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Allele-specific CRISPR/Cas9 genome editing of the single-base P23H mutation for rhodopsin associated dominant retinitis pigmentosa

Pingjuan Li, Benjamin P. Kleinstiver, Mihoko Y. Leon, Michelle S. Prew, Daniel Navarro-Gomez, Scott H. Greenwald, Eric A. Pierce, J. Keith Joung, Qin Liu
doi: https://doi.org/10.1101/197962
Pingjuan Li
1Ocular Genomics Institute, Massachusetts Eye & Ear Infirmary, Boston, Massachusetts 02114;
2Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts 02114;
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Benjamin P. Kleinstiver
3Molecular Pathology Unit & Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts 02129;
4Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, Massachusetts 02114;
5Department of Pathology, Harvard Medical School, Boston, Massachusetts 02114
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Mihoko Y. Leon
1Ocular Genomics Institute, Massachusetts Eye & Ear Infirmary, Boston, Massachusetts 02114;
2Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts 02114;
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Michelle S. Prew
3Molecular Pathology Unit & Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts 02129;
4Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, Massachusetts 02114;
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Daniel Navarro-Gomez
1Ocular Genomics Institute, Massachusetts Eye & Ear Infirmary, Boston, Massachusetts 02114;
2Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts 02114;
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Scott H. Greenwald
1Ocular Genomics Institute, Massachusetts Eye & Ear Infirmary, Boston, Massachusetts 02114;
2Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts 02114;
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Eric A. Pierce
1Ocular Genomics Institute, Massachusetts Eye & Ear Infirmary, Boston, Massachusetts 02114;
2Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts 02114;
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J. Keith Joung
3Molecular Pathology Unit & Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts 02129;
4Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, Massachusetts 02114;
5Department of Pathology, Harvard Medical School, Boston, Massachusetts 02114
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Qin Liu
1Ocular Genomics Institute, Massachusetts Eye & Ear Infirmary, Boston, Massachusetts 02114;
2Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts 02114;
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  • For correspondence: Qin_Liu@meei.harvard.edu
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Abstract

Treatment strategies for dominantly inherited disorders typically involve silencing or ablating the pathogenic allele. CRISPR/Cas nucleases have shown promise in allele-specific knockout approaches when the dominant allele creates unique protospacer adjacent motifs (PAMs) that can lead to allele restricted targeting. Here, we present a spacer-mediated allele-specific knockout approach that utilizes both SpCas9 variants and truncated single guide RNAs (trusgRNAs) to achieve efficient discrimination of a single-nucleotide mutation in rhodopsin (Rho)-P23H mice, a model of dominant retinitis pigmentosa (RP). We found that approximately 45% of the mutant P23H allele was edited at DNA level, and that the relative RNA expression of wild-type Rho was about 2.8 times more than that of mutant Rho in treated retinas. Furthermore, the progression of photoreceptor cell degeneration in outer nuclear layer was significantly delayed in treated regions of the Rho-P23H retinas at five weeks of age. Our proof-of-concept study therefore outlines a general strategy that could potentially be expanded to examine the therapeutic benefit of allele-specific gene editing approach to treat human P23H patient. Our study also extends allele-specific editing strategies beyond discrimination within the PAM sites, with potentially broad applicability to other dominant diseases.

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Posted January 29, 2018.
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Allele-specific CRISPR/Cas9 genome editing of the single-base P23H mutation for rhodopsin associated dominant retinitis pigmentosa
Pingjuan Li, Benjamin P. Kleinstiver, Mihoko Y. Leon, Michelle S. Prew, Daniel Navarro-Gomez, Scott H. Greenwald, Eric A. Pierce, J. Keith Joung, Qin Liu
bioRxiv 197962; doi: https://doi.org/10.1101/197962
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Allele-specific CRISPR/Cas9 genome editing of the single-base P23H mutation for rhodopsin associated dominant retinitis pigmentosa
Pingjuan Li, Benjamin P. Kleinstiver, Mihoko Y. Leon, Michelle S. Prew, Daniel Navarro-Gomez, Scott H. Greenwald, Eric A. Pierce, J. Keith Joung, Qin Liu
bioRxiv 197962; doi: https://doi.org/10.1101/197962

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