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. 2016 Nov 22;5(11):e389.
doi: 10.1038/mtna.2016.92.

In vivo Editing of the Human Mutant Rhodopsin Gene by Electroporation of Plasmid-based CRISPR/Cas9 in the Mouse Retina

Maria Carmela Latella  1 , Maria Teresa Di Salvo  2 , Fabienne Cocchiarella  1 , Daniela Benati  1 , Giulia Grisendi  3 , Antonella Comitato  2 , Valeria Marigo  2 , Alessandra Recchia  1
Affiliations

In vivo Editing of the Human Mutant Rhodopsin Gene by Electroporation of Plasmid-based CRISPR/Cas9 in the Mouse Retina

Maria Carmela Latella et al. Mol Ther Nucleic Acids. .

Abstract

The bacterial CRISPR/Cas system has proven to be an efficient tool for genetic manipulation in various organisms. Here we show the application of CRISPR-Cas9 technology to edit the human Rhodopsin (RHO) gene in a mouse model for autosomal dominant Retinitis Pigmentosa. We designed single or double sgRNAs to knock-down mutant RHO expression by targeting exon 1 of the RHO gene carrying the P23H dominant mutation. By delivering Cas9 and sgRNAs in a single plasmid we induced an efficient gene editing in vitro, in HeLa cells engineered to constitutively express the P23H mutant RHO allele. Similarly, after subretinal electroporation of the CRISPR/Cas9 plasmid expressing two sgRNAs into P23H RHO transgenic mice, we scored specific gene editing as well as significant reduction of the mutant RHO protein. Successful in vivo application of the CRISPR/Cas9 system confirms its efficacy as a genetic engineering tool in photoreceptor cells.

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Figures

Figure 1
Figure 1
CRISPR/Cas9 targeting of the human RHO gene. Schematic representation of human chromosome 3 and RHO gene. The magnified view illustrates the sgRNAs and the PAM sequences (underlined) tailored to exon 1. The C to A conversion resulting in the P23H mutation present in the first exon of the RHO gene is highlighted in bold. Start codon of RHO gene is in italic. Black arrowheads indicate cleavage sites.
Figure 2
Figure 2
NHEJ-mediated knock-out of human RHO gene using the CRISPR/Cas9 system. (a) The Surveyor (Cel-I) nuclease assay on exon 1 of P23H RHO showed targeted cleavage of the digested PCR products in P23H HeLa clone #78 transfected with sgRNA1, sgRNA3, 2sgRNA but not with pX330 (no sgRNA) or in untransfected cells (negative control, NC; full-length, FL; short-edited, SE). Cells transfected with 2sgRNA show the short edited PCR product. (b) Sequence analysis of PCR products surrounding the Cas9 target sites in the genome of HeLa clone #78 transfected with sgRNA1, sgRNA3, and 2sgRNA (in bold) showed a wide variety of Indel mutations mediated by NHEJ at the targeted exon 1. The top sequence in red is the unmodified sequence, underlined are the PAMs. The mismatches/insertions are indicated in cyan. The number of PCR amplicons for each sequence is indicated in parentheses and the modified length is indicated.
Figure 3
Figure 3
In vitro knock-down of human P23H RHO expression. (a) RT-PCR analysis on RHO expression in P23H HeLa clone #78 transfected with pX330, sgRNA1, sgRNA3, and 2sgRNA and untransfected cells (negative control, NC). FL indicates the full-length transcript. SE indicates the short edited transcript. RT-PCR was normalized by analysis of GAPDH. (b) Real time TaqMan PCR on RHO expression in untransfected P23H HeLa clone #78 (NC) or transfected with sgRNA1, sgRNA3, 2sgRNA, and pX330. The experiment was performed in triplicate and is presented as mean ± SEM (*P-value < 0.05, **P < 0.01). (c) Western blottings on total protein extracts to analyse RHO expression in untransfected (NC, negative control) and nuclease-treated P23H HeLa clone #78. The 35 kDa monomer RHO protein is strongly reduced after exposure to sgRNA1, sgRNA3, and 2sgRNA but not after exposure to the control pX330. The western blotting was normalized using anti-α-tubulin antibodies (lower panel). GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Figure 4
Figure 4
Evaluation of CRISPR/Cas9 off-target effects for sgRNAs designed to knock-down the human RHO gene. (a) and (b) Surveyor analysis at the top five potential off-target sites for sgRNA1 or sgRNA3 in P23H HeLa clone #78 transfected with pX330 (−) or with the corresponding sgRNA1 or 3 (+). OT: off-target locus.
Figure 5
Figure 5
Electroporation of the CRISPR/Cas9 plasmids into mice retinas. (a) Microphotographs of electroporated retinas showing transfected areas labelled by GFP expression (green). Positive areas were cut at the stereoscope (dashed lines) and used for further analyses shown in b. (b) RT-PCR analysis on Cas9 and GFP expression in eight mouse retinas injected with sgRNA1 (left eye, L). Mice 1, 2, and 4 show also expression analyses in the not injected contralateral (right, R) retinas. No expression of Cas9 as well as GFP was detectable in these samples.
Figure 6
Figure 6
In vivo knock-down of human P23H RHO in the retina. (a) RT-PCR analysis of Pde6b expression in GFP+ and GFP− cells sorted from mouse retinas electroporated with 2sgRNAs (#1–#5) or with pX330 (#6–#8). Ribosomal s26 RNA was used for normalization. Similar expression of the rod photoreceptor specific gene Pde6b was observed in GFP+ and GFP− sorted cells. (b) Sequences of PCR products surrounding the target sites amplified from the genomic DNA of GFP+ cells sorted from all mouse retinas injected with 2sgRNA. The top sequence in red is the unmodified sequence, underlined are the PAMs. The mismatches/insertions are indicated in cyan. The number of PCR amplicons for each sequence is reported in parentheses and the modified length is indicated. (c) End-point RT-PCR analysis on human RHO mRNA from GFP+ and GFP− cells sorted from mouse retinas electroporated with 2sgRNAs (#1–#5), or with pX330 (#6–#8). FL indicates full-length P23H RHO transcript, SE indicates, short-edited transcript. Unspecific bands amplified in GFP- cells from 2sgRNA or pX330 electroporated retinas are indicated by stars. (d) Immunoblot analysis of RHO on total protein extracts from GFP+ areas dissected as in Figure 6a. Four left 2sgRNA electroporated retinas (L-2sgRNA) were compared with the contralateral not-injected right retinas (R-NI) and showed reduction of RHO visible as monomer at 35 kDa and dimers/multimers at higher molecular weights. Four control left pX330 electroporated retinas (L-pX330) were compared with the contralateral not-injected right retinas (R-NI) and showed no significant change of RHO. The western blotting was normalized using antirecoverin antibodies, detecting a protein expressed in photoreceptors (lower panel).
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