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Comprehensive interrogation of the ADAR2 deaminase domain for engineering enhanced RNA base-editing activity, functionality and specificity

ABSTRACT: Comprehensive interrogation of the ADAR2 deaminase domain for engineering enhanced RNA base-editing activity, functionality and specificity

PROVIDER: PRJNA666136 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR12729451_1.fastq.gz	Fastqsanger.gz
	SRR12729451_2.fastq.gz	Fastqsanger.gz
	SRR12729452_1.fastq.gz	Fastqsanger.gz
	SRR12729452_2.fastq.gz	Fastqsanger.gz
	SRR12729453_1.fastq.gz	Fastqsanger.gz

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Comprehensive interrogation of the ADAR2 deaminase domain for engineering enhanced RNA base-editing activity, functionality and specificity

Project description:We use RNA-seq to study the transcriptome wide specificity profiles of A-to-I RNA editing by split-ADAR2 constructs.

2022-01-19 | GSE158656 | GEO

Comprehensive interrogation of the ADAR2 deaminase domain for engineering enhanced RNA editing activity and specificity.

Project description:Adenosine deaminases acting on RNA (ADARs) can be repurposed to enable programmable RNA editing, however their exogenous delivery leads to transcriptome-wide off-targeting, and additionally, enzymatic activity on certain RNA motifs, especially those flanked by a 5' guanosine is very low thus limiting their utility as a transcriptome engineering toolset. Towards addressing these issues, we first performed a novel deep mutational scan of the ADAR2 deaminase domain, directly measuring the impact of every amino acid substitution across 261 residues, on RNA editing. This enabled us to create a domain-wide mutagenesis map while also revealing a novel hyperactive variant with improved enzymatic activity at 5'-GAN-3' motifs. As overexpression of ADAR enzymes, especially hyperactive variants, can lead to significant transcriptome-wide off-targeting, we next engineered a split-ADAR2 deaminase which resulted in >100-fold more specific RNA editing as compared to full-length deaminase overexpression. Taken together, we anticipate this systematic engineering of the ADAR2 deaminase domain will enable broader utility of the ADAR toolset for RNA biotechnology applications.

| S-EPMC8809894 | biostudies-literature

Knockdown of RNA deaminase ADAR2 in Th17 cells

Project description:Knockdown of RNA deaminase ADAR2 in Th17 cells

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Overexpression of RNA deaminase ADAR2 in Th17 cells

Project description:Overexpression of RNA deaminase ADAR2 in Th17 cells

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Overexpression of RNA deaminase ADAR2 in Th17 cells

Project description:ADAR2 is induced upon T cell activation and its immunological function is not known. Here, we performed RNA-seq on control and Th17 cells overexpressing wildtype or catalytic dead ADAR2 to identify A-to-I edited transcripts that are targets of ADAR2.

2024-12-31 | GSE227153 | GEO

Knockdown of RNA deaminase ADAR2 in Th17 cells

Project description:ADAR2 is induced upon T cell activation and its immunological function is not known. Here, we performed RNA-seq on Cas9+ control and Th17 cells expressing the PSIN empty vector control or guide RNAs against the ATG start codon or ADAR2 to identify ADAR2 dependent A-to-I edited transcripts.

2024-12-31 | GSE227152 | GEO

Next Generation Sequencing Analysis of Wild Type, ADAR2 over expressing and ADAR2+SRSF9 overexpresssing SH-Sy5y cells Transcriptomes

Project description:Purpose: A-to-I RNA editing is critical for many cellular processes. The sites of A-I RNA editing can be identified through RNA-seq and matching the reads to the annotated database like RADAR. The goals of this study is to compare A-I RNA editing profile among wild type, ADAR2 overexpressing and ADAR2+SRSF9 overexpressing 293T cells to evaluate the influence of SRSF9 repressive action on A-I RNA editing Methods: A-I RNA editing profile and gene expression profiles were generated by deep sequencing from all the samples, using NEBNext® Ultra™ Directional RNA Library Prep Kit. The sequence reads that passed quality filters were analyzed for identifying the A-I RNA editing sites using RADAR Database Results: Upon overexpression of ADAR2, we found that 18,174 sites were differentially edited compared to control cells, of which 97.0% showed a significant increase in their editing levels as expected (P < 0.05, Fisher’s test). Furthermore, when we overexpressed both ADAR2 and SRSF9 together, we found that 6,994 sites were differentially edited compared to overexpression of the deaminase alone (P < 0.05, Fisher’s test). Importantly, the editing of 92.5% of these sites was down-regulated, consistent with the function of SRSF9 as a repressor of editing. Conclusions: Our study reports a detailed analysis of the effect of SRSF9 on A-I RNA editing of ADAR2-specific targets. We Conclude that this SRSF9 regulon is significantly enriched for brain-specific editing sites despite our unbiased approach.

2018-06-21 | GSE114970 | GEO

Next Generation Sequencing Analysis of Wild Type, ADAR2 over expressing and ADAR2+SRSF9 overexpresssing HEK 293T cells Transcriptomes

2018-06-21 | GSE108126 | GEO

MiRNAinU118_Cells_Inactive_vs_ active_ Adar2

Project description:MicroRNA expression profile of NB U118 cells transfected with a plasmid directing an inactive Adar2 vs an active Adar2 - 3 biological replicates 1 Condition experiment, 3 biological replicates, 2 replicates/array

2015-02-28 | E-GEOD-63694 | biostudies-arrayexpress

MiRNAinU118_Cells_Inactive_vs_ active_ Adar2

Project description:MicroRNA expression profile of NB U118 cells transfected with a plasmid directing an inactive Adar2 vs an active Adar2 - 3 biological replicates

2015-02-28 | GSE63694 | GEO

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