Supplementary Figure 4: Systemically delivered AAV9-Cas9-gRNAs genetically modify multiple organs, with editing frequency reflecting viral transduction efficiency.
From: A multifunctional AAV–CRISPR–Cas9 and its host response
(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).
