2017
DOI: 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

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-… Show more

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Cited by 25 publications
(37 citation statements)
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References 48 publications
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“…In addition to the new functional assays, the recent entrance of CRISPR/Cas opens great opportunities for IRD research, as it enables a more efficient introduction of specific mutations into animal models or cells [ 72 ]. This technology also offers a new therapeutic potential [ 73 ].…”
mentioning
confidence: 99%
“…In addition to the new functional assays, the recent entrance of CRISPR/Cas opens great opportunities for IRD research, as it enables a more efficient introduction of specific mutations into animal models or cells [ 72 ]. This technology also offers a new therapeutic potential [ 73 ].…”
mentioning
confidence: 99%
“…However, the proposed strategy was designed to be allele specific relating to the murine gene without distinguishing the WT and the mutant allele, thus could not be translated to future clinical applications. More practical and physiological approaches to knock down dominant mutation in rhodopsin gene were described by Broccoli41 and Liu teams 82. Both groups designed a P23H-specific gRNA to knock down the mutant murine allele in P23H knock-in mice, and employed the engineered SpCas9 VQR variant coupled to the mutation-specific gRNA generating indels predominantly at the mutant allele 41.…”
Section: Nhej For Disruption Of Mutant Genesmentioning
confidence: 99%
“…Both groups designed a P23H-specific gRNA to knock down the mutant murine allele in P23H knock-in mice, and employed the engineered SpCas9 VQR variant coupled to the mutation-specific gRNA generating indels predominantly at the mutant allele 41. Li et al 82 obtained a higher and more efficient discrimination between the mutant and the WT allele by combining a 5′-truncated 17-nucleotide gRNA together with the SpCas9 VRQR, by subretinal injected plasmids followed by electroporation. However, clinical translation to humans of this approach may be difficult due to the requirement of invasive surgery to place microelectrodes in close proximity of target cells to limit tissue damage.…”
Section: Nhej For Disruption Of Mutant Genesmentioning
confidence: 99%
“…So far, allele-specific CRISPR has been increasingly employed in treating various diseases such as retinitis pigmentosa [5][6][7][8], corneal dystrophy [9], dominant progressive hearing loss [10] and multiple cancers [11][12][13], as well as genome imprinting diseases [14]. And it was also used to alleviate haploinsufficiency by allele-specific CRISPR activation of wildtype alleles [15], and even was designed for manipulating human leukocyte antigen (HLA) locus [16].…”
Section: Introductionmentioning
confidence: 99%