Optoribogenetic control of regulatory RNA molecules

Pilsl, Sebastian; Morgan, Charles H.; Choukeife, Moujab; Möglich, Andreas; Mayer, Günter

doi:10.1038/s41467-020-18673-5

Cited by 18 publications

(22 citation statements)

References 44 publications

(49 reference statements)

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“…This photo-controlled PAL-RNA interaction has been further used to reversibly regulate the cellular functions of micro RNAs and short hairpin RNAs in HEK293 cells. 170 Our group has also recently demonstrated a genetically encoded RNA aptamer-based photosensitizer system, termed GRAP, for targeted cell regulation in both prokaryotic and eukaryotic cells. 171 These photosensitizers can generate reactive oxygen species (ROS) upon light irradiation and lead to cell structure damage and photodynamic therapy.…”

Section: Genetically Encoded Photo-responsive Rna Nanodevicesmentioning

confidence: 99%

Genetically encoded RNA nanodevices for cellular imaging and regulation

You

2021

Nanoscale

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Section: Genetically Encoded Photo-responsive Rna Nanodevicesmentioning

confidence: 99%

Genetically encoded RNA nanodevices for cellular imaging and regulation

You

2021

Nanoscale

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“…Only few light‐regulated RNA modification tools currently exist, mostly based on RNA interference or short regulatory RNAs, which are CRISPR‐independent. [ 20–23 ]…”

Section: Figurementioning

confidence: 99%

“…Only few light-regulated RNA modification tools currently exist, mostly based on RNA interference or short regulatory RNAs, which are CRISPR-independent. [20][21][22][23] We devised here an optogenetic tool to destabilize cellular mRNA by enabling optical control of RNA-targeting CRISPR/ Cas13 systems. For this, we combined a blue light-inducible gene expression switch [24] with the Prevotella sp.-derived Cas13b effector (PspCas13b), [9] resulting in a system termed Lockdown (blue light-operated CRISPR/Cas13b-mediated mRNA knockdown).…”

mentioning

confidence: 99%

“…[42,43] However, only a few optogenetic tools for the control of mRNA exist. [20][21][22][23] The here-described Lockdown system combines optogenetic regulation of transcription with RNA-guided RNA processing by the PspCas13b ribonucleoprotein to generate optogenetic control of RNA degradation. Lockdown is compatible and synergistically active with the Blue-OFF system, achieving nearly complete depletion of a protein through combined blue light-triple-induction of transcriptional repression, and mRNA and protein degradation.…”

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confidence: 99%

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Blue Light‐Operated CRISPR/Cas13b‐Mediated mRNA Knockdown (Lockdown)

et al. 2021

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that derive from clustered regularly interspaced short palindromic repeats (CRISPR)-based techniques, currently positively reshapes biological studies. [2] Key applications of the specific DNA-recognizing CRISPR/Cas9 systems encompass for example genome editing and transcriptional regulation with high sequence specificity. [2][3][4][5][6] In recent years, these technologies led to major advances in synthetic biology, gene therapy, and gene modification in almost every model organism. Various Cas-variants were subsequently derived from different microorganisms and utilized to overcome some of the limitations restricting in vivo applications, or enabled the recognition of RNA instead of DNA. [2,[7][8][9] Among those, the discovery of the RNA-targeting Cas13 proteins yielded in powerful RNA-editing tools. [9,10] Cas13 belongs to type VI CRISPR effectors and has been utilized for specific knockdown of endogenous RNAs in human cells and manipulation of alternative RNA splicing. [9,11] While the DNAtargeting CRISPR/Cas9 systems require the presence of a short protospacer adjacent motif (PAM) sequence at the editing site, Cas13 is PAM-independent. [9,10] Recent engineering efforts of CRISPR/Cas tools for optogenetic regulation expanded their capabilities including high spatial and temporal control precision, [12][13][14][15][16][17][18][19] however, these systems exclusively target DNA. Only few light-regulated RNA modification tools currently exist, mostly based on RNA interference or short regulatory RNAs, which are CRISPR-independent. [20][21][22][23] We devised here an optogenetic tool to destabilize cellular mRNA by enabling optical control of RNA-targeting CRISPR/ Cas13 systems. For this, we combined a blue light-inducible gene expression switch [24] with the Prevotella sp.-derived Cas13b effector (PspCas13b), [9] resulting in a system termed Lockdown (blue light-operated CRISPR/Cas13b-mediated mRNA knockdown). Blue light activates the gene switch to induce the expression of PspCas13b which, in the presence of a gRNA targeting the mRNA of interest, leads to downregulation of said RNA. Our results demonstrate how Lockdown can be used to regulate cellular processes with blue light through specific mRNA degradation. In our tests, these induced G2 cell cycle arrest and inhibition of cell growth under blue light. [25,26] We further combined Lockdown with the recently published "Blue-OFF" system for synergistic triple-targeted downregulation of proteins. [27,28] To engineer the Lockdown system, we used a split transcription factor based on a modified light-oxygen-voltage domain The introduction of optogenetics into cell biology has furnished systems to control gene expression at the transcriptional and protein stability level, with a high degree of spatial, temporal, and dynamic light-regulation capabilities. Strategies to downregulate RNA currently rely on RNA interference and CRISPR/Cas-related methods. However, these approaches lack the key characteristics and advantages provided by optical control. "Lockdown" introd...

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“…Another approach, light-inducible knock-downs, has only been achieved in cell lines, for example with photo-caged oligonucleotides/siRNAs (e.g. Mikat et al, 2007), or more recently with a genetically encoded optogenetic RNA interference (RNAi) system (Pilsl et al, 2020). While these methods are efficient and allow for precise temporal control in cell lines, their efficacy is unknown in more complex tissues and may be limited.…”

Section: Introductionmentioning

confidence: 99%

Spatio-temporal, optogenetic control of gene expression in organoids

Legnini

Emmenegger

Wurmus

et al. 2021

Preprint

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Quantifying gene expression in space, for example by spatial transcriptomics, is essential for describing the biology of cells and their interactions in complex tissues. Perturbation experiments, at single-cell resolution and conditional on both space and time, are necessary for dissecting the molecular mechanisms of these interactions. To this aim, we combined optogenetics and CRISPR technologies to activate or knock-down RNA of target genes, at single-cell resolution and in programmable spatial patterns. As a proof of principle, we optogenetically induced Sonic Hedgehog (SHH) signaling at a distinct spatial location within human neural organoids. This robustly induced known SHH spatial domains of gene expression, cell-autonomously and across the entire organoid. In principle, our approach can be used to induce or knock down RNAs from any gene of interest in specific spatial locations or patterns of complex biological systems.

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Optoribogenetic control of regulatory RNA molecules

Cited by 18 publications

References 44 publications

Genetically encoded RNA nanodevices for cellular imaging and regulation

Genetically encoded RNA nanodevices for cellular imaging and regulation

Blue Light‐Operated CRISPR/Cas13b‐Mediated mRNA Knockdown (Lockdown)

Spatio-temporal, optogenetic control of gene expression in organoids

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