USH2A Gene Editing Using the CRISPR System

Fuster-García, Carla; García‐García, Gema; González-Romero, Elisa; Jaijo, Teresa; Sequedo, María Dolores; Ayuso, Carmen; Vázquez‐Manrique, Rafael P.; Millán, José María; Aller, Elena

doi:10.1016/j.omtn.2017.08.003

Cited by 58 publications

(57 citation statements)

References 83 publications

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“…All these evidences suggest that this variant may play a specific role in the pathogenesis of non-syndromic RP or USH2. However, further functional and gene editing studies [ 49 ] could shed additional light on the role of this USH2A variant.…”

Section: Discussionmentioning

confidence: 99%

Unravelling the pathogenic role and genotype-phenotype correlation of the USH2A p.(Cys759Phe) variant among Spanish families

et al. 2018

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IntroductionMutations in USH2A cause both isolated Retinitis Pigmentosa (RP) and Usher syndrome (that implies RP and hearing impairment). One of the most frequent variants identified in this gene and among these patients is the p.(Cys759Phe) change. However, the pathogenic role of this allele has been questioned since it was found in homozygosity in two healthy siblings of a Spanish family. To assess the causative role of USH2A p.(Cys759Phe) in autosomal recessive RP (ARRP) and Usher syndrome type II (USH2) and to establish possible genotype-phenotype correlations associated with p.(Cys759Phe), we performed a comprehensive genetic and clinical study in patients suffering from any of the two above-mentioned diseases and carrying at least one p.(Cys759Phe) allele.Materials and methodsDiagnosis was set according to previously reported protocols. Genetic analyses were performed by using classical molecular and Next-Generation Sequencing approaches. Probands of 57 unrelated families were molecularly studied and 63 patients belonging to these families were phenotypically evaluated.ResultsMolecular analysis characterized 100% of the cases, identifying: 11 homozygous patients for USH2A p.(Cys759Phe), 42 compound heterozygous patients (12 of them with another missense USH2A pathogenic variant and 30 with a truncating USH2A variant), and 4 patients carrying the p.(Cys759Phe) allele and a pathogenic variant in another RP gene (PROM1, CNGB1 or RP1). No additional causative variants were identified in symptomatic homozygous patients. Statistical analysis of clinical differences between zygosity states yielded differences (p≤0.05) in age at diagnosis of RP and hypoacusis, and progression of visual field loss. Homozygosity of p.(Cys759Phe) and compound heterozygosity with another USH2A missense variant is associated with ARRP or ARRP plus late onset hypoacusis (OR = 20.62, CI = 95%, p = 0.041).ConclusionsThe present study supports the role of USH2A p.(Cys759Phe) in ARRP and USH2 pathogenesis, and demonstrates the clinical differences between different zygosity states. Phenotype-genotype correlations may guide the genetic characterization based upon specific clinical signs and may advise on the clinical management and prognosis based upon a specific genotype.

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Section: Discussionmentioning

confidence: 99%

Unravelling the pathogenic role and genotype-phenotype correlation of the USH2A p.(Cys759Phe) variant among Spanish families

et al. 2018

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“…In addition to the eye, the ear is another site that also allows ready access for genome editing. While the studies in the ear have not progressed as far as the eye, correction of the USH2A gene responsible for both visual and hearing impairment in Usher syndrome has been shown in patient cells utilizing Cas9 and an DNA template [118]. Beyond in vitro cell manipulation, Cas9 nickases with paired sgRNAs in combination with an oligonucleotide donor template editing of zygotes were able to prevent age-related hearing loss in an mouse model [119].…”

Section: Looking Forward: Upcoming Areas For Gene Editor Clinical Trialsmentioning

confidence: 99%

Gene editing and CRISPR in the clinic: current and future perspectives

et al. 2020

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Genome editing technologies, particularly those based on zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR (clustered regularly interspaced short palindromic repeat DNA sequences)/Cas9 are rapidly progressing into clinical trials. Most clinical use of CRISPR to date has focused on ex vivo gene editing of cells followed by their re-introduction back into the patient. The ex vivo editing approach is highly effective for many disease states, including cancers and sickle cell disease, but ideally genome editing would also be applied to diseases which require cell modification in vivo. However, in vivo use of CRISPR technologies can be confounded by problems such as off-target editing, inefficient or off-target delivery, and stimulation of counterproductive immune responses. Current research addressing these issues may provide new opportunities for use of CRISPR in the clinical space. In this review, we examine the current status and scientific basis of clinical trials featuring ZFNs, TALENs, and CRISPR-based genome editing, the known limitations of CRISPR use in humans, and the rapidly developing CRISPR engineering space that should lay the groundwork for further translation to clinical application.

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“…To facilitate the in vivo assessment of the function of Ush2a without exon 12 (Ush2a-∆Ex12), we knocked out the exon 12 (homologous to exon 13 in human) from the Ush2a locus in mouse zygotes using an NHEJ-mediated CRISPR/Cas9 and dual-sgRNAs targeting flanking introns approach (Fig. 5a) [42][43][44][45] . Initial genotypic screening of 40 founder mice revealed that, in addition to a wild type band (2.3 kb), 29 founders (71%) carried a smaller PCR fragment that was predicted to be amplified from the edited Ush2a alleles missing 3'-portion of intron 11, exon 12, and 5'-end of intron 12 ( Fig.…”

Section: Generation Of Ush2a-∆ex12 Micementioning

confidence: 99%

Exon 13-skipped USH2A protein retains functional integrity in mice, suggesting an exo-skipping therapeutic approach to treat USH2A-associated disease

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Lamas

Maeder

et al. 2020

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Mutations in the USH2A gene are the most common cause of non-syndromic inherited retinal degeneration and Usher syndrome, which is characterized by congenital deafness and progressive vision loss. Development of a vector mediated therapy for USH2A-associated disease has been challenging due to its large size of coding sequence (~15.6kb). Therefore, there is an unmet need to develop alternative therapeutic strategies. The USH2A protein (Usherin) contains many repetitive domains, and it has been hypothesized that some domains may be dispensable with regard to protein function. Here, we show that skipping of exon 13 of the human USH2A gene or the equivalent exon 12 of the mouse Ush2a gene results in an in-frame transcript that produces functional Usherin protein. This nearly full length Usherin rescues the ciliogenesis in Ush2a null cells as well as the cochlear and retinal phenotypes in Ush2a null mice. Together, our results support the development of exon-skipping strategies to treat both visual and hearing loss in patients with USH2A-associated disease due to mutations in exon 13.

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USH2A Gene Editing Using the CRISPR System

Cited by 58 publications

References 83 publications

Unravelling the pathogenic role and genotype-phenotype correlation of the USH2A p.(Cys759Phe) variant among Spanish families

Unravelling the pathogenic role and genotype-phenotype correlation of the USH2A p.(Cys759Phe) variant among Spanish families

Gene editing and CRISPR in the clinic: current and future perspectives

Exon 13-skipped USH2A protein retains functional integrity in mice, suggesting an exo-skipping therapeutic approach to treat USH2A-associated disease

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