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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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.).
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
(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.
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.
(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.
(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.
(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).
(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.
(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.
Supplementary Figures 1–14, Supplementary Table 4, Supplementary Note and Supplementary Sequences. (PDF 3274 kb)
Total-mRNA sequencing dataset. (XLS 37889 kb)
Epitope mapping dataset. (XLS 1824 kb)
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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