Nature Biotechnology: doi: /nbt Supplementary Figure 1. In vitro validation of OTC sgrnas and donor template.
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1 Supplementary Figure 1 In vitro validation of OTC sgrnas and donor template. (a) In vitro validation of sgrnas targeted to OTC in the MC57G mouse cell line by transient transfection followed by 4-day puromycin enrichment and SURVEYOR nuclease assays. sgrna1 showed the highest efficiency in inducing indels in the targeted loci and was therefore chosen for subsequent studies. Arrows denote SURVEYOR nuclease cleaved fragments of the OTC PCR products. Results were replicated in 2 independent experiments. (b) In vitro validation of OTC donor template. MC57G cells were transiently transfected with a plasmid co-expressing OTC sgrna1, SaCas9, and an AgeI restriction site tagged OTC donor plasmid followed by 4-day puromycin enrichment. RFLP analysis was performed following AgeI digestion to detect HDR in vitro. Co-transfection of the AgeItagged OTC donor template with an SaCas9 plasmid without OTC sgrna1 did not result in detectable HDR. Arrows denote AgeIsensitive cleavage products resulting from HDR. Results were replicated in 2 independent experiments. Indel and HDR frequency were calculated based on band intensities 31.
2 Supplementary Figure 2 Vector dose optimization to improve in vivo gene correction. Postnatal day 2 spf ash pups received temporal vein injection of 5x10 10 GC AAV8.SaCas9 and either 5x10 10 (n=5), 1x10 11 (n=3), or 5x10 11 (n=5) GC of AAV8.sgRNA1.donor vector. Liver samples were collected 3 weeks post vector treatment for analysis. (a) Quantification of gene correction based on the percentage of area on liver sections expressing OTC by immunostaining. (b) Quantification of OTC mrna levels in the liver by RT-qPCR using primers spanning exons 4 5 to amplify wild-type OTC. Mean ± SEM are shown. ** P<0.01, Dunnett s test.
3 Supplementary Figure 3 Time course of gene expression by Western analysis and HDR analysis by RFLP. (a) HDR analysis by RFLP. OTC target region was PCR amplified from the liver genomic DNA isolated from untreated spf ash mice or spf ash mice treated with the dual AAV vectors. Untreated spf ash control samples were collected at 8 weeks of age. Samples from the treated spf ash mice were collected at 1, 3, and 8 weeks (n=3 animals per time point) following neonatal injection of the dual AAV8 vectors. Targeted animals received AAV8.SaCas9 (5x10 10 GC/pup) and AAV8.sgRNA1.donor (5x10 11 GC/pup). Untargeted animals received AAV8.SaCas9 (5x10 10 GC/pup) and AAV8.control.donor (5x10 11 GC/pup). AgeI digestion was performed and estimated HDR percentages are shown. (b) Western blot analysis. Liver lysates were prepared from untreated WT and spf ash mice or spf ash mice treated with the dual AAV vectors for detection of FLAG-SaCas9 and OTC protein.
4 Supplementary Figure 4 Examination of liver toxicity in animals treated with AAV8.CRISPR-SaCas9 dual vectors. (a) Histological analysis on livers harvested 3 and 8 weeks following the dual vector treatment. Scale bar, 100 µm. (b) Liver transaminase levels in untreated spf ash mice (n=9) or 8 weeks following dual vector treatment. Untargeted mice received 5x10 10 GC AAV8.SaCas9 and 5x10 11 of AAV8.control.donor vectors (n=8), while gene-targeted mice received 5x10 10 GC AAV8.SaCas9 and 5x10 11 GC of AAV8.sgRNA1.donor (n=7). Mean ± SEM are shown. There were no statistically significant differences between groups, Dunnett s test.
5 Supplementary Figure 5 Examination of liver toxicity in adult animals treated with AAV8.CRISPR-SaCas9 dual vectors. (a) Histological analysis on livers harvested 3 weeks (low-dose) or 2 weeks (high-dose) following dual vector treatment. Scale bar, 100 µm. (b) Liver transaminase levels in untreated spf ash mice, or 3 weeks following low-dose dual vector treatment, or 2 weeks following high-dose dual vector treatment (n=3 for each group). Low-dose, untargeted mice received 1x10 11 GC AAV8.SaCas9 and 1x10 12 GC of AAV8.control.donor vectors, while low-dose, gene-targeted mice received 1x10 11 GC AAV8.SaCas9 and 1x10 12 GC of AAV8.sgRNA1.donor. High-dose, untargeted mice received 1x10 12 GC AAV8.SaCas9 and 5x10 12 GC of AAV8.control.donor vectors, while high-dose, gene-targeted mice received 1x10 12 GC AAV8.SaCas9 and 5x10 12 GC of AAV8.sgRNA1.donor. Mean ± SEM are shown. Adult animals received high-dose, gene-targeted vectors showed a trend of elevated ALT and AST levels, although not statistically different when compared with other groups (Dunnett s test).
6 Supplementary Figure 6 Comparison of SaCas9 vector DNA and mrna levels in the livers of neonatal treated and adult treated mice. Neonatal spf ash mice received 5x10 10 GC AAV8.SaCas9 and 5x10 11 GC of AAV8.sgRNA1.donor vectors and were sacrificed at 1 (n=5), 3 (n=6), and 8 weeks (n=7) after injection. Low-dose adult spf ash mice received 1x10 11 GC AAV8.SaCas9 and 1x10 12 GC of AAV8.sgRNA1.donor vectors and were sacrificed at 3 weeks (n=3) after injection. High-dose adult spf ash mice received 1x10 12 GC AAV8.SaCas9 and 5x10 12 GC of AAV8.sgRNA1.donor vectors and were sacrificed at 2 weeks (n=3) after injection. (a) Quantification of SaCas9 vector DNA in the liver by qpcr. (b) Quantification of SaCas9 mrna in the liver by RT-qPCR. Mean ± SEM are shown.
7 Supplementary Table 1. Summary of the frequencies of indels, correction of OTC spf ash mutation, and HDR in animals treated with the dual AAV gene-targeting vectors. ID Treatment Time after treatment (week) Indel (%) OTC reads with a G (%) Reads with Perfect HDR (%) Reads with Partial HDR (%) 4001 Neonatal, sgrna Neonatal, sgrna Neonatal, sgrna Neonatal, sgrna Neonatal, sgrna Neonatal, sgrna Adult, untargeted, low dose Adult, sgrna1, low dose Adult, sgrna1, low dose Adult, sgrna1, low dose Adult, untargeted, high dose Adult, sgrna1, high dose Adult, sgrna1, high dose Adult, sgrna1, high dose Untreated spf ash n/a
8 Supplementary Table 2. A full list of simple indels (insertions or deletions) detected by deep sequencing on the OTC target region in the liver of two spf ash mice (#4001 and #4003) 3 weeks after neonatal injection of the sgrna1 dual AAV vectors, and two adult spf ash mice (#648 and #653) 2 weeks after injection of high-dose sgrna1 dual AAV vectors. The rates of complex indels (insertions + deletions) are also indicated. Please see attached Excel file.
9 Supplementary Table 3. Deletion sizes identified by on-target deep sequencing and their distribution in neonatal-treated and adult-treated animals. Size range Neonatal (3w) Adult (high-dose, 2w) #4001 #4003 #648 # bp bp bp bp bp bp bp bp bp bp >100 bp Indel overlap with Exon
10 Supplementary Table 4. Off-target analysis. Potential off-target sequences for sgrna1 identified and scored by Benchling s offtarget analysis. ID Sequence PAM Score Chromosome Strand Position Mismatches On-target Indel (treated) Indel (untreated ctl) sgrna1 TCTCTTTTAAACTAACCCAT CAGAG 100 X TRUE OT1 TATCTTTTCAACTAACCCAA TAGGA FALSE OT2 TTTCTTTTATACTAGCCCAT AGGGG X FALSE OT3 TCTTTTTTTAAATAACCCAT TAGAA FALSE OT4 TGGCATTTAAACTAACCCAA CTGGG FALSE OT5 TCTTCATGAAACTAACCCAT CTGAA FALSE OT6 ATTTTTTTAAACAAACCCAT CTGGA FALSE OT7 GATCTTGTAATCTAACCCAT GTGAA FALSE OT8 CCTTTTATAATCTAACCCAT GTGAA FALSE OT9 TCTGTTTTAAAGTAACCCTT CAGGG FALSE OT10 TTGCTTTTCAACTAACCCAA GTGGG FALSE OT11 AATATTTTAAACTATCCCAT GTGAG FALSE OT12 TTACTTTAAAACTATCCCAT CTGAA X FALSE OT13 ACACTTTTAAAATAACCCAG ATGAA FALSE OT14 AATCTTCTAAACCAACCCAT TGGAG FALSE OT15 TCTTTATTAGACTAACCCAG CTGAG X FALSE OT16 TTTCATTTAACATAACCCAT ATGGA FALSE
11 Supplementary Table 5. Primers and sequences for construction and analysis of the donor template and sgrna plasmids. Name Sequence (5'->3') Note WT sequence GAAAGTCTCACAGACACCGCTCGGTTTGTAAAACTTTTCTTCCTTCCAAAGTTT ATTTCAAACTCTGATGGGTTAGTTTAAAAGAGAAGATG Part of OTC exon 4 and intron 4 spf ash sequence OTC donor sequence for sgrna1 ( bp) OTC sgrna1_fwd OTC sgrna1_rev OTC sgrna2_fwd OTC sgrna2_rev OTC sgrna3_fwd OTC sgrna3_rev HDR-Fwd HDR-Rev OTC_PointM_F OTC_PointM_R GAAAGTCTCACAGACACCGCTCAGTTTGTAAAACTTTTCTTCCTTCCAAAGTTT ATTTCAAACTCTGATGGGTTAGTTTAAAAGAGAAGATG GAAAGTCTCACAGACACCGCTCGGTTTGTAAAACTTTTCTTCCTTCCAAAGTTTACCGGT ATTTCAATTGGTGATGGGTTAGTTTAAAAGAGAAGATG CACCGTCTCTTTTAAACTAACCCAT AAACATGGGTTAGTTTAAAAGAGAC CACCGCACAAGACATTCACTTGGGT AAACACCCAAGTGAATGTCTTGTGC CACCGAAAGTTTATTTCAAACTCTG AAACCAGAGTTTGAAATAAACTTTC TGGAGCAATTCTGCACATGGA CTTACTGAACATGGCAGTTTCCC GGCTATGCTTGGGAATGTCCT GCTACAGAATGAAAGAGAGGCG spf ash G->A mutation PAM mutation; AgeI Restriction site insertion OTC target sequence 1 OTC target sequence 2 OTC target sequence 3 OTC PCR for RFLP analysis OTC PCR for Surveyor assay
12 Supplementary Table 6. PCR primer sequences for detecting potential off-target effects by deep sequencing. Primer Name Sequence (5'->3') Note OTC_OT1F1 OTC_OT1R1 OTC_OT1F2 OTC_OT1R2 OTC_OT2F1 OTC_OT2R1 OTC_OT2F2 OTC_OT2R2 OTC_OT3F1 OTC_OT3R1 OTC_OT3F2 OTC_OT3R2 OTC_OT4F1 OTC_OT4R1 OTC_OT4F2 OTC_OT4R2 OTC_OT5F1 OTC_OT5R1 OTC_OT5F2 OTC_OT5R2 OTC_OT6F1 OTC_OT6R1 OTC_OT6F2 OTC_OT6R2 OTC_OT7F1 OTC_OT7R1 OTC_OT7F2 OTC_OT7R2 OTC_OT8F1 OTC_OT8R1 OTC_OT8F2 OTC_OT8R2 OTC_OT9F1 OTC_OT9R1 OTC_OT9F2 OTC_OT9R2 CTGGTGCCTTTTTCTATCGCC CCAAGAGCAACTACAATGGCTT GCATTTTCATGAGCATTCCA CATGTTGTGCCTGCATCTCT GCAGACTCCAAGATGCAAGAC GATGTTGTTCCACCCGCATCT CACTGAGCCAAGTCACTGGA AGGGACAAAACCAAACAGCA TGGCCTTCTAAAGCAACCAA CCGTCTCCCAGATCACATGAC ATAACTCATAATCTATGCATGGCACAA TTTGATCATGGTGTTTATCAGAGC TTGAGACCTAGCTCATGCCC TAACGCAGAACTGGCACAGG TCAGCTTCGAATCACACCAG GAATGTGGCATTGGCTTTTT GTCCCCGACAAACCAAGCTA TGAACTGGCAGTATGCAGGG AACATGGTTTCTGCCCTCAG GGACCATGCCGAACTCTTAC GAGAGAGCCAATCTGCCCAT CACCGGAAACGTGTGAGAGA ACTTCCCATGATCCCATTGA AGCTTCCCTCCAAGTGTCCT GATGGGCATAAGCCCGAAGTA TAAGGCCCAGTGTTGTTGTGT GTAGCAGGGGCTCTGTGAAG TGGCCTGAAATACCCAGAAC GTTGAATTCGCGTGTCCAGG TCCCATGGCGAGAATGTCAC CCCTGTAGGAAACACAGAGGA TGCTTTGGATGTTGATTCTAAA GGCAAAGGACTAGCTTGCAC GGGTGCTATGAGGACCAGTG CAGTGGTGTGTGGAGAGCTG AGAGAGAGCGCTTGACTTTGA Primers for OT1 Primers for OT2 Primers for OT3 Primers for OT4 Primers for OT5 Primers for OT6 Primers for OT7 Primers for OT8 Primers for OT9
13 Primer Name Sequence (5'->3') Note OTC_OT10F1 OTC_OT10R1 OTC_OT10F2 OTC_OT10R2 OTC_OT11F1 OTC_OT11R1 OTC_OT11F2 OTC_OT11R2 OTC_OT12F OTC_OT12R OTC_OT13F1 OTC_OT13R1 OTC_OT13F2 OTC_OT13R2 OTC_OT14F1 OTC_OT14R1 OTC_OT14F2 OTC_OT14R2 OTC_OT15F1 OTC_OT15R1 OTC_OT15F2 OTC_OT15R2 OTC_OT16F1 OTC_OT16R1 OTC_OT16F2 OTC_OT16R2 CTTTGACTCCCGGCGAAAGA TTGTCCATCCGGGTCATTGC AAGTCCTTCTTGCCCAACTT CAGCCCCAATGCATTTTT ATTACAGGTCCTGGTTGGGC ACTGAGCCTGGTAGAGCCTT GGAAGGTGAAGGAAGGAAGG TTTTCTAGGAATTCAGGACATACA TCATGGTCCTTAAAATTTTTGC TCCAGGTATGCAAAGTGGAT TTCAGTTGTACTTTGGATGCTCTGA CATCTGAATAGCAGCAGGCG TAGCACAGCCCAAATGACTT TCATGAAACCCCATAATCAGAA AGTGGGTCATCCTTTGTTACCC TGCCAGTTATCAGCCAAGCA CCCAGGAACTTAACTCAGGTG TGCCATTTGACCTCATAAGTCT TTCAGCCCCCTTGAGTGTTTA GTCTCTGAGCACAAAGAGACGA TTGCCTGTCCCAACTAGAGC GGCCCAAGAATGCACATTTA CCACACACTGGCTAGGACTG ACTGGCAGCACTTGAGACAA GATGGCATGCTGTGGTTTTT CAATGCTTCCACACAGAACC Primers for OT10 Primers for OT11 Primers for OT12 Primers for OT13 Primers for OT14 Primers for OT15 Primers for OT16
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