Journal: Molecular Therapy

Article Title: Long-Term Effects of In Vivo Genome Editing in the Mouse Retina Using Campylobacter jejuni Cas9 Expressed via Adeno-Associated Virus

doi: 10.1016/j.ymthe.2018.10.009

Figure Lengend Snippet: Histologic Evaluation of Retinal Tissues at 14 Months after Intravitreal Injection of AAV-CjCas9 in Mice (A) Representative H&E images of the murine retina at 14 months after intravitreal injection of PBS (No AAV), AAV-CjCas9: Rosa26, and AAV-CjCas9: Hif1a. (B) The number of apoptotic cells in retinal tissues observed at 400× magnification (n = 6). Error bars indicate SEM. (C) Representative immunofluorescence images of the murine retina at 14 months after intravitreal injection of PBS (No AAV), AAV-CjCas9: Rosa26, and AAV-CjCas9: Hif1a. GCL, ganglion cell layer; INL, inner nuclear layer; IS, inner segment; ONL, outer nuclear layer; OS, outer segment. Scale bars, 25 μm.

Article Snippet: Construction of CjCas9 and sgRNA Plasmids A human codon-optimized CjCas9 coding sequence, derived from Campylobacter jejuni subsp. Jejuni NCTC 11168, was synthesized with a nuclear localization signal and an HA epitope at its C-terminal end (GeneArt Gene Synthesis, Thermo Fisher Scientific) and cloned into the p3s plasmid.

Techniques: Injection, Immunofluorescence

Journal: Molecular Therapy

Article Title: Long-Term Effects of In Vivo Genome Editing in the Mouse Retina Using Campylobacter jejuni Cas9 Expressed via Adeno-Associated Virus

doi: 10.1016/j.ymthe.2018.10.009

Figure Lengend Snippet: Electrical Properties of Retinal Neuronal Cells at 14 Months after Intravitreal Injection of AAV-CjCas9 in Mice Amplitudes of scotopic and photopic light responses of mice treated with PBS (No AAV), AAV-CjCas9: Rosa26, and AAV-CjCas9: Hif1a on ERG analyses (ns = 6–10). (A) Amplitudes of the scotopic b-wave at −24 dB. (B) Amplitudes of the scotopic a-wave at 0 dB. (C) Amplitudes of the scotopic b-wave at 0 dB. (D) Amplitudes of the photopic b-wave at 5 dB. (E) Amplitudes of the flicker response at 0 dB. (F) Amplitudes of the oscillatory potential at 5 dB. One-way ANOVA. NS, not significant. Error bars indicate SEM.

Article Snippet: Construction of CjCas9 and sgRNA Plasmids A human codon-optimized CjCas9 coding sequence, derived from Campylobacter jejuni subsp. Jejuni NCTC 11168, was synthesized with a nuclear localization signal and an HA epitope at its C-terminal end (GeneArt Gene Synthesis, Thermo Fisher Scientific) and cloned into the p3s plasmid.

Techniques: Injection

Journal: Molecular Therapy

Article Title: Long-Term Effects of In Vivo Genome Editing in the Mouse Retina Using Campylobacter jejuni Cas9 Expressed via Adeno-Associated Virus

doi: 10.1016/j.ymthe.2018.10.009

Figure Lengend Snippet: Long-Term Genome Editing Efficacy in Retinal and RPE Cells at 14 Months after Intravitreal Injection of AAV-CjCas9 in Mice Mutation frequencies at Hif1a and Rosa26 target sites in (A) retinal and (B) RPE cells determined using deep sequencing at 14 months after intravitreal injection of AAV-CjCas9. Error bars indicate SEM. n = 4 for AAV-uninjected control (mock); n = 7 and n = 3 for AAV-CjCas9: Hif1a and AAV-CjCas9: Rosa26, respectively. 6W, 6 weeks post-injection; 14Mo, 14 months post-injection. **p < 0.01, Student’s t test.

Article Snippet: Construction of CjCas9 and sgRNA Plasmids A human codon-optimized CjCas9 coding sequence, derived from Campylobacter jejuni subsp. Jejuni NCTC 11168, was synthesized with a nuclear localization signal and an HA epitope at its C-terminal end (GeneArt Gene Synthesis, Thermo Fisher Scientific) and cloned into the p3s plasmid.

Techniques: Injection, Mutagenesis, Sequencing, Control

Journal: Molecular Therapy

Article Title: Long-Term Effects of In Vivo Genome Editing in the Mouse Retina Using Campylobacter jejuni Cas9 Expressed via Adeno-Associated Virus

doi: 10.1016/j.ymthe.2018.10.009

Figure Lengend Snippet: Long-Term Genome Editing Specificity in Retinal and RPE Cells at 14 Months after Intravitreal Injection of AAV-CjCas9 in Mice Indel frequencies at potential off-target sites of CjCas9 targeted to Hif1a identified by (A) Digenome-seq and (B) Cas-OFFinder. Genomic DNA isolated from retinal and RPE cells at 14 months after intravitreal injection of AAV-CjCas9 was subjected to targeted deep sequencing. Mismatched nucleotides are indicated in blue, and protospacer-adjacent motifs are indicated in orange. On, on-target site; OT, off-target sites. Error bars indicate SEM. n = 3 for AAV-uninjected control (-AAV); n = 7 for AAV-CjCas9: Hif1a.

Article Snippet: Construction of CjCas9 and sgRNA Plasmids A human codon-optimized CjCas9 coding sequence, derived from Campylobacter jejuni subsp. Jejuni NCTC 11168, was synthesized with a nuclear localization signal and an HA epitope at its C-terminal end (GeneArt Gene Synthesis, Thermo Fisher Scientific) and cloned into the p3s plasmid.

Techniques: Injection, Isolation, Sequencing, Control