genomes. Nat. Biotechnol. 32, 347355 (2014).
  6. Aponte, J.L. et al. Point mutations in the murine fumarylacetoacetate hydrolase gene: animal models for the human genetic disorder hereditary tyrosinemia type 1. Proc. Natl. Acad. Sci. USA 98, 641645 (2001).
  7. Azuma, H. et al. Robust expansion of human hepatocytes in Fah-/-/Rag2-/-/Il2rg-/- mice. Nat. Biotechnol. 25, 903910 (2007).
  8. Paulk, N.K. et al. Adeno-associated virus gene repair corrects a mouse model of hereditary tyrosinemia in vivo. Hepatology 51, 12001208 (2010).
  9. Schwank, G. et al. Functional repair of CFTR by CRISPR-Cas9 in intestinal stem cell organoids of cystic fibrosis patients. Cell Stem Cell 13, 653658 (2013).
  10. Wu, Y. et al. Correction of a genetic disease in mouse via use of CRISPR-Cas9. Cell Stem Cell 13, 659662 (2013).
  11. Long, C. et al. Prevention of muscular dystrophy in mice by CRISPR-Cas9-mediated editing of germline DNA. Science 345, 11841188 (2014).
  12. Ran, F.A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186191 (2015).
  13. Swiech, L. et al. In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9. Nat. Biotechnol. 33, 102106 (2015).
  14. Zuris, J.A. et al. Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat. Biotechnol. 33, 7380 (2015).
  15. Chu, V.T., Weber, T. & Wefers, B. Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat. Biotechnol. 33, 543548 (2015).
  16. Yin, H. et al. Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nat. Biotechnol. 32, 551553 (2014).
  17. Khorsandi, S.E. et al. Minimally invasive and selective hydrodynamic gene therapy of liver segments in the pig and human. Cancer Gene Ther. 15, 225230 (2008).
  18. Kay, M.A. State-of-the-art gene-based therapies: the road ahead. Nat. Rev. Genet. 12, 316328 (2011).
  19. Yin, H. et al. Non-viral vectors for gene-based therapy. Nat. Rev. Genet. 15, 541555 (2014).
  20. Love, K.T. et al. Lipid-like materials for low-dose, in vivo gene silencing. Proc. Natl. Acad. Sci. USA 107, 18641869 (2010).
  21. Semple, S.C. et al. Rational design of cationic lipids for siRNA delivery. Nat. Biotechnol. 28, 172176 (2010).
  22. Chen, D. et al. Rapid discovery of potent siRNA-containing lipid nanoparticles enabled by controlled microfluidic formulation. J. Am. Chem. Soc. 134, 69486951 (2012).
  23. Kormann, M.S. et al. Expression of therapeutic proteins after delivery of chemically modified mRNA in mice. Nat. Biotechnol. 29, 154157 (2011).
  24. Fu, Y. et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat. Biotechnol. 31, 822826 (2013).
  25. Kauffman, K.J. et al. Optimization of lipid nanoparticle formulations for mRNA delivery in vivo with fractional factorial and definitive screening designs. Nano Lett. 15, 73007306 (2015).
  26. Sundararajan, S., Wakamiya, M., Behringer, R.R. & Rivera-Pérez, J.A. A fast and sensitive alternative for β-galactosidase detection in mouse embryos. Development 139, 44844490 (2012).
  27. Kim, H. et al. A co-CRISPR strategy for efficient genome editing in Caenorhabditis elegans. Genetics 197, 10691080 (2014).
  28. Barzel, A. et al. Promoterless gene targeting without nucleases ameliorates haemophilia B in mice. Nature 517, 360364 (2015).
  29. Xue, W. et al. CRISPR-mediated direct mutation of cancer genes in the mouse liver. Nature 514, 380384 (2014).
  30. Tsai, S.Q. et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat. Biotechnol. 33, 187197 (2014).
  31. Mahiny, A.J. et al. In vivo genome editing using nuclease-encoding mRNA corrects SP-B deficiency. Nat. Biotechnol. 33, 584586 (2015).
  32. Hendel, A. et al. Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nat. Biotechnol. 33, 985989 (2015).
  33. Li, H. et al. In vivo genome editing restores haemostasis in a mouse model of haemophilia. Nature 475, 217221 (2011).
  34. Wu, X. et al. Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells. Nat. Biotechnol. 32, 670676 (2014).
  35. Chen, H.Z. et al. Canonical and atypical E2Fs regulate the mammalian endocycle. Nat. Cell Biol. 14, 11921202 (2012).
  36. Elliott, B., Richardson, C., Winderbaum, J., Nickoloff, J.A. & Jasin, M. Gene conversion tracts from double-strand break repair in mammalian cells. Mol. Cell. Biol. 18, 93101 (1998).
  37. Goldberg, A.D. et al. Distinct factors control histone variant H3.3 localization at specific genomic regions. Cell 140, 678691 (2010).
  38. Findlay, G.M., Boyle, E.A., Hause, R.J., Klein, J.C. & Shendure, J. Saturation editing of genomic regions by multiplex homology-directed repair. Nature 513, 120123 (2014).
  39. Cox, D.B., Platt, R.J. & Zhang, F. Therapeutic genome editing: prospects and challenges. Nat. Med. 21, 121131 (2015).
  40. Atabai, K. et al. Mfge8 is critical for mammary gland remodeling during involution. Mol. Biol. Cell 16, 55285537 (2005).
  41. Gilbert, L.A. et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, 442451 (2013).
  42. Hsu, P.D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827832 (2013).
  43. Bolukbasi, M.F. et al. DNA-binding-domain fusions enhance the targeting range and precision of Cas9. Nat. Methods 12, 11501156 (2015).
  44. Zhu, L.J. et al. ChIPpeakAnno: a Bioconductor package to annotate ChIP-seq and ChIP-chip data. BMC Bioinformatics 11, 237 (2010).
  45. Zhu, L. Overview of guide RNA design tools for CRISPR-Cas9 genome editing technology. Front. Biol. 10, 289296 (2015).
  46. Zhu, L.J., Holmes, B.R., Aronin, N. & Brodsky, M.H. CRISPRseek: a bioconductor package to identify target-specific guide RNAs for CRISPR-Cas9 genome-editing systems. PLoS One 9, e108424 (2014).

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Author information

Affiliations

  1. David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Hao Yin,
    • Joseph R Dorkin,
    • Qiongqiong Wu,
    • Junghoon Yang,
    • Sneha Suresh,
    • Stephen Walsh,
    • Roman L Bogorad,
    • Robert Langer &
    • Daniel G Anderson
  2. RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, Massachusetts, USA.

    • Chun-Qing Song,
    • Angela Park,
    • Aizhan Bizhanova &
    • Wen Xue
  3. Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, USA.

    • Chun-Qing Song,
    • Lihua J Zhu &
    • Wen Xue
  4. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Joseph R Dorkin
  5. Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.

    • Lihua J Zhu,
    • Ankit Gupta,
    • Mehmet F Bolukbasi,
    • Scot A Wolfe &
    • Wen Xue
  6. Program in Bioinformatics and Integrative Biology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.

    • Lihua J Zhu &
    • Zhiping Weng
  7. Department of Bioinformatics, School of Life Science and Technology, Tongji University, Shanghai, P.R. China.

    • Yingxiang Li
  8. Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.

    • Mehmet F Bolukbasi &
    • Scot A Wolfe
  9. Gene Therapy Center, University of Massachusetts Medical School, Worcester, Massachusetts, USA.

    • Guangping Gao
  10. College of Pharmacy, the Ohio State University, Columbus, Ohio, USA.

    • Yizhou Dong
  11. Skolkovo Institute of Science and Technology, Skolkovo, Russia.

    • Victor Koteliansky
  12. Department of Chemistry, M.V. Lomonosov Moscow State University, Leninskie Gory, Russia.

    • Victor Koteliansky
  13. Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Robert Langer &
    • Daniel G Anderson
  14. Harvard-MIT Division of Health Sciences & Technology, Cambridge, Massachusetts, USA.

    • Robert Langer &
    • Daniel G Anderson
  15. Institute of Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Robert Langer &
    • Daniel G Anderson

Contributions

H.Y., W.X. and D.G.A. designed the study. H.Y. and W.X. directed the project. H.Y., C.-Q.S., J.R.D., L.J.Z., Y.L., Q.W., J.Y., S.S., A.B., A.G., M.F.B., A.P., S.W. and R.L.B. performed experiments and analyzed data. G.G., Z.W., Y.D., V.K., S.A.W. and R.L. provided reagents and conceptual advice. H.Y., W.X. and D.G.A. wrote the manuscript with comments from all authors.

Competing financial interests

D.G.A., H.Y., J.R.K. and W.X. have applied for patents on the subject matter of this paper. D.G.A. is a scientific co-founder of CRISPR Therapeutics.

Corresponding authors

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Supplementary information

Supplementary Figures

  1. Supplementary Figure 1: Cas9 mRNA nanoparticles characterization. (79 KB)

    (a) nano.Cas9 formulation scheme. Cas9 mRNA was mixed with C12-200, DOPE, Cholesterol, C14PEG2000 and arachidonic acid in a microfluidic chamber. (b) nano.Cas9 structure is characterized by cryo-TEM. Scale bar indicates 100nm. (c) Average diameter of nano.Cas9 was measured by dynamic light scattering. The size of nano.Cas9 (d) and the polydispersity index (PDI) (e) were measured 0, 7, 11 or 18 days after formulation and storage at 4˚C.

  2. Supplementary Figure 2: The expression of proteins in mouse liver after mRNA nanoparticles treatment. (83 KB)

    (a) C57bl/6 mice were i.v. injected with nanoparticles encapsulated with β-gal (b and c) or Cas9 mRNA (nano.Cas9, d and e), and livers taken. (b) The expression of β-gal protein was measured in liver lysate at 14 hours after injection. (c) The activity of β-gal in liver sections was determined by salmon-gal assay. Scale bar indicates 200 µm. (d) The expression of Cas9 protein was measured in liver lysate 14 hours after injection. 50μg negative control samples mixed with 10, 1 or 0.1ng Cas9 protein served as positive controls. β-actin served as a loading control in (b) and (d). (e) The Cas9 mRNA level in liver was determined by qRT-PCR at 4, 14, and 24 hours after injection (n=3 mice).

  3. Supplementary Figure 3: Cas9 mRNA nanoparticles are well tolerated. (125 KB)

    C57/Bl6 mice were treated with 2mg/kg nano.Cas9, and histology (a), the levels of liver damage markers (b) and plasma cytokines (c) were determined after 24 hours. Scale bar indicates 50μM. (n=4 mice).

  4. Supplementary Figure 4: The time course of sgRNA expression in mouse liver. (42 KB)

    Mice were injected with AAV-HDR and livers taken at 0, 3, 7 and 14 days after injection. qRT-PCR was performed to determine sgRNA expression in liver. The expression levels were normalized to Day 3 (n = 4 mice).

  5. Supplementary Figure 5: A PCR approach proves substitution of the correct sequence. (78 KB)

    (a) Design of the PCR primers. Blue arrow indicates the reverse PCR primer, which is outside the repair template. The sequence of the forward primer is presented, and “G” and “CC” in the corrected sequence are highlighted. (b) Genomic DNA of the liver tissue was extracted, and PCR was performed using the primers in (a). The predicted size of PCR product is 1.02kb. A representative sample from each group is shown (n = 3 mice). (c) The PCR product from (b) was cloned to a TA cloning vector and Sanger sequenced. The corrected “G” and “CC” are highlighted.

  6. Supplementary Figure 6: Viral delivery of Cas9 does not increase HDR rate compared to mRNA delivery. (108 KB)

    (a) Design of AAV-HDR template. Four “G” point mutations resulting in stabilization of β-Catenin are highlighted. Ad.Cas9 is an adenovirus expressing Cas9. (b) β-Catenin IHC. AAV-HDR-Ctnnb1 alone serves as a control. Arrows denote β-Catenin positive hepatocytes. (c-d) The Ctnnb1 locus in the liver total DNA of Ad.Cas9+ AAV-HDR-Ctnnb1 treated mice (n=2) were deep sequenced to measure indels. (e) β-Catenin positive hepatocytes were counted to determine the percentage of HDR. P < 0.01 (n = 3 mice). mRNA delivery of Cas9 yields higher rate of HDR for Fah (>6%).

  7. Supplementary Figure 7: Cas9 mRNA delivery has minimal off-target effects at assayed sites in vivo. (63 KB)

    (a) Top ranking off-target sites (OT1, OT3 and OT4) for sgFah and the predicted score (Hsu et al, 2013). Mismatch bases are in red. Score for the wildtype sgFah.2 targets the mutant Fah which has one mismatch with wildtype Fah (wt Fah). (b) Indel frequency is low and is comparable between control mouse and nano-Cas9+ AAV-HDR mouse. OT1, OT3 and OT4 regions were PCR amplified from mouse liver genomic DNA and analyzed by deep sequencing. (c) Surveyor assay did not detect indels at OT1, OT3 and OT4. Predicted size of uncut and cut bands are indicated.

  8. Supplementary Figure 8: Indel rate measured by deep sequencing for GUIDE-Seq off-target sites. (51 KB)

    OT1 is the strongest off-target sites identified by GUIDE-Seq. GOT1-11 are additional genomic sites that displayed GUIDE-Seq oligonucleotide insertions. (a) Mouse Hepa1-6 liver cells transfected with pX330.sgFah.2. #1 and #2 are replicates. (b) Mouse livers treated with nano.Cas9 and AAV-HDR (treated) or control treated (control). Fah is the on-target site. See Table S9 for details.

PDF files

  1. Supplementary Text and Figures (3,988 KB)

    Supplementary Figures 1–8, Supplementary Tables 1–6 and Supplementary Sequences

  2. Supplementary Table 7 (41,297 KB)

    sgRNA2 GUIDE-seq +&- strand peaks

  3. Supplementary Table 8 (77 KB)

    sgRNA2 GUIDE-seq merged peaks

  4. Supplementary Table 9 (196 KB)

    Deep sequencing of off-target sites

Additional data