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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jun 27, 2024 |
| Title |
Precise in-vivo RNA base editing with a wobble-enhanced circular CLUSTER guide RNA |
| Organism |
Mus musculus |
| Experiment type |
Other
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| Summary |
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.
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| Overall design |
Four weeks post injection, brains were dissected in 5 mM HEPES in HBSS and total RNA was isolated from individual brain regions using QIAzol reagent (Qiagen, #79306) and the Qiagen miRNeasy Kit reagent (Qiagen, #217004) according to the manufacturer’s instructions. Purified RNA was delivered to CeGaT for total RNA‐seq. The library was prepared with the KAPA RNA HyperPrep Library Prep Kit with RiboErase (HMR) and KAPA Globin Depletion Hybridization Oligos (Roche) and sequenced with a NovaSeq 6000 (50 M reads, 2 × 100‐bp paired‐end; Illumina).
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| Web link |
https://pubmed.ncbi.nlm.nih.gov/38997581/
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| Contributor(s) |
Reautschnig P, Fruhner C, Wahn N, Wiegand CP, Kragness S, Yung JF, Hofacker DT, Fisk J, Eidelman M, Waffenschmidt N, Feige M, Pfeiffer LS, Schulz AE, Füll Y, Levanon EY, Mandel G, Stafforst T |
| Citation(s) |
38997581 |
| BioProject |
PRJNA1100948 |
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| Submission date |
Apr 25, 2024 |
| Last update date |
Sep 29, 2024 |
| Contact name |
Philipp Reautschnig |
| Organization name |
University of Tübingen
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| Street address |
Auf der Morgenstelle
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| City |
Tübingen |
| ZIP/Postal code |
72076 |
| Country |
Germany |
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| Platforms (1) |
| GPL24247 |
Illumina NovaSeq 6000 (Mus musculus) |
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| Samples (4)
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| Supplementary file |
Size |
Download |
File type/resource |
| GSE265898_A2GEditingIndex.csv.gz |
98 b |
(ftp)(http) |
CSV |
| GSE265898_A2GEditingIndex_S9664Nr3.csv.gz |
82 b |
(ftp)(http) |
CSV |
| GSE265898_RAW.tar |
130.0 Kb |
(http)(custom) |
TAR (of CSV) |
SRA Run Selector |
| Raw data are available in SRA |
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