Modular, programmable RNA sensing using ADAR editing in living cells

Kaseniit, Kristjan Eerik; Katz, Noa; Kolber, Natalie S.; Call, Connor C.; Wengier, Diego L.; Cody, Will B; Sattely, Elizabeth S.; Gao, Xiaojing

doi:10.1038/s41587-022-01493-x

Cited by 61 publications

(33 citation statements)

References 34 publications

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“…Circuit-level analyses of the mouse brain have relied upon the availability of genetically delivered molecular tools to excite, inhibit, and record from individual neuronal populations. These tools have historically been delivered to specific subpopulations of neurons through the use of recombinase-based systems, but more recently, RNA editing-based strategies have been developed to enable translation of transgenes only in the presence of specific endogenous mRNA transcripts [25][26][27] . Both strategies require nominating small numbers of high-value marker genes that can optimally distinguish amongst many distinct clusters.…”

Section: Variation In Neuronal Diversity Across Neuroanatomical Struc...mentioning

confidence: 99%

The cell type composition of the adult mouse brain revealed by single cell and spatial genomics

Langlieb

Sachdev

Balderrama

et al. 2023

Preprint

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The function of the mammalian brain relies upon the specification and spatial positioning of diversely specialized cell types. Yet, the molecular identities of the cell types, and their positions within individual anatomical structures, remain incompletely known. To construct a comprehensive atlas of cell types in each brain structure, we paired high-throughput single-nucleus RNA-seq with Slide-seq, a recently developed spatial transcriptomics method with near-cellular resolution, across the entire mouse brain. Integration of these datasets revealed the cell type composition of each neuroanatomical structure. Cell type diversity was found to be remarkably high in the midbrain, hindbrain, and hypothalamus, with most clusters requiring a combination of at least three discrete gene expression markers to uniquely define them. Using these data, we developed a framework for genetically accessing each cell type, comprehensively characterized neuropeptide and neurotransmitter signaling, elucidated region-specific specializations in activity-regulated gene expression, and ascertained the heritability enrichment of neurological and psychiatric phenotypes. These data, available as an online resource (BrainCellData.org) should find diverse applications across neuroscience, including the construction of new genetic tools, and the prioritization of specific cell types and circuits in the study of brain diseases.

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Section: Variation In Neuronal Diversity Across Neuroanatomical Struc...mentioning

confidence: 99%

The cell type composition of the adult mouse brain revealed by single cell and spatial genomics

Langlieb

Sachdev

Balderrama

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

“…In such systems, the low levels of endogenous ADAR carry out editing of a subset of sensor molecules. (ii) Other implementations rely on constitutive overexpression of exogenous ADAR 9,11 , which efficiently mediates editing of sensor molecules while sacrificing ease-of-delivery and increasing the unnecessary consumption of cellular resources. (iii) A potential solution that builds on these approaches is based on conditional expression of exogenous ADAR.…”

Section: Identification Of Design Rules For Dart Vadar Sensorsmentioning

confidence: 99%

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Recently, the repertoire of transcript-sensing riboregulators was broadened to eukaryotes in a technology known as eToeholds, which relies on engineered mRNA internal ribosome entry sites (IRES) 8 . In eToeholds, inhibitory loops of IRES structures are disrupted upon hybridization with target RNAs, thereby restoring ribosome recruitment and enabling RNA-responsive translational control.Most recently, three groups have independently described convergent approaches to design RNA-based sensors [9][10][11] . In these preliminary reports, base editing by adenosine deaminases acting on RNAs (ADARs) couples the detection of an RNA trigger to the translation of a user-defined genetic payload (Fig.

…”

mentioning

confidence: 99%

“…Most recently, three groups have independently described convergent approaches to design RNA-based sensors [9][10][11] . In these preliminary reports, base editing by adenosine deaminases acting on RNAs (ADARs) couples the detection of an RNA trigger to the translation of a user-defined genetic payload (Fig.…”

mentioning

confidence: 99%

“…Therefore, circuits using endogenous levels of ADARs might not be as effective in other tissues. Overexpression of exogenous ADAR has been explored as a possible solution to enhance the performance of this class of circuits in cells with a lower supply of endogenous ADAR 9,11 (Fig. 1b).…”

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confidence: 99%

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Autocatalytic base editing for RNA-responsive translational control

et al. 2023

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Genetic circuits that control transgene expression in response to pre-defined transcriptional cues would enable the development of smart therapeutics. To this end, here we engineer programmable single-transcript RNA sensors in which adenosine deaminases acting on RNA (ADARs) autocatalytically convert target hybridization into a translational output. Dubbed DART VADAR (Detection and Amplification of RNA Triggers via ADAR), our system amplifies the signal from editing by endogenous ADAR through a positive feedback loop. Amplification is mediated by the expression of a hyperactive, minimal ADAR variant and its recruitment to the edit site via an orthogonal RNA targeting mechanism. This topology confers high dynamic range, low background, minimal off-target effects, and a small genetic footprint. We leverage DART VADAR to detect single nucleotide polymorphisms and modulate translation in response to endogenous transcript levels in mammalian cells.

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Harnessing Catalytic RNA Circuits for Construction of Artificial Signaling Pathways in Mammalian Cells

Wu,

Zhang

et al. 2024

Angewandte Chemie

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Engineering of genetic networks with artificial signaling pathways (ASPs) can reprogram cellular responses and phenotypes under different circumstances for a variety of diagnostic and therapeutic purposes. However, construction of ASPs between originally independent endogenous genes in mammalian cells is highly challenging. Here we report an amplifiable RNA circuit that can theoretically build regulatory connections between any endogenous genes in mammalian cells. We harness the system of catalytic hairpin assembly with combination of controllable CRISPR‐Cas9 function to transduce the signals from distinct messenger RNA expression of trigger genes into manipulation of target genes. Through introduction of these RNA‐based genetic circuits, mammalian cells are endowed with autonomous capabilities to sense the changes of RNA expression either induced by ligand stimuli or from various cell types and control the cellular responses and fates via apoptosis‐related ASPs. Our design provides a generalized platform for construction of ASPs inside the genetic networks of mammalian cells based on differentiated RNA expression.

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Modular, programmable RNA sensing using ADAR editing in living cells

Cited by 61 publications

References 34 publications

The cell type composition of the adult mouse brain revealed by single cell and spatial genomics

The cell type composition of the adult mouse brain revealed by single cell and spatial genomics

Autocatalytic base editing for RNA-responsive translational control

Harnessing Catalytic RNA Circuits for Construction of Artificial Signaling Pathways in Mammalian Cells

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