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

Reautschnig, Philipp; Wahn, Nicolai; Wettengel, Jacqueline; Schulz, Annika E; Latifi, Ngadhnjim; Vogel, P.; Kang, Tae‐Won; Pfeiffer, Laura S.; Zarges, Christine; Naumann, Ulrike; Zender, Lars; Li, Jin Billy; Stafforst, Thorsten

doi:10.1038/s41587-021-01105-0

Cited by 66 publications

(47 citation statements)

References 40 publications

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“…In the future, large-scale screening efforts can probe the determinants of sensor editing, including secondary structure and sequence properties that affect readthrough, in the presence and absence of targets, ultimately yielding versatile guide design rules. Here we find that MS2 loops can block readthrough, recruit MCP-ADAR2dd, and potentially stabilize the multivalent guide binding regions and prevent self-folding, as has been seen with similar therapeutic RNA editing systems 20 , and it is likely that additional engineering improvements at both the ADAR protein and guide level could further increase editing and sensor output. For applications in real-time RNA monitoring, more work is needed on tuning the RADARS kinetic properties.…”

Section: Discussionsupporting

confidence: 60%

Programmable eukaryotic protein expression with RNA sensors

Jiang

Koob

Chen

et al. 2022

Preprint

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The diversity of cell types and states can be scalably measured and defined by expressed RNA transcripts. However, approaches to programmably sense and respond to the presence of specific RNAs within living biological systems with high sensitivity are lacking. RNA sensors that gate expression of reporter or cargo genes would have diverse applications for basic biology, diagnostics and therapeutics by enabling cell-state specific control of transgene expression. Here, we engineer a novel programmable RNA-sensing technology, Reprogrammable ADAR Sensors (RADARS), which leverages RNA editing by adenosine deaminases acting on RNA (ADAR) to gate translation of a protein payload on the presence of endogenous RNA transcripts. In mammalian cells, we engineer RADARS with diverse payloads, including luciferase and fluorescent proteins, with up to 164-fold activation and quantitative detection in the presence of target RNAs. We show RADARS are functional either expressed from DNA or as synthetic mRNA. Importantly, RADARS can function with endogenous cellular ADAR. We apply RADARS to multiple contexts, including RNA-sensing induced cell death via caspases, cell type identification, and in vivo control of synthetic mRNA translation, demonstrating RADARS as a tool with significant potential for gene and cell therapy, synthetic biology, and biomedical research.

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Section: Discussionsupporting

confidence: 60%

Programmable eukaryotic protein expression with RNA sensors

Jiang

Koob

Chen

et al. 2022

Preprint

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“…Some early approaches to retargeting ADAR RNA editing used fusions of other RNAbinding proteins to an ADAR deaminase domain and some of these gave efficient target editing; however, they also identified a critical problem arising from high off-target editing (Montiel-Gonzalez et al 2019). This, and difficulties expected with expression of proteins and possible immunogenicity of foreign proteins in humans mean that current approaches concentrate on retargeting endogenous ADAR proteins by providing an ADAR-recruiting 'guide' RNA that will also base pair across the mutation site and make the target adenosine available for editing (Katrekar et al 2022;Reautschnig et al 2022;Yi et al 2022). Studies on ADAR2 have been the predominant source of information on how ADARs recognize RNA substrates for highly efficient site-specific RNA editing and on how we might design guide RNAs to retarget ADARs for gene therapy.…”

Section: Applications Of Adars In Gene Therapymentioning

confidence: 99%

ADAR2 enzymes: efficient site-specific RNA editors with gene therapy aspirations

Hajji

Sedmik

Cherian

et al. 2022

RNA

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The adenosine deaminase acting on RNA (ADAR) enzymes are essential for neuronal function and innate immune control. ADAR1 RNA editing prevents aberrant activation of antiviral dsRNA sensors, through editing of long double-stranded RNAs (dsRNAs). In this review we focus on the ADAR2 proteins involved in the efficient, highly site-specific RNA editing to recode open reading frames first discovered in the GRIA2 transcript encoding the key GLUA2 subunit of AMPA receptors; ADAR1 proteins also edits many sites. We summarize the history of ADAR2 protein research and give an up to date review of ADAR2 structural studies, human ADARBI(ADAR2) mutants causing severe infant seizures and mouse disease models. Structural studies on ADARs and their RNA substrates facilitate current efforts to develop ADAR RNA editing gene therapy to edit disease-causing single nucleotide polymorphisms (SNPs). Artificial ADAR guide RNAs are being developed to retarget ADAR RNA editing to new target transcripts in order to correct SNP mutations in them at the RNA level. Site-specific RNA editing has been expanded to recode hundreds of sites in CNS transcripts in Drosophila and cephalopods. In Drosophila and C. elegans ADAR RNA editing also suppresses responses to self dsRNA.

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“…Second, motivated by work showing that ADAR enzymes are tolerant of or even benefited by disruptions in the target dsRNA 25 , we hypothesized that we could rescue the signal from the non-functional 36 bp sensor if we introduced an additional 54 bp sequence complementary to another part of the long Bdnf 3' UTR, so that a 90 bp discontiguous dsRNA would be formed between the sensor and trigger. A similar strategy of split complementarity has recently been described to benefit RNA editing with endogenous ADARs 23 . A suitable short, 36 bp sensor sequence can be more readily identified in a transcript compared to a longer sequence, and nearly all transcripts have at least one 54 bp stretch without an in-frame stop in the reverse complement.…”

Section: Resultsmentioning

confidence: 99%

“…ADAR editing also tends to bias the cellular response away from the two other pathways, as A-to-I edited dsRNA stretches are poor substrates for the immune receptors and RNAi 13,14 , further reducing unintended interactions between the sensor and the cellular context. In addition to the intrinsic benefits of ADAR, our engineering efforts are facilitated by the rich knowledge from an active field that has investigated the basic biology of ADAR [15][16][17][18][19] and is applying it to the editing of disease-causing transcripts in gene therapies [20][21][22][23] .…”

Section: Introductionmentioning

confidence: 99%

Modular and programmable RNA sensing using ADAR editing in living cells

Kaseniit

Katz

Kolber

et al. 2022

Preprint

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With the increasing availability of single-cell transcriptomes, RNA signatures offer a promising basis for targeting living cells. Molecular RNA sensors would enable the study of and therapeutic interventions for specific cell types/stats in diverse contexts, particularly in human patients and non-model organisms. Here we describe a modular and programmable design for live RNA sensing using adenosine deaminases acting on RNA (RADAR). We validated and then expanded our basic design, characterized its performance, and thoroughly analyzed its compatibility with the human/mouse transcriptomes. We also identified strategies to further boost output levels and improve the dynamic range. We show that RADAR is programmable and modular, and uniquely enables compact AND logic. In addition to being quantitative, RADAR can distinguish disease-relevant point mutations. Finally, we demonstrate that RADAR is a self-contained system with the potential to function in diverse organisms.

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

Cited by 66 publications

References 40 publications

Programmable eukaryotic protein expression with RNA sensors

Programmable eukaryotic protein expression with RNA sensors

ADAR2 enzymes: efficient site-specific RNA editors with gene therapy aspirations

Modular and programmable RNA sensing using ADAR editing in living cells

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