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Mark Ellsworth Kleinman, Sushil Kumar Dubey, Kyung Jung, Jacob Roney, Dingyuan Lou, Jennifer Brown, Kabhilan Mohan; Safety and efficiency of CRISPR-Cas9 gene editing in the retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1981. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The recently discovered CRISPR-Cas9 system has emerged as an easy, efficient, cost effective and broadly useful tool for genome editing. However, the therapeutic potential of this technique is uncertain due to the lack of a comprehensive understanding of the toxicity and immune responses that may occur with expression Cas9 in the retinal pigment epithelium (RPE). In this study, off-target cytotoxicity and efficiency of CRISPR-Cas9 was evaluated in the RPE.
CRISPR–SpCas9n plasmids (Addgene) with flanking guide RNAs (gRNAs targeting HDAC1/2, CRISPR Design Toolkit, MIT) were transfected (Lipofectamine 3000) in ARPE19 (ATCC, n=3) and primary human RPE (Lonza, n=3). Control transfections included vectors with scrambled non-genomic gRNA and vectors without Cas9 or gRNA. After sorting (GFP) and puromycin selection (1μg/mL) of transfected cultures, DNA was Sanger sequenced to confirm target excision. Quantitative PCR and Western blotting (WB) were used to confirm loss of target gene expression (n=3). RPE cytotoxicity was evaluated by an MTT assay (Promega) and annexin/PI (BD, n=6 ). Globally expressing Cas9 mice (Gt(Rosa)26Sor, Jackson) were evaluted for retinal degeneration by color fundoscopy, SD-OCT (Spectralis) and ZO-1 immunofluorescence (Abcam) of flatmounts. Cas9 gene insert reporter (FLAG) was studied by immunofluorescence (Cell Signaling) of cryosections and RPE/choroid flatmounts (n=3). Statistical tests used included Mann-Whitney U, ANOVA, and Fisher's exact.
Annexin/PI and MTT assays demonstrated no evidence of loss of RPE viability with pCas9 or scrambled gRNA expression (p>0.05). Expression of targeted genes (HDAC1/2) in RPE after CRISPR-Cas gene editing was non-detectable by qPCR or WB (p<0.01) compared to controls. Retinal phenotype of the globally expressing Cas9 appeared normal at 6-8 weeks of age by color fundoscopy, SD-OCT, and ZO-1 staining (Fig. 1A, p>0.05). Cryosections and RPE/choroidal flatmounts showed moderate levels of FLAG reporter in Cas9 versus Wt mice (Fig. 1B, n=3).
Our studies suggest that CRISPR-Cas9 gene editing is well-tolerated by RPE and did not induce cytotoxicity. In vitro data suggested that targeted editing is possible with excellent efficiency. Future studies will be directed towards investigating off-target immune responses with CRISPR-Cas9 expression and the development of constructs of RPE specific gRNA expression in the global Cas9 mouse.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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