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Self-cleaving guide RNAs enable pharmacological selection of precise gene editing events in vivo

ABSTRACT: Self-cleaving guide RNAs enable pharmacological selection of precise gene editing events in vivo

PROVIDER: PRJNA894207 | ENA |

REPOSITORIES: ENA

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Self-cleaving guide RNAs enable pharmacological selection of precise gene editing events in vivo

Project description:In this study we compared the gene expression of mice that had received the rAAV-GR-Cypor scgRNA and rAAV-spCas9 with and without APAP selection. APAP selected mice were also compared to mice which had received the rAAV-GR-Hpd miRNA.

2022-10-25 | GSE216550 | GEO

CLUSTER guide RNAs enable precise and efficient RNA editing with endogenous ADAR enzymes in vivo

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CLUSTER guide RNAs enable precise and efficient RNA editing with endogenous ADAR enzymes in vivo

Project description:RNA base editing represents a promising alternative for genome editing. Recent approaches harness the endogenous RNA editing enzyme ADAR to circumvent problems related to the ectopic expression of an editing enzyme, but they suffer from sequence restriction, lack of efficiency, and bystander editing. Here, we present in‐silico optimized CLUSTER guide RNAs, which bind their target mRNAs in a multivalent fashion and thereby enable editing with unprecedented precision as shown by next generation sequencing. CLUSTER guide RNAs can be genetically encoded and manufactured into viruses to work in various cell lines. They achieve on‐target editing on endogenous transcripts like GUSB and NUP43 with yields up to 45% without bystander editing and have been shown to recruit endogenous ADAR in vivo. The CLUSTER approach tremendously enlarges the sequence space available for guide RNA design and opens new avenues for drug development in the field of RNA base editing.

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Precise in-vivo RNA base editing with a wobble-enhanced circular CLUSTER guide RNA

Project description:Adenosine-to-inosine RNA base editing is a strategy developed to safely manipulate genetic information at the RNA level. Particularly promising for clinical implementation is the use of the ubiquitously expressed endogenous editing enzyme ADAR (adenosine deaminase acting on RNA) with tailored guide RNAs. However, the precision of editing could be compromised by global off-target events that can potentially occur throughout the transcriptome. In this study, we introduce a novel circular CLUSTER guide RNA design that recruits endogenous ADAR in vivo. The goals of the whole transcriptome sequencing experiment were to evaluate the A-to-G RNA editing index and the global editing precision of this novel design. To achieve this, Rett syndrome mice harboring a Mecp2 W104Amber mutation were treated either with a circular CLUSTER guide RNA targeting the mutant Mecp2 transcript or a scrambled and thus non-targeting control guide RNA. Both the targeting and the non-targeting guide RNA were encoded as AAV and delivered via retro-orbital injection of 4x10^12 viral genomes per mouse. The used AAV serotype PHP.eB allows cargo delivery to the mouse brain after systemic administration. Four weeks after injection the thalamus was isolated for NGS analysis. Whole transcriptome sequencing showed that the A-to-G RNA editing index was unaffected by treatment with the targeting guide RNA compared to the scrambled non-targeting control. We were unable to identify any global off-target events, excluding mouse to mouse variability, which suggests a very high precision of our approach on the transcriptome-wide level. Harnessing endogenous ADAR with permanent, AAV-driven CLUSTER guide RNAs in the CNS is an important next step towards the development of novel drug modalities that fight neurological diseases.

2024-06-27 | GSE265898 | GEO

Precise Genome Editing in vivo with Single, Canonical AAV Vectors. Encoding Guide RNA, Cas9, and Homologous Repair Donor

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2023-01-10 | GSE222531 | GEO

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