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.2017 Mar 14:8:14716.
doi: 10.1038/ncomms14716.

Nrl knockdown by AAV-delivered CRISPR/Cas9 prevents retinal degeneration in mice

Affiliations

Nrl knockdown by AAV-delivered CRISPR/Cas9 prevents retinal degeneration in mice

Wenhan Yu et al. Nat Commun..

Abstract

In retinitis pigmentosa, loss of cone photoreceptors leads to blindness, and preservation of cone function is a major therapeutic goal. However, cone loss is thought to occur as a secondary event resulting from degeneration of rod photoreceptors. Here we report a genome editing approach in which adeno-associated virus (AAV)-mediated CRISPR/Cas9 delivery to postmitotic photoreceptors is used to target the Nrl gene, encoding for Neural retina-specific leucine zipper protein, a rod fate determinant during photoreceptor development. Following Nrl disruption, rods gain partial features of cones and present with improved survival in the presence of mutations in rod-specific genes, consequently preventing secondary cone degeneration. In three different mouse models of retinal degeneration, the treatment substantially improves rod survival and preserves cone function. Our data suggest that CRISPR/Cas9-mediated NRL disruption in rods may be a promising treatment option for patients with retinitis pigmentosa.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. EGFP knockdown in photoreceptors by an AAV-CRISPR/Cas9 system.
(a) Schematic representation of the AAV vectors delivering SpCas9 and sgRNA. (b) Schematic representation of the EGFP locus showing the location of the sgRNA target. The targeted genomic site is indicated in blue. Protospacer adjacent motif (PAM) sequence is marked in red. (c) Timeline for the EGFP knockdown experiments. EachNrl-L-EGFP mouse was subretinally injected with 2.5 × 109 vector genomes (vg) AAV-Cas9 and 2.5 × 109 vg AAV-sgRNA-EGFP in one eye, and the same doses of AAV-Null and AAV-sgRNA-EGFP (control) in the fellow eye. A total of eight mice including both genders were used. (d) SURVEYOR nuclease assay revealing indel formation at the EGFP locus in flow-sorted tdTomato-expressing cells. Each lane contained sample from an individual mouse (n=2). DNA fragments digested by SURVEYOR nuclease are indicated by arrowhead. Indel rate of each sample is shown below the gel image. (e) Representative FACS plots of dissociated cells from retinas receiving control vectors (upper panel) or CRISPR-EGFP vectors (middle and lower panels). Dissociated cells from two retinas were used in each group. (f) Statistical analysis of flow-sorted retinal cells of three independent experiments. Data are represented as mean±s.d. PI, post injection.
Figure 2
Figure 2. EGFP knockdown by AAV-CRISPR/Cas9 in mouse retina.
FourNrl-L-EGFP mice including both genders received subretinal codelivery of 2.5 × 109 vector genomes (vg) AAV-Cas9 and 2.5 × 109 vg AAV-sgRNA-EGFP per eye at P14. Same doses of AAV-Null and AAV-sgRNA-EGFP were coinjected to the fellow eyes as controls. Representative retinal whole-mounts (left panels) and sections (right panels) of a treated mouse at 3 months of age are shown. TdTomato expression (red) indicates the transduction by the AAV-sgRNA vector. IS, inner segments; N, nasal; ONL, outer nuclear layer; OS, outer segments; S, superior. Scale bars, 50 μm.
Figure 3
Figure 3.Nrl gene knockdown in postmitotic photoreceptors using AAV-CRISPR/Cas9.
(a) Schematic representation of mouseNrl locus showing the location of the sgRNA target. The targeted genomic site is indicated in blue. Protospacer adjacent motif (PAM) sequence is marked in red. (b) Timeline for theNrl knockdown experiments. Each wild-type (WT) C57bl/6j mouse was subretinally injected with 5 × 109 vector genomes (vg) AAV-Cas9 and 2.5 × 109 vg AAV-sgRNA-Nrl (CRISPR-Nrl) in one eye and same doses of AAV-Cas9 and AAV-sgRNA-EGFP (CRISPR-EGFP) in the fellow eye. A total of eight mice including both genders were used. (c) SURVEYOR nuclease assay showing indel formation in theNrl locus in flow-sorted tdTomato-expressing cells. Each lane contained sample from an individual mouse (n=3). DNA fragments digested by SURVEYOR nuclease are indicated by red arrow. Indel rate of each sample is shown below the gel image. (d) Rate of sequence change at the target site ofNrl locus in flow-sorted tdTomato-expressing cells by deep sequencing analysis. Schematic positions of indels in the amplicon (left) and total non-homologous end joining (NHEJ) frequencies (right) of untreated (green), CRISPR-EGFP-treated (red) and CRISPR-Nrl-treated (blue) eyes were compared. Two retinas were used for each treatment. (e) Representative mutation patterns and corresponding ratios in total reads detected by deep sequencing ofNrl locus. Top, wild-type sequence; red dashes, deleted bases; red bases: insertions; the red triangle indicates CRISPR/Cas9 cutting site. (f) Immunoblot analysis of NRL protein at 12 weeks post vector injection. Retinas fromNrl-knockout (KO) and C57bl/6j (C57) mice served as negative and positive controls, respectively. Histone H3 served as loading control. (g) Immunostaining of NRL in retina sections. IS, inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; Scale bar, 50 μm.
Figure 4
Figure 4. Morphological and functional changes of retina followingNrl knockdown.
(a) Toluidine blue-stained semi-thin retinal sections (0.5 μm) of untreated, CRISPR-EGFP-treated and CRISPR-Nrl-treated eyes of C57bl/6j mice. Insets show the magnified images of the marked areas. (b) Electron micrographs of photoreceptor nuclei in CRISPR-EGFP- and CRISPR-Nrl-treatedCrxp-Nrl (rod-only) mice. Ina,b, sample nuclei are outlined in yellow. Heterochromatin (h) and euchromatin (e) are labeled. Red arrows indicate cone-like nuclei. Scale bar, 2 μm. (c) Representative ERG forms of CRISPR-EGFP- and CRISPR-Nrl-treated eyes from a single C57bl/6j mouse at 6 weeks post vector injection. (d) Statistical analysis of ERG amplitudes. Significantly lower amplitudes of dark-adapted a- and b-waves were obtained in response to increasing intensities of flash stimuli in the CRISPR-Nrl-treated eyes, whereas the amplitude of light-adapted b-wave was not affected (n=12, including both genders). Error bars show s.e.m. and the significance between the CRISPR-EGFP- and CRISPR-Nrl-treated eyes was calculated using two-tailed pairedt-test. *P<0.05; **P<0.01.
Figure 5
Figure 5. Expression alterations following CRISPR/Cas9-mediatedNrl knockdown.
(a–c) RNA sequencing analysis of flow-sorted tdTomato-expressing cells isolated from C57bl/6j mice receiving subretinal administration of CRISPR-Nrl or CRISPR-EGFP (control) vectors, conducted at 2.5 months post vector injection. Four mice including both genders received vector administration. Two retinas were used in each group and the data were averaged from two independent experiments. Fold differences in expression of (a) rod- and (b) cone-specific genes between CRISPR-Nrl- and CRISPR-EGFP-treated eyes are shown. Statistical significance is shown by volcano plot (c). Gene expression with fold difference of >2 (absolute log fold difference of >1, indicated by dash lines ina,b) andP value <0.05 (indicated by dash line inc) is considered to be significantly changed, and is shown as green dot. (d) Immunoblot analysis of C57bl/6j mouse retinas receiving CRISPR vectors, performed at 2.5 month post vector injection. Combined lysate from two retinas for each treatment was used. (e) Immunostaining of GNB3 in retina sections ofNrl-L-GFP mice receiving CRISPR-Nrl vectors. Control sections were from mice injected with AAV-Null and AAV-sgRNA-Nrl vectors. EGFP expressing cells indicate rods. Two mice including both genders were used. Scale bar, 50 μm.
Figure 6
Figure 6. Rescue of retinal degeneration inRho−/− mice followingNrl knockdown.
(a) Time course of photoreceptor degeneration inRho−/− mouse (upper) and timeline for CRISPR/Cas9-mediatedNrl knockdown experiments (lower). Seven mice including both genders received CRISPR-Nrl vector treatment in the right eyes and the control CRISPR-EGFP vector treatment in the left eyes. (b) Time-course changes of light-adapted b-wave amplitude in response to the highest stimulus intensity (100 cd s m−2) (n=7). (c) Light-adapted b-wave amplitude in response to a series of flash stimuli at P90 (n=7). (d) Representative ERG waveforms from a single mouse. (e) Immunofluorescence images showing better preserved ONL and S-opsin expression in CRISPR-Nrl-treated eyes than CRISPR-EGFP-treated control eyes. Scale bar, 50 μm. (f) Quantification of ONL cells in 300 μm segments of retina (n=3). Error bars show s.e.m., and the significance between the CRISPR-EGFP- and CRISPR-Nrl-treated eyes was calculated using two-tailed pairedt-test. *P<0.05; **P<0.01; ***P<0.001.
Figure 7
Figure 7. Rescue of retinal degeneration inRd10/Nrl-L-EGFP mice followingNrl knockdown.
(a) Time course of photoreceptor degeneration inRd10/Nrl-L-EGFP (upper) and timeline for CRISPR/Cas9-mediatedNrl knockdown experiments (lower). Seven mice including both genders received CRISPR-Nrl vector treatment in the right eyes and the control CRISPR-EGFP vector treatment in the left eyes. (b) Light-adapted b-wave amplitude in response to a series of flash stimuli at P84 to P90 (n=7). (c) Representative ERG waveforms from a single mouse. (d) Haematoxylin and eosin (H&E) staining and immunofluorescence images revealing better preserved ONL structure, cone morphology and S-opsin expression in CRISPR-Nrl-treated eyes than CRISPR-EGFP-treated control eyes. Scale bar, 50 μm. (e) Quantification of ONL cells in 300 μm segments of retina (n=3). Error bars show s.e.m. and the significance between CRISPR-EGFP- and CRISPR-Nrl-treated eyes was calculated using two-tailed pairedt-test. *P<0.05; **P<0.01; ***P<0.001.
Figure 8
Figure 8. Rescue of retinal degeneration in humanRHO P347S mice byNrl knockdown.
(a) Time course of photoreceptor degeneration in humanRHO P347S transgenic mouse (upper) and timeline for CRISPR/Cas9-mediatedNrl knockdown experiments (lower). Ten mice including both genders received CRISPR-Nrl vector treatment in the right eyes and the control CRISPR-EGFP vector treatment in the left eyes. (b) Time-course changes of light-adapted b-wave amplitude in response to the highest stimulus intensity (100 cd s m−2) (n=10). (c) Light-adapted b-wave amplitude in response to a series of flash stimuli at P90 (n=10). (d) Representative ERG waveforms from a single mouse. (e) Schematic of optomotor test (upper) and the spatial resolution (expressed as cycles/degree, lower) from CRISPR-EGFP- and CRISPR-Nrl-treated eyes at P90 (n=6). (f) Immunofluorescence images revealing better preserved ONL and S-opsin expression in CRISPR-Nrl-treated eyes than CRISPR-EGFP-treated control eyes. Scale bar, 50 μm. (g) Quantification of ONL cells in 300 μm segments of retina (n=3). Error bars show s.e.m. and paired two-tailedt-test was performed for statistical analyses. **P<0.01; ***P<0.001.
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