Research ArticleHEALTH AND MEDICINE

CRISPR-Cas9–mediated therapeutic editing of Rpe65 ameliorates the disease phenotypes in a mouse model of Leber congenital amaurosis

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Science Advances  30 Oct 2019:
Vol. 5, no. 10, eaax1210
DOI: 10.1126/sciadv.aax1210
  • Fig. 1 Genome editing of Rpe65 by CRISPR-Cas9 in vitro.

    (A) A schematic drawing of Rpe65 in rd12 MEF. Red text, sequence of the premature stop codon; orange arrow, TS4 sgRNA target sequence; blue text, protospacer adjacent motif (PAM). (B) TS4 sgRNA–induced indel frequencies measured by targeted deep sequencing (n = 4). ssODN dose (in microgram scale) are indicated in parenthesis. (C) Correction frequencies in the rd12 mutation measured by targeted deep sequencing (n = 4). (D) The mean values of the number of deep sequencing reads in different categories show the pattern of indels induced by the TS4 sgRNA (n = 4). Of the total reads, about 61% contain deletions. Ins, insertion; Del, deletion; WT, wild type. (E) Mean percent values of different types of in-frame and out-of-frame indels induced by the TS4 sgRNA (n = 4). 3N, one codon; 3N + 1, one codon + 1 nucleotide; 3N + 2, one codon + 2 nucleotides. (F) Nucleotide sequences showing types of editing induced by the TS4 sgRNA and 1.5 μg of ssODN in rd12 MEF (n = 4). Violet triangle, position of the DSB induced by the TS4 sgRNA; red text, sequences of the stop codon in rd12 MEF; blue text, PAM sequences; green text, synonymous mutations in the donor template; underlined sequences, nucleotides encompassing the premature stop codon and their corresponding amino acid sequences. Error bars indicate SEM. **P < 0.01; ***P < 0.001 by Kruskal-Wallis test with Dunn’s multiple comparison test.

  • Fig. 2 HDR-mediated genome editing induced by CRISPR-Cas9 in the retina and RPE of rd12 mice.

    (A) A schematic drawing of the dual AAV strategy for CRISPR-Cas9–mediated therapeutic correction of the nonsense mutation in Rpe65 in rd12 mice. The premature stop codon in Rpe65 (red text) is generated by a C-to-T mutation in exon 3. The U6 and EFS promoters in the AAV vector were used to express the sgRNA together with the donor template and SpCas9, respectively. Green text, synonymous mutations in the donor template. Therapeutic gene correction could be derived from HDR-mediated precise correction (HDR) or in-frame NHEJ (Inf-NHEJ). (B and C) Indel frequencies measured by targeted deep sequencing in the retina (B) and RPE (C) of rd12 mice at 4 weeks after injection of high-dose (a total of 2 × 1011 vg per eye) and low-dose (a total of 2 × 1010 vg per eye) AAV. SpCas9–to–TS4 sgRNA-Rpe65-donor ratios are indicated in parentheses. (D) Correlation between the indel frequency and AAV copy number per diploid cell (n = 10). A high dose of 1:1 ratio of SpCas9 and TS4 sgRNA-Rpe65-donor was injected subretinally. (E) Frequencies of therapeutic HDR at the site of the C-to-T mutation in the retina and RPE of rd12 mice at 4 weeks after injection. (F) Ratio of HDR to indel frequencies. A high or low dose of SpCas9 and TS4 sgRNA-Rpe65-donor at a 1:1 ratio was injected subretinally (n = 4). (G) Mean values of the number of deep sequencing reads including insertions, deletions, or HDR in AAV-treated RPE (n = 10). A high dose of 1:1 ratio of SpCas9 and TS4 sgRNA-Rpe65-donor was injected subretinally. Rpe65mut, nonedited; Ins, insertion; Out-f-Del, out-of-frame deletion; Inf-Del, in-frame deletion. (H) Nucleotide sequences showing the Rpe65 donor template and changes induced by the TS4 sgRNA in AAV-treated RPE (n = 10). A high dose of 1:1 ratio of SpCas9 and TS4 sgRNA-Rpe65-donor was injected subretinally. Violet triangle, position of the DSB induced by the TS4 sgRNA; red text, sequences corresponding to the premature stop codon in rd12 mice; blue text, PAM sequences; green text, synonymous mutations in the donor template; underlined sequences, nucleotides encompassing the premature stop codon and their corresponding amino acid sequences. *P < 0.05; **P < 0.01; ***P < 0.001 by Kruskal-Wallis test with Dunn’s multiple comparison test (B, C, and E) and Mann-Whitney U test (F).

  • Fig. 3 Therapeutic effects of HDR-mediated correction of the Rpe65 gene in rd12 mice.

    (A) Overview of animal experiments. Time points for subretinal injection and electroretinography are indicated. P0, postnatal day 0; ERG, electroretinography; w, weeks. (B) RPE65 protein expression in the RPE layer of rd12 mice at 6 weeks after the injection. Scale bar, 10 μm. (C) The relative expression of Rpe65 mRNA in the RPE cells of rd12 mice at 7 months after the injection (n = 4). (D) Representative electroretinographic responses to bright stimuli (0 dB) after dark adaptation from normal C57BL/6 and rd12 mice at 6 weeks after the injection. (E to H) Amplitudes of electroretinographic responses to bright stimuli (0 dB) after dark adaptation (n = 4). (E and G) Amplitudes of the a-wave at 0 dB at 6 weeks (E) and 7 months (G) after the injection. (F and H) Amplitudes of the b-wave at 0 dB at 6 weeks (F) and 7 months (H) after the injection. (I) Representative hematoxylin and eosin (H&E) images of retinal tissues from rd12 mice at 7 months after the injection. Scale bar, 20 μm. (J) The number of layers of outer nuclear layer nuclei in rd12 mice at 7 months after the injection (n = 4). Normal, C57BL/6; rd12, rd12 mice without treatment; rd12-AAV, rd12 mice treated with subretinal injection of a high dose of 1:1 ratio of SpCas9 and TS4 sgRNA-Rpe65-donor. *P < 0.05; **P < 0.01; ***P < 0.001 by Kruskal-Wallis test with Dunn’s multiple comparison test. DAPI, 4′,6-diamidino-2-phenylindole; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer.

  • Fig. 4 Analysis of potential off-target sites.

    (A) Genome-wide Circos plot showing in vitro cleavage sites in the rd12 mouse genome in the absence (gray) or in the presence (blue) of TS4 sgRNA. On-target cleavage is indicated by the red arrow. (B) Targeted deep sequencing results for the top 29 Digenome-seq candidates in AAV-treated retina (n = 3). Nucleotides in red and orange indicate sequences that are mismatched relative to the on-target site, and nucleotides in blue indicate PAM sequences. (C) Reanalysis of the active off-targets by targeted deep sequencing in AAV-treated RPE (n = 3). (D) Representative whole mount of RPE tissues from rd12 mice at 7 months after treatment with or without the dual AAV system encoding SpCas9 and the donor template. Scale bar, 10 μm.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/10/eaax1210/DC1

    Fig. S1. Screening of sgRNAs targeting the Rpe65 gene in rd12 MEFs.

    Fig. S2. Delivered AAV copies between retina and RPE.

    Fig. S3. Analysis of human RPE65 editing.

    Fig. S4. Electroretinographic responses to bright stimuli (0 dB) after dark adaptation from normal C57BL/6 and rd12 mice at 6 weeks after the injection (n = 4).

    Fig. S5. Electroretinographic responses to dim stimuli (−25 dB) after dark adaptation from normal C57BL/6 and rd12 mice at 6 weeks after the injection.

    Fig. S6. Analysis of TS4 sgRNA off-target effects.

    Fig. S7. Comparison of off-target frequency between wild-type SpCas9 and SniperCas9.

    Table S1. Summary of predictions at the target site with sgRNA by the inDelphi approach.

    Table S2. Active off-target sites.

    Table S3. Primers used in this study.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Screening of sgRNAs targeting the Rpe65 gene in rd12 MEFs.
    • Fig. S2. Delivered AAV copies between retina and RPE.
    • Fig. S3. Analysis of human RPE65 editing.
    • Fig. S4. Electroretinographic responses to bright stimuli (0 dB) after dark adaptation from normal C57BL/6 and rd12 mice at 6 weeks after the injection (n = 4).
    • Fig. S5. Electroretinographic responses to dim stimuli (−25 dB) after dark adaptation from normal C57BL/6 and rd12 mice at 6 weeks after the injection.
    • Fig. S6. Analysis of TS4 sgRNA off-target effects.
    • Fig. S7. Comparison of off-target frequency between wild-type SpCas9 and SniperCas9.
    • Table S1. Summary of predictions at the target site with sgRNA by the inDelphi approach.
    • Table S2. Active off-target sites.
    • Table S3. Primers used in this study.

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