Dataset Information

CRISPR Interference-Potential Application in Retinal Disease.

ABSTRACT: The treatment of dominantly inherited retinal diseases requires silencing of the pathogenic allele. RNA interference to suppress gene expression suffers from wide-spread off-target effects, while CRISPR-mediated gene disruption creates permanent changes in the genome. CRISPR interference uses a catalytically inactive 'dead' Cas9 directed by a guide RNA to block transcription of chosen genes without disrupting the DNA. It is highly specific and potentially reversible, increasing its safety profile as a therapy. Pre-clinical studies have demonstrated the versatility of CRISPR interference for gene silencing both in vivo and in ex vivo modification of iPSCs for transplantation. Applying CRISPR interference techniques for the treatment of autosomal dominant inherited retinal diseases is promising but there are few in vivo studies to date. This review details how CRISPR interference might be used to treat retinal diseases and addresses potential challenges for clinical translation.

SUBMITTER: Peddle CF

PROVIDER: S-EPMC7177328 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Publications

CRISPR Interference-Potential Application in Retinal Disease.

Peddle Caroline F CF Fry Lewis E LE McClements Michelle E ME MacLaren Robert E RE

International journal of molecular sciences 20200327 7

The treatment of dominantly inherited retinal diseases requires silencing of the pathogenic allele. RNA interference to suppress gene expression suffers from wide-spread off-target effects, while CRISPR-mediated gene disruption creates permanent changes in the genome. CRISPR interference uses a catalytically inactive 'dead' Cas9 directed by a guide RNA to block transcription of chosen genes without disrupting the DNA. It is highly specific and potentially reversible, increasing its safety profil ...[more]

PMID: 32230903

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Project description:BackgroundCRISPR/Cas is a widespread adaptive immune system in prokaryotes. This system integrates short stretches of DNA derived from invading nucleic acids into genomic CRISPR loci, which function as memory of previously encountered invaders. In Escherichia coli, transcripts of these loci are cleaved into small RNAs and utilized by the Cascade complex to bind invader DNA, which is then likely degraded by Cas3 during CRISPR interference.ResultsWe describe how a CRISPR-activated E. coli K12 is cured from a high copy number plasmid under non-selective conditions in a CRISPR-mediated way. Cured clones integrated at least one up to five anti-plasmid spacers in genomic CRISPR loci. New spacers are integrated directly downstream of the leader sequence. The spacers are non-randomly selected to target protospacers with an AAG protospacer adjacent motif, which is located directly upstream of the protospacer. A co-occurrence of PAM deviations and CRISPR repeat mutations was observed, indicating that one nucleotide from the PAM is incorporated as the last nucleotide of the repeat during integration of a new spacer. When multiple spacers were integrated in a single clone, all spacer targeted the same strand of the plasmid, implying that CRISPR interference caused by the first integrated spacer directs subsequent spacer acquisition events in a strand specific manner.ConclusionsThe E. coli Type I-E CRISPR/Cas system provides resistance against bacteriophage infection, but also enables removal of residing plasmids. We established that there is a positive feedback loop between active spacers in a cluster--in our case the first acquired spacer--and spacers acquired thereafter, possibly through the use of specific DNA degradation products of the CRISPR interference machinery by the CRISPR adaptation machinery. This loop enables a rapid expansion of the spacer repertoire against an actively present DNA element that is already targeted, amplifying the CRISPR interference effect.

Dataset Information

CRISPR Interference-Potential Application in Retinal Disease.

Publications

CRISPR Interference-Potential Application in Retinal Disease.

Similar Datasets

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