A multifunctional AAV–CRISPR–Cas9 and its host response

Abstract

CRISPR–Cas9 delivery by adeno-associated virus (AAV) holds promise for gene therapy but faces critical barriers on account of its potential immunogenicity and limited payload capacity. Here, we demonstrate genome engineering in postnatal mice using AAV–split-Cas9, a multifunctional platform customizable for genome editing, transcriptional regulation, and other previously impracticable applications of AAV–CRISPR–Cas9. We identify crucial parameters that impact efficacy and clinical translation of our platform, including viral biodistribution, editing efficiencies in various organs, antigenicity, immunological reactions, and physiological outcomes. These results reveal that AAV–CRISPR–Cas9 evokes host responses with distinct cellular and molecular signatures, but unlike alternative delivery methods, does not induce extensive cellular damage in vivo. Our study provides a foundation for developing effective genome therapeutics.

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Figure 1: Postnatal genome editing with AAV9–Cas9–gRNAs and transcriptional activation with AAV9–Cas9–VPR–gRNAs.
Figure 2: AAV9 and Cas9 evoke host immune responses.
Figure 3: Immunological phenotypes toward AAV–CRISPR–Cas9.

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References

  1. 1

    Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).

    CAS  Article  Google Scholar 

  2. 2

    Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. 3

    Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. 4

    Gao, G. et al. Clades of adeno-associated viruses are widely disseminated in human tissues. J. Virol. 78, 6381–6388 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. 5

    Boutin, S. et al. Prevalence of serum IgG and neutralizing factors against adeno-associated virus (AAV) types 1, 2, 5, 6, 8, and 9 in the healthy population: implications for gene therapy using AAV vectors. Hum. Gene Ther. 21, 704–712 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. 6

    Zincarelli, C., Soltys, S., Rengo, G. & Rabinowitz, J.E. Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection. Mol. Ther. 16, 1073–1080 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. 7

    Ran, F.A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186–191 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. 8

    Nelson, C.E. et al. In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy. Science 351, 403–407 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. 9

    Tabebordbar, M. et al. In vivo gene editing in dystrophic mouse muscle and muscle stem cells. Science 351, 407–411 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. 10

    Long, C. et al. Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy. Science 351, 400–403 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. 11

    Yang, Y. et al. A dual AAV system enables the Cas9-mediated correction of a metabolic liver disease in newborn mice. Nat. Biotechnol. 34, 334–338 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. 12

    Yin, H. et al. Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo. Nat. Biotechnol. 34, 328–333 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. 13

    Senís, E. et al. CRISPR/Cas9-mediated genome engineering: an adeno-associated viral (AAV) vector toolbox. Biotechnol. J. 9, 1402–1412 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. 14

    Swiech, L. et al. In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9. Nat. Biotechnol. 33, 102–106 (2015).

    Article  CAS  Google Scholar 

  15. 15

    Mays, L.E. & Wilson, J.M. The complex and evolving story of T cell activation to AAV vector-encoded transgene products. Mol. Ther. 19, 16–27 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. 16

    Esvelt, K.M. et al. Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nat. Methods 10, 1116–1121 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. 17

    Zetsche, B. et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163, 759–771 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. 18

    Nishimasu, H. et al. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 156, 935–949 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. 19

    Jinek, M. et al. Structures of Cas9 endonucleases reveal RNA-mediated conformational activation. Science 343, 1247997 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. 20

    Nishimasu, H. et al. Crystal structure of Staphylococcus aureus Cas9. Cell 162, 1113–1126 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. 21

    Hirano, H. et al. Structure and engineering of Francisella novicida Cas9. Cell 164, 950–961 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. 22

    Li, J., Sun, W., Wang, B., Xiao, X. & Liu, X.Q. Protein trans-splicing as a means for viral vector-mediated in vivo gene therapy. Hum. Gene Ther. 19, 958–964 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. 23

    Zetsche, B., Volz, S.E. & Zhang, F. A split-Cas9 architecture for inducible genome editing and transcription modulation. Nat. Biotechnol. 33, 139–142 (2015).

    Article  CAS  Google Scholar 

  24. 24

    Wright, A.V. et al. Rational design of a split-Cas9 enzyme complex. Proc. Natl. Acad. Sci. USA 112, 2984–2989 (2015).

    CAS  Article  Google Scholar 

  25. 25

    Nihongaki, Y., Kawano, F., Nakajima, T. & Sato, M. Photoactivatable CRISPR-Cas9 for optogenetic genome editing. Nat. Biotechnol. 33, 755–760 (2015).

    Article  CAS  Google Scholar 

  26. 26

    Truong, D.J. et al. Development of an intein-mediated split-Cas9 system for gene therapy. Nucleic Acids Res. 43, 6450–6458 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. 27

    Fine, E.J. et al. Trans-spliced Cas9 allows cleavage of HBB and CCR5 genes in human cells using compact expression cassettes. Sci. Rep. 5, 10777 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  28. 28

    Madisen, L. et al. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat. Neurosci. 13, 133–140 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. 29

    Chavez, A. et al. Highly efficient Cas9-mediated transcriptional programming. Nat. Methods 12, 326–328 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. 30

    Kiani, S. et al. Cas9 gRNA engineering for genome editing, activation and repression. Nat. Methods 12, 1051–1054 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. 31

    Dahlman, J.E. et al. Orthogonal gene knockout and activation with a catalytically active Cas9 nuclease. Nat. Biotechnol. 33, 1159–1161 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. 32

    Konermann, S. et al. Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature 517, 583–588 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. 33

    Kleinstiver, B.P. et al. Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature 523, 481–485 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. 34

    Xu, G.J. et al. Viral immunology. Comprehensive serological profiling of human populations using a synthetic human virome. Science 348, aaa0698 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. 35

    Adachi, K., Enoki, T., Kawano, Y., Veraz, M. & Nakai, H. Drawing a high-resolution functional map of adeno-associated virus capsid by massively parallel sequencing. Nat. Commun. 5, 3075 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. 36

    Altboum, Z. et al. Digital cell quantification identifies global immune cell dynamics during influenza infection. Mol. Syst. Biol. 10, 720 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. 37

    Pipkin, M.E. et al. Interleukin-2 and inflammation induce distinct transcriptional programs that promote the differentiation of effector cytolytic T cells. Immunity 32, 79–90 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. 38

    Wang, D. et al. Adenovirus-mediated somatic genome editing of Pten by CRISPR/Cas9 in mouse liver in spite of Cas9-specific immune responses. Hum. Gene Ther. 26, 432–442 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. 39

    Zajac, A.J. et al. Viral immune evasion due to persistence of activated T cells without effector function. J. Exp. Med. 188, 2205–2213 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. 40

    Curtsinger, J.M., Lins, D.C. & Mescher, M.F. Signal 3 determines tolerance versus full activation of naive CD8 T cells: dissociating proliferation and development of effector function. J. Exp. Med. 197, 1141–1151 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. 41

    Mendell, J.R. et al. Dystrophin immunity in Duchenne muscular dystrophy. N. Engl. J. Med. 363, 1429–1437 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. 42

    Lin, S.W., Hensley, S.E., Tatsis, N., Lasaro, M.O. & Ertl, H.C. Recombinant adeno-associated virus vectors induce functionally impaired transgene product-specific CD8+ T cells in mice. J. Clin. Invest. 117, 3958–3970 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43

    Velazquez, V.M., Bowen, D.G. & Walker, C.M. Silencing of T lymphocytes by antigen-driven programmed death in recombinant adeno-associated virus vector-mediated gene therapy. Blood 113, 538–545 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. 44

    Kay, M.A., He, C.Y. & Chen, Z.Y. A robust system for production of minicircle DNA vectors. Nat. Biotechnol. 28, 1287–1289 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. 45

    Balazs, A.B. et al. Antibody-based protection against HIV infection by vectored immunoprophylaxis. Nature 481, 81–84 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. 46

    Matsuda, T. & Cepko, C.L. Electroporation and RNA interference in the rodent retina in vivo and in vitro. Proc. Natl. Acad. Sci. USA 101, 16–22 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. 47

    Grieger, J.C., Choi, V.W. & Samulski, R.J. Production and characterization of adeno-associated viral vectors. Nat. Protoc. 1, 1412–1428 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. 48

    Zolotukhin, S. et al. Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield. Gene Ther. 6, 973–985 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. 49

    Aurnhammer, C. et al. Universal real-time PCR for the detection and quantification of adeno-associated virus serotype 2-derived inverted terminal repeat sequences. Hum. Gene Ther. Methods 23, 18–28 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. 50

    Aihara, H. & Miyazaki, J. Gene transfer into muscle by electroporation in vivo. Nat. Biotechnol. 16, 867–870 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. 51

    Hsu, P.D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827–832 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. 52

    Mamedov, I.Z. et al. Preparing unbiased T-cell receptor and antibody cDNA libraries for the deep next generation sequencing profiling. Front. Immunol. 4, 456 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. 53

    Shugay, M. et al. Towards error-free profiling of immune repertoires. Nat. Methods 11, 653–655 (2014).

    Article  CAS  Google Scholar 

  54. 54

    Bolotin, D.A. et al. MiXCR: software for comprehensive adaptive immunity profiling. Nat. Methods 12, 380–381 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. 55

    Love, M.I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. 56

    R Development Core Team. A language and environment for statistical computing (R Foundation for Statistical Computing, 2014).

  57. 57

    DiMattia, M.A. et al. Structural insight into the unique properties of adeno-associated virus serotype 9. J. Virol. 86, 6947–6958 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. 58

    Mandell, D.J. et al. Biocontainment of genetically modified organisms by synthetic protein design. Nature 518, 55–60 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. 59

    Trapnell, C. et al. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat. Biotechnol. 28, 511–515 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. 60

    Trapnell, C. et al. Differential analysis of gene regulation at transcript resolution with RNA-seq. Nat. Biotechnol. 31, 46–53 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. 61

    Bindea, G. et al. ClueGO: a Cytoscape plug-in to decipher functionally grouped gene ontology and pathway annotation networks. Bioinformatics 25, 1091–1093 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. 62

    Shannon, P. et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 13, 2498–2504 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. 63

    Carpenter, A.E. et al. CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biol. 7, R100 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank R. Chari, H. Lee, D. Mandell, R. Kalhor, A. Chavez, S. Bryne, S. Shipman, V. Busskamp, K. Esvelt, L. Gu, N. Eroshenko, J. Aach, Y. Mayshar, B. Stranges, B. Bauer, K. Hsu, T.G. Tan, A. Castiglioni, T. Serwold, and L. Vandenberghe for discussions; and J. Goldstein for technical assistance. W.L.C. is supported by the National Science Scholarship from the Agency for Science, Technology and Research (A*STAR), Singapore. M.T. is an Albert J. Ryan fellow. This work was supported in part by grants from the Howard Hughes Medical Institute and NIH (UO1 HL100402 and PN2 EY018244 to A.J.W.; and P50 HG005550 to G.M.C.).

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Contributions

W.L.C., M.T., A.J.W., and G.M.C. conceived of and designed the study and interpreted results. W.L.C. conducted in vitro experiments, viral production, mouse handling, genotyping, qPCR, western blot, TCR-β clonotyping, epitope mapping, fluorescent immunoassay, histology, immunostaining, microscopy, mRNA sequencing, and data analyses. M.T. conducted mouse handling, intramuscular electroporation, single myofiber isolation, FACS and its analysis, ELISA and its analysis, histology, and immunostaining. P.M. assisted in initial in vitro CRISPR–Cas9 optimization. J.K.W.C. and E.Y.W. conducted mouse handling, histology, and immunostaining. A.H.M.N. conducted mRNA sequencing and its analysis. K.Z. conducted mouse handling. W.L.C. wrote the manuscript with input from the other authors. G.M.C. and A.J.W. supervised the project and edited the manuscript.

Corresponding authors

Correspondence to Amy J Wagers or George M Church.

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Competing interests

W.L.C., M.T., A.J.W., and G.M.C. have filed for patent applications regarding in vivo genetic modifications (PCT/US2015/063181 and WO/2016/089866). W.L.C. and G.M.C. have filed for patent applications regarding split-Cas9 (PCT/US2016/012570 and WO/2016/112242) and AAV-CRISPR-Cas9. M.T. is a current employee of Editas Medicine. G.M.C. is a founder of Editas Medicine and has equity in Caribou/Intellia (for full disclosure list, see http://arep.med.harvard.edu/gmc/tech.html).

Integrated supplementary information

Supplementary Figure 1 Split-Cas9 retains full biological activity of full-length Cas9.

(a) SpCas9 (canonical PAM: NGG) broadly targets the human exome and transcriptional start sites (TSS), while orthologs suffer from restrictive PAMs (Sa: NNGRRT; St1: NNAGAAW; Nm: NNNNGATT). Sp* and Sa* denote engineered Cas9 variants and include non-canonical PAMs (see Supplementary Note). (b) Schematic of the split-Cas9 strategy and of plasmids encoding split-Cas9. SMVP = promoter; IntN/IntC = split-inteins; NLS = nuclear localization signal; polyA = SV40 polyadenylation signal; P2A = co-translating linker for ribosomal skipping. (c) Split-Cas9 achieves equivalent editing frequencies as full-length Cas9 (Cas9FL) in C2C12 myoblasts. C2C12 cells were transfected with equal total plasmid amounts (400 ng of total Cas9-encoding plasmids and 400 ng of total gRNAs-encoding plasmids) and with Cas9N:Cas9C at the indicated ratios. Deep-sequencing indicates that mutation frequencies induced by split-Cas9 and Cas9FL were not significantly different across the three targeted genes (one-way ANOVA). Left panels: Cas9 without P2A-turboGFP (n = 3 independent transfections); Right panels: Cas9 with P2A-turboGFP (n = 2 independent transfections). Error bars denote s.e.m. (d) Split-Cas9 targets Ai9 fibroblasts equivalently to Cas9FL, activating tdTomato fluorescence by excision of the 3×Stop terminators cassette. TdTomato+ cells were rarely observed with single-gRNA, or with paired-gRNAs both targeting the same side of 3×Stop (n = 2 independent transfections). Td5 and TdL target 5’ of 3×Stop; Td3 and TdR target 3’ of 3×Stop. Gray, tdTomato. Scale bar, 200 μm.

Supplementary Figure 2 AAV-Cas9-gRNAs direct gene-editing in differentiated myotubes, tail-tip fibroblasts, and spermatogonial cells.

(a) Schematic of AAV-Cas9-gRNAs. ITR = AAV inverted terminal repeat; SMVP and CASI = promoters; IntN/IntC = split-inteins; NLS = nuclear localization signal; polyA = SV40 polyadenylation signal; P2A = co-translating linker for ribosomal skipping. (b) Time course of GFP epifluorescence following transduction of C2C12 myotubes with AAV-Cas9C-P2A-turboGFP. Expression was detected by 1-2 days post-transduction. (c) Both unpurified AAV-Cas9-gRNAs-containing lysates (100 μl per well) or 1E10 (vg, vector genomes) of chloroform-ammonium sulfate purified AAV-Cas9-gRNAs edited the targeted endogenous loci in differentiated C2C12 myotubes. AAV-Cas9N-gRNAs:AAV-Cas9C-P2A-turboGFP ratio of 1:1 was used, with each locus targeted by two adjacent gRNAs. Each dot represents the mutation frequency detected per transduction per condition (one-tailed Wilcoxon rank-sum against no-gRNA controls, Bonferroni corrected). (d) Dose-dependency of AAV-Cas9-gRNAs in C2C12 myotubes. At each functional Cas9N:Cas9C, mutation frequency increased with AAV dose, but began to plateau at ~6% (n.s., not significant between 1E11 and 1E12) (one-way ANOVA, followed by Holm-Šídák test). (e) Transduction of Ai9 tail-tip fibroblasts with 1E12 (total vg) of AAV-Cas9-gRNAs targeting the 3×Stop cassette induced excision-dependent fluorescence activation (n = 2 transductions). gRNA pairs and AAV-Cas9N-gRNAs:AAV-Cas9C-P2A-turboGFP ratios are indicated. Td5 and TdL target 5’ of 3×Stop; Td3 and TdR target 3’ of 3×Stop. TdTomato was not observed in negative controls transduced with 6.7E11 (total vg) of AAV-Cas9C-P2A-turboGFP only. Images were taken 7 days post-transduction. (f) AAV-Cas9-gRNAs edited the Mstn gene in GC-1 spermatogonial cells (Cas9N:Cas9C, 1:1) (n-way ANOVA, followed by Holm-Šídák test). Error bars denote s.e.m. Scale bars, 500 μm.

Supplementary Figure 3 Paired Cas9FL-gRNAs excise intervening genomic sequences in DNA-electroporated skeletal muscles.

(a) Schematic of gRNAs targeting the Mstn and Acvr2b loci in vivo. DNA vectors encoding Cas9FL, gRNA pairs (targeting adjacent sites), and GFP were co-electroporated into the tibialis anterior (TA) muscle of adult mice. Deep-sequencing of isolated single GFP+ myofibers indicated that Cas9FL-gRNAs modified 0.21-6.6% of Mstn and 0.18-7.6% of Acvr2b alleles in these multi-nucleated cells, with frequent precise genomic excision delimited by the two predicted cut-sites (2-4 bp 5’ from the PAM3,7,64,65). Each bar depicts data from a single myofiber, colored according to fractions of mutation types. pSp, plasmid-SpCas9; MCSp, minicircle-SpCas9; pSpPG, plasmid-SpCas9-P2A-turboGFP; Horizontal dashed lines, sequencing error rate. n denotes mice injected. Representative deep-sequencing alignments are shown with dotted vertical lines demarcating Cas9 cut-sites. The fractions of sequencing reads harbouring precise excisions from each myofiber are shown in the histograms with grey bars. (b) Schematic of the Ai9 allele and in situ detection of gene-edited cells with the excision-dependent Ai9 reporter mice. (c) Transverse muscle sections from mice electroporated with Cas9FL-gRNAs targeting the 3×Stop cassette (n = 4 mice), Cas9FL-only no-gRNA control (n = 4 mice), or Cre (n = 3 mice). TdTomato+ cells were induced by Cas9FL-gRNAs or Cre, and not by Cas9FL only (no-gRNA). All conditions included 15 μg of co-electroporated pCAG-GFP to demarcate transduced myofibers. Red = tdTomato; Green = GFP; Blue = DAPI. Each image comprises 4 × 4 tiles. Scale bar, 500 μm. (d) TdTomato intensity correlates with GFP intensity in GFP+ myofibers of muscles electroporated with DNA encoding Cas9FL-gRNAs and GFP (n = 4 mice), or Cre and GFP (n = 3 mice). Dots depict individual transduced myofibers, color-coded to each mouse; all transduced myofibers within the transverse sections were quantified.3 Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816-821, doi:10.1126/science.1225829 (2012).7 Ran, F. A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186-191, doi:10.1038/nature14299 (2015).64 Maddalo, D. et al. In vivo engineering of oncogenic chromosomal rearrangements with the CRISPR/Cas9 system. Nature 516, 423-427, doi:10.1038/nature13902 (2014).65 Xu, L. et al. CRISPR-mediated genome editing restores dystrophin expression and function in mdx mice. Mol Ther, doi:10.1038/mt.2015.192 (2015).

Supplementary Figure 4 Systemically delivered AAV9-Cas9-gRNAs genetically modify multiple organs, with editing frequency reflecting viral transduction efficiency.

(a) Deep-sequencing of tissues indicates mean Mstn gene-targeting rates ranging from 7.8% to 0.25% (n = 4 mice, 4E12 vg of AAV9-Cas9-gRNAsM3+M4) (*, P < 0.05, Wilcoxon rank-sum, Bonferroni corrected). Error bars denote s.e.m. Black dashed line denotes sequencing error. (b) Predicted off-target sites were assessed by deep-sequencing. The bona fide off-target locus (chr16:+3906202) contains two mismatches (in red) compared to the on-target sequence. (n = 4 mice, 4E12 vg of AAV9-Cas9-gRNAsM3+M4; and n = 2 control mice, 4E12 vg of AAV9-Cas9-gRNAsTdL+TdR for determination of baseline sequencing error rates). (c) Triple-AAV9s co-transduce to generate double edits on the same chromatin, as assessed by deep-sequencing (n = 4 mice, each co-injected with 2E12 vg of AAV9-Cas9C-P2A-turboGFP, 1E12 vg of AAV9-Cas9N-gRNAM3, and 1E12 vg of AAV9-Cas9N-gRNAM4). Mutation types are classified as: M3 or M4, single-site edits; M3+M4, double-site edits; Precise excision, subset of M3+M4 with deletions delimited by the Cas9-gRNAs cut-sites. (d) AAV9-Cas9-gRNAs preferentially transduce the liver, heart and skeletal muscle (gastrocnemius and diaphragm) (***, P < 0.001; Wilcoxon rank-sum, Bonferroni corrected) (n = 7 mice, 4E12 vg). Red, means ± s.e.m.; black dashed line with gray box, qPCR false positive rate (2.5 vg/dg) with s.d. (e) Transduction efficiency with 5E11 vg of AAV9-Cas9-gRNAs (**, P < 0.01; ***, P < 0.001; Wilcoxon rank-sum, Bonferroni corrected) (n = 9 mice). (f) Correlation of gene-targeting rates with vg/dg is maintained at lower dosage (n = 2 mice, 5E11 vg of AAV9-Cas9-gRNAsM3+M4). Data from mice injected with 4E12 vg of AAV9-Cas9-gRNAsM3+M4, as shown in Figure 1b, is reproduced here for comparison. (g) Recombinase-activated tdTomato fluorescence by AAV9-GFP-Cre (n = 2 mice per condition, 2.5E11 vg). Mean vg/dg shown. All examined cells within the liver, heart and muscle recombined, indicating ~100% transduction efficiency within these organs. Within the testis, absence of tdTomato+ cells in the germline-residing seminiferous tubules argues against AAV9 transmission to the male germline. (h) Dual-AAV9s co-transduce multiple organs (n = 2 mice per condition, 2E12 vg each of AAV9-GFP and AAV9-mCherry).

Supplementary Figure 5 Whole-mount epifluorescence images from neonatal mice injected intraperitoneally with AAV9-Cas9-gRNAs (5E11 vg) targeting the 3×Stop cassette and controls.

Numerous tdTomato+ cells were observed in mice injected with AAV9-Cas9-gRNAs targeting the genomic 3×Stop cassette (Td5+Td3 and TdL+TdR), but not in negative control vehicle-injected mice, indicating that fluorescence activation resulted from 3×Stop excision. TdTomato+ cells were also observed, at low frequencies, in mice injected with AAV9s encoding two gRNAs both targeting one side of the 3×Stop cassette (AAV9-Cas9-gRNAsTd5+TdL or AAV9-Cas9-gRNAsTd3+TdR), suggesting the rare introduction of large deletions that removed the 3×Stop terminators. All injected mice are shown. Gray, tdTomato; scale bar, 5 mm.

Supplementary Figure 6 Tissue sections from mice injected with AAV9-Cas9-gRNAs.

AAV9-Cas9-gRNAsTdL+TdR (n = 3 mice injected with 4E12 vg) transduced multiple organs, excising the 3×Stop cassette from the Ai9 genomic locus, as indicated by tdTomato activation in (a) liver, (b) heart, and (c) skeletal muscle. TdTomato+ cells were not detected in control mice injected with AAV9-Cas9-gRNAsM3+M4 (n = 4 mice injected with 4E12 vg). Scale bars, 500 μm.

Supplementary Figure 7 Transcriptional activation with AAV-Cas9-VPR-gRNAs.

(a) AAV-Cas9-VPR-gRNAs (cyan) exhibit reduced endonucleolytic activity on the Mstn gene in GC-1 spermatogonial cells compared to AAV-Cas9-gRNAs (black). Data for AAV-Cas9-gRNAs is reproduced from Supplementary Figure 2f for comparison, and included in statistical tests. (b) AAV-Cas9-VPR-gRNAs upregulated target genes in GC-1 spermatogonial cells, as determined by qRT-PCR. gRNA 1 and gRNA 2 are on-target gRNAs for the indicated genes, non-target gRNAs consist of all other gRNAs used in the experiments. (c) AAV-Cas9-VPR-gRNAs upregulated target genes in C2C12 myotubes. (d) Postnatal exposure to AAV9-Cas9-VPR-gRNAs results in global transcriptome perturbations alongside target gene activation (n = 3 mice per group, FDR = 0.05). Top MA-plot depicts differential expression between muscles co-injected with AAV9-Cas9-VPR-gRNAs targeting Mstn, Fst, Pd-l1, and Cd47 (gRNAsset 1, 4E12 vg) and AAV9-turboRFP (1E11 vg) versus muscles injected with AAV9-turboRFP only (group R, 1E11 vg). Middle MA-plot depicts differential expression between muscles injected with AAV9-Cas9-VPR-gRNAs targeting Mstn and Fst (gRNAsset 2, 4E12 vg) and AAV9-turboRFP (1E11 vg) versus muscles injected with AAV9-turboRFP only (group R, 1E11 vg). Bottom MA-plot depicts differential expression comparing AAV9-Cas9-VPR-gRNAsset 1 against AAV9-Cas9-VPR-gRNAsset 2, both at 4E12 vg and co-injected with 1E11 vg of AAV9-turboRFP. FPKM values for Mstn, Fst, Pd-l1, and Cd47 from samples: AAV9-turboRFP only (R), AAV9-Cas9-VPR-gRNAsset 2 (2), and AAV9-Cas9-VPR-gRNAsset 1 (1). *, q < 0.05; **, q < 0.01; FDR = 0.05. (e) AAV9-Cas9-VPR-gRNAs activated the target Pd-l1 and Cd47 genes in adult skeletal muscles, as assessed by qRT-PCR and calculated as 2-∆∆Ct (n = 3 mice per group). Fold-change in gene expression was quantified between AAV9-Cas9-VPR-gRNAs-treated samples that differed only in the gRNA spacer sequences (one-tailed t-test). Samples treated with AAV9-Cas9-VPR-gRNAs and AAV9-turboRFP showed transcriptional alterations against samples treated with AAV9-turboRFP only, due to immunity-associated transcriptome perturbation. For panels a to c, *, P < 0.05; ***, P < 0.001; n-way ANOVA followed by Holm-Šídák test. AAV-Cas9N-gRNAs:AAV-Cas9C-VPR ratio of 1:1 was used in all experiments. Error bars denote s.e.m.

Supplementary Figure 8 Differential expression of immune-related genes following AAV9-Cas9-VPR-gRNAs treatment.

Genes differentially expressed (q < 0.05, FDR = 0.05) following treatment are enriched for immunological gene ontology (GO) terms (n = 3 mice injected with 4E12 vg of AAV9-Cas9-VPR-gRNAsset 2 and 1E11 vg of AAV9-turboRFP compared to n = 3 mice injected with 1E11 vg of AAV9-turboRFP only). Nodes denote GO terms, edges denote interactions. Sizes of nodes are scaled according to GO-level q-values, while color intensities are scaled according to the percentage of genes differentially expressed within each GO term. Parent GO terms are colored and in bold, while child GO terms are in gray.

Supplementary Figure 9 Enrichment of immune cells in Cas9-expressing muscles.

(a) Full Western blot image corresponding to Figure 2a. (b) Cas9 induces lymphocyte infiltration in both the draining lymph nodes and Cas9-expressing muscles (n = 4 mice per condition) (*, P < 0.05; ***, P < 0.001; n-way ANOVA). Checkmarks denote injected vectors and conditions. (c) CD45+ immunostaining is enriched around transgene-expressing myofibers. Mice were electroporated with minicircle-Cas9FL and pCAG-GFP. Part of a histological section is shown to depict quantification method. Mean CD45 fluorescence intensity on the edge of myofibers was calculated for GFP+ myofibers, 1° and 2° neighboring myofibers, and distal (> 2°) myofibers, followed by normalization to the mean intensity around distal (> 2°) myofibers within the same section (n = 4 mice, 2 sections from each). (*, P < 0.001, Wilcoxon rank-sum against distal myofibers, Bonferroni corrected). Black lines, means. (d) Schematic of FACS gating for immune cell surface markers. (e) Fractions of immune cell types within all live cells in injected muscles as assessed by FACS (n = 4 mice per condition). Myeloid and T-cell fractions increased in Cas9-treated muscles (*, P < 0.05, **, P < 0.01; ***, P < 0.001; n.s., not significant; one-way ANOVA, followed by Dunnett’s test against vehicle-injected muscles). (f) AAV9-Cas9-VPR-gRNAs elicit immune cell infiltration/expansion irrespective of gRNAs employed (n = 3 mice per condition). gRNA set 1 targets Mstn, Fst, Pd-l1, and Cd47, while gRNA set 2 targets Mstn and Fst, all at 4E12 vg of 1:1 AAV9-Cas9N-gRNAs:AAV9-Cas9C-VPR and 1E11 vg of AAV9-turboRFP (*, P < 0.05; n.s., not significant; one-way ANOVA, followed by Dunnett’s test against AAV9-turboRFP-injected muscles). Error bars denote s.e.m.

Supplementary Figure 10 Additional data for epitope-mapping and recoding of AAV9-CRISPR-Cas9.

(a) ELISA indicate Cas9-specific IgG1 antibodies elicited by Cas9-exposure (n = 4 mice per condition). (b) Mapped epitopes for monoclonal (mAb) and polyclonal (pAb) Cas9-specific antibodies titrated at 200, 20, and 2 μg ml-1. P-values from Wald test, Benjamini-Hochberg adjusted for FDR = 0.1. (c) Structural representation of mapped epitopes from Cas9-exposed animals. Immunodominant epitopes reside in the REC1 and PI domains of Cas9 (PDB ID: 4CMP19) (n = 4 electroporated and n = 4 AAV9-delivered). Red, immunodominant epitopes; Black, private epitopes; Cyan, REC1 domain; Pink, PI domain. (d) Known functional variants of Cas919,33 can be combined to recode identified epitopes. Recoded Cas9 retains endonucleolytic function, whereas deletion of the epitope (Δ1126-1135) abolishes Cas9 activity. Ai9 fibroblasts were lipofected with wild-type or variant Cas9-encoding plasmids, and tdTomato fluorescence was assayed 4 days post-transfection (n = 2 transfections). Scale bar, 500 μm. (e) AAV9-specific antibodies were elicited by two weeks post-injection, as determined by fluorescent immunoassay (FIAX). Two groups of mice injected with 4E12 vg AAV9-Cas9-VPR-gRNAs are shown, differing only in the gRNA spacers employed (n = 3 mice per condition) (**, P < 0.01; one-way ANOVA, followed by Dunnett’s test against vehicle-injected mice). (f) Mapped AAV9 epitopes reside predominantly on the capsid surface. Red bar, mean. Antigenicity, ranging from 0 to 8, denotes number of animals in which a particular residue is part of a linear epitope. Epitopes are represented on the AAV9 VP3 structure (PDB ID: 3UX157). (g) Capsid residues within identified epitopes preferentially confer loss of viral blood persistency when mutated, suggesting their association with maintaining blood persistency. Each dot represents a double-alanine mutated AAV9 capsid variant, plotted according to its measured blood persistency35 and antigenicity of the residue (this study). Red bar, mean. (h) Capsid residues within identified epitopes preferentially de-targets the liver when mutated, suggesting their association with hepatotropism. Each dot represents a double-alanine mutated AAV9 capsid variant, plotted according to its measured tropism35 and antigenicity of the residue (this study). Blue bar, mean liver transduction efficiency; Magenta bar, mean global transduction efficiency, excluding the liver. Antigenicity, ranging from 0 to 8, denotes number of animals in which a particular residue is part of a linear epitope.19 Jinek, M. et al. Structures of Cas9 endonucleases reveal RNA-mediated conformational activation. Science 343, 1247997, doi:10.1126/science.1247997 (2014).33 Kleinstiver, B. P. et al. Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature 523, 481-485, doi:10.1038/nature14592 (2015).35 Adachi, K., Enoki, T., Kawano, Y., Veraz, M. & Nakai, H. Drawing a high-resolution functional map of adeno-associated virus capsid by massively parallel sequencing. Nat Commun 5, 3075, doi:10.1038/ncomms4075 (2014).57 DiMattia, M. A. et al. Structural insight into the unique properties of adeno-associated virus serotype 9. Journal of virology 86, 6947-6958, doi:10.1128/JVI.07232-11 (2012).

Supplementary Figure 11 Deconvoluting admixture transcriptomes into immunological cell types.

(a) Deconvoluted hematopoietic lineage tree from draining lymph node samples (n = 3 mice injected with 4E12 vg of AAV9-Cas9-VPR-gRNAsset 2 and 1E11 vg of AAV9-turboRFP compared to n = 3 mice injected with 1E11 vg of AAV9-turboRFP only). Node sizes scale with gene signature fold-differences, and are labelled according to the ImmGen annotation. (b) DCQ recalls gene signatures from the > 200 ImmGen reference immune cell transcriptomes.

Supplementary Figure 12 IL-2 and perforin protein levels were unremarkable in muscles injected with AAV9-Cas9-VPR-gRNAs.

Mice were targeted with gRNAs set 1 (7 gRNAs against Mstn, Fst, Pd-l1 and Cd47) or with gRNAs set 2 (3 gRNAs against Mstn and Fst), all at 4E12 vg total of 1:1 AAV9-Cas9N-gRNAs:AAV9-Cas9C-VPR (n = 3 mice per condition, including data presented in Figure 3c). All injections include 1E11 vg of AAV9-turboRFP to demarcate transduction. Scale bar, 500 μm.

Supplementary Figure 13 IL-2 and perforin protein levels were elevated in muscles electroporated with Cas9-encoding DNA.

Immunosuppression by FK506 reduced IL-2 and perforin levels (n = 3 mice per condition). Scale bar, 500 μm.

Supplementary Figure 14 Immunosuppression by FK506 administration reduces the host immune response.

(a) Cellular damage that causes myofiber degeneration and repair typically results in centrally nucleated myofibers under histological examination. Part of a histological section is shown to depict the quantification method. Delivery of minicircle-Cas9FL or pCAG-GFP via DNA electroporation induced an increase in the fraction of centrally nucleated myofibers, compared to controls electroporated with vehicle only (n = 4 mice per condition) (*, P < 0.05; **, P < 0.01; ***, P < 0.001; one-way ANOVA, followed by Tukey-Kramer test). FK506 reduced but did not fully mitigate the elevated fraction of centrally nucleated myofibers. (b) FK506 reduces CD45+ immune cell infiltration in muscles electroporated with minicircle-Cas9FL and/or pCAG-GFP as assessed by immunofluorescence. Gray lines, histograms of CD45 fluorescence intensity around each myofiber per muscle histological section; black solid lines, mean distributions of histograms (n = 4 mice per condition, 2 sections per mouse). (c) FK506 reduces immune cell infiltration in transgene-expressing muscles as assessed by FACS (n = 4 mice per condition) (*, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., not significant; one-way ANOVA, followed by Dunnett’s test against uninjected muscles). Checkmarks denote injected vectors and conditions. (d) FK506 reduces the elevated intramuscular IgG and IgM antibody levels induced by electroporation of vectors expressing Cas9 and/or GFP (n = 4 mice per condition). Scale bar, 200 μm. (e) FK506-treated mice show significantly lower body weights compared to vehicle-injected mice, manifesting signs of expected adverse reactions towards broad-spectrum immunosuppression (n = 3 mice per condition) (one-tailed Welch’s t-test, assuming unequal variances). Red lines, means.

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Supplementary Figures 1–14, Supplementary Table 4, Supplementary Note and Supplementary Sequences. (PDF 3274 kb)

Supplementary Table 1

Total-mRNA sequencing dataset. (XLS 37889 kb)

Supplementary Table 2

Epitope mapping dataset. (XLS 1824 kb)

Supplementary Table 3

Admixture transcriptomes deconvolution dataset. (XLS 39 kb)

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Chew, W., Tabebordbar, M., Cheng, J. et al. A multifunctional AAV–CRISPR–Cas9 and its host response. Nat Methods 13, 868–874 (2016). https://doi.org/10.1038/nmeth.3993

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