Homologous recombination occurs frequently at innate GT microsatellites in normal somatic and germ cells in vivo

Zheng, Jianbo; Li, Heng; Zhang, Qi; Sun, Lei; Liu, Xiangfang; Luo, Chen

doi:10.1186/s12864-018-4758-y

Cited by 1 publication

(1 citation statement)

References 31 publications

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“…In view of the highly repetitive sequence context, our findings raised the intriguing possibility that deletion of the 32-bp segment was mediated by an alternative DNA repair mechanism such as microhomology-mediated end joining (MMEJ) [ 39 , 40 ]. These results are consistent with the notion that the outcome of genome editing events is not random and instead can be related to intrinsic features of the target sequence [ 38 , 41 ]. In the case of our current findings, we postulate that neighboring repetitive sequences within ORF15 may be used as a template for homologous recombination following a double-strand break in the DNA.…”

Section: Discussionsupporting

confidence: 90%

Restoration of RPGR expression in vivo using CRISPR/Cas9 gene editing

Gumerson

Alsufyani

et al. 2021

Gene Ther

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Mutations in the gene for Retinitis Pigmentosa GTPase Regulator (RPGR) cause the X-linked form of inherited retinal degeneration, and the majority are frameshift mutations in a highly repetitive, purine-rich region of RPGR known as the OFR15 exon. Truncation of the reading frame in this terminal exon ablates the functionally important C-terminal domain. We hypothesized that targeted excision in ORF15 by CRISPR/Cas9 and the ensuing repair by non-homologous end joining could restore RPGR reading frame in a portion of mutant photoreceptors thereby correcting gene function in vivo. We tested this hypothesis in the rd9 mouse, a naturally occurring mutant line that carries a frameshift mutation in RPGRORF15, through a combination of germline and somatic gene therapy approaches. In germline gene-edited rd9 mice, probing with RPGR domain-specific antibodies demonstrated expression of full length RPGRORF15 protein. Hallmark features of RPGR mutation-associated early disease phenotypes, such as mislocalization of cone opsins, were no longer present. Subretinal injections of the same guide RNA (sgRNA) carried in AAV sgRNA and SpCas9 expression vectors restored reading frame of RPGRORF15 in a subpopulation of cells with broad distribution throughout the retina, confirming successful correction of the mutation. These data suggest that a simplified form of genome editing mediated by CRISPR, as described here, could be further developed to repair RPGRORF15 mutations in vivo.

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