Source 1primary paper2022
Toolkit/mOptoT7
mOptoT7
Also known as: mammalian OptoT7, mOptoT7, Opto-T7RNAPs
Taxonomy: Mechanism Branch / Architecture. Workflows sit above the mechanism and technique branches rather than replacing them.
Summary
mOptoT7 is a mammalian optogenetic transcription system composed of a split T7 RNA polymerase fused to the blue-light-inducible nMag/pMag Magnets photodimerization system. Blue light drives reconstitution of the split polymerase to activate transcription from orthogonal T7 promoters in mammalian cells, and the system has been used to produce protein-coding mRNA, shRNA, and the Pepper RNA aptamer.
Usefulness & Problems
Why this is useful
mOptoT7 provides light-gated control of an orthogonal transcriptional program in mammalian cells, enabling inducible expression without relying on endogenous mammalian transcription machinery. Reported applications include protein expression, protein inhibition via shRNA, and RNA visualization via Pepper aptamer, and the system was also reported to mitigate gene-expression burden relative to another optogenetic construct.
Source:
mOptoT7 is used here to generate mRNA for protein expression, shRNA for protein inhibition, and Pepper aptamer for RNA visualization
Problem solved
mOptoT7 addresses the need for temporally controlled, light-responsive gene expression in mammalian cells using a transcription system that is orthogonal to host transcriptional machinery. It also addresses the challenge of improving responsiveness at low light intensity through transfer of evolved Magnets variants, which the authors state can reduce potential phototoxicity in long-term experiments.
Source:
mOptoT7 is used here to generate mRNA for protein expression, shRNA for protein inhibition, and Pepper aptamer for RNA visualization
Taxonomy & Function
Primary hierarchy
Mechanism Branch
Architecture: A composed arrangement of multiple parts that instantiates one or more mechanisms.
Techniques
No technique tags yet.
Target processes
recombinationtranscriptionInput: Light
Implementation Constraints
The system consists of a split T7 RNA polymerase coupled by domain fusion to the blue-light-inducible nMag/pMag Magnets pair and is implemented in mammalian cells. Performance was improved by expression tuning, and additional low-light-responsive variants were obtained by transferring Magnets variants generated through directed evolution and high-throughput screening.
The supplied evidence does not report absolute expression levels, kinetics, reversibility, or validation across multiple mammalian cell types or in vivo settings. Claims about reduced phototoxicity are presented as an inferred benefit of low-light operation rather than direct phototoxicity measurements in the provided evidence.
Validation
Cell-freeBacteriaMammalianMouseHumanTherapeuticIndep. Replication
Observations
successMammalian Cell Lineapplication demo
Inferred from claim c2 during normalization. mOptoT7 reached almost 20-fold light activation over dark control after expression tuning. Derived from claim c2. Quoted text: reaching up to an almost 20-fold change activation over the dark control
Source:
fold change activation over dark control20 fold(up to)
Supporting Sources
Source 2primary paper2022ACS Synthetic Biology
Ranked Claims
mOptoT7 reached almost 20-fold light activation over dark control after expression tuning.
reaching up to an almost 20-fold change activation over the dark control
fold change activation over dark control 20 fold
mOptoT7 was used to generate mRNA for protein expression, shRNA for protein inhibition, and Pepper aptamer for RNA visualization.
mOptoT7 is used here to generate mRNA for protein expression, shRNA for protein inhibition, and Pepper aptamer for RNA visualization
mOptoT7 can mitigate gene expression burden compared with another optogenetic construct.
we show that mOptoT7 can mitigate the gene expression burden when compared to another optogenetic construct
The authors developed and applied a directed evolution and high-throughput screening strategy to alter the light sensitivity of the nMag/pMag photodimerization system.
we develop and apply a simple, yet powerful, directed evolution and high-throughput screening strategy that allows us to alter the most fundamental property of the widely used nMag/pMag photodimerization system: its light sensitivity
The authors developed and applied a directed evolution and high-throughput screening strategy to alter the light sensitivity of the nMag/pMag photodimerization system.
we develop and apply a simple, yet powerful, directed evolution and high-throughput screening strategy that allows us to alter the most fundamental property of the widely used nMag/pMag photodimerization system: its light sensitivity
The authors developed and applied a directed evolution and high-throughput screening strategy to alter the light sensitivity of the nMag/pMag photodimerization system.
we develop and apply a simple, yet powerful, directed evolution and high-throughput screening strategy that allows us to alter the most fundamental property of the widely used nMag/pMag photodimerization system: its light sensitivity
The authors developed and applied a directed evolution and high-throughput screening strategy to alter the light sensitivity of the nMag/pMag photodimerization system.
we develop and apply a simple, yet powerful, directed evolution and high-throughput screening strategy that allows us to alter the most fundamental property of the widely used nMag/pMag photodimerization system: its light sensitivity
The authors developed and applied a directed evolution and high-throughput screening strategy to alter the light sensitivity of the nMag/pMag photodimerization system.
we develop and apply a simple, yet powerful, directed evolution and high-throughput screening strategy that allows us to alter the most fundamental property of the widely used nMag/pMag photodimerization system: its light sensitivity
The authors developed and applied a directed evolution and high-throughput screening strategy to alter the light sensitivity of the nMag/pMag photodimerization system.
we develop and apply a simple, yet powerful, directed evolution and high-throughput screening strategy that allows us to alter the most fundamental property of the widely used nMag/pMag photodimerization system: its light sensitivity
The authors developed and applied a directed evolution and high-throughput screening strategy to alter the light sensitivity of the nMag/pMag photodimerization system.
we develop and apply a simple, yet powerful, directed evolution and high-throughput screening strategy that allows us to alter the most fundamental property of the widely used nMag/pMag photodimerization system: its light sensitivity
mOptoT7 is orthogonal to mammalian cellular transcriptional machinery.
The tool is orthogonal to the cellular machinery for transcription
Transferred variants in mOptoT7 increased gene expression levels at low light intensities, which the authors state results in reduced potential phototoxicity in long-term experiments.
We demonstrate increased gene expression levels for low light intensities, resulting in reduced potential phototoxicity in long-term experiments.
Transferred variants in mOptoT7 increased gene expression levels at low light intensities, which the authors state results in reduced potential phototoxicity in long-term experiments.
We demonstrate increased gene expression levels for low light intensities, resulting in reduced potential phototoxicity in long-term experiments.
Transferred variants in mOptoT7 increased gene expression levels at low light intensities, which the authors state results in reduced potential phototoxicity in long-term experiments.
We demonstrate increased gene expression levels for low light intensities, resulting in reduced potential phototoxicity in long-term experiments.
Transferred variants in mOptoT7 increased gene expression levels at low light intensities, which the authors state results in reduced potential phototoxicity in long-term experiments.
We demonstrate increased gene expression levels for low light intensities, resulting in reduced potential phototoxicity in long-term experiments.
Transferred variants in mOptoT7 increased gene expression levels at low light intensities, which the authors state results in reduced potential phototoxicity in long-term experiments.
We demonstrate increased gene expression levels for low light intensities, resulting in reduced potential phototoxicity in long-term experiments.
Transferred variants in mOptoT7 increased gene expression levels at low light intensities, which the authors state results in reduced potential phototoxicity in long-term experiments.
We demonstrate increased gene expression levels for low light intensities, resulting in reduced potential phototoxicity in long-term experiments.
Transferred variants in mOptoT7 increased gene expression levels at low light intensities, which the authors state results in reduced potential phototoxicity in long-term experiments.
We demonstrate increased gene expression levels for low light intensities, resulting in reduced potential phototoxicity in long-term experiments.
For some variants, photosensitivity and expression levels could be changed independently, enabling tuning of the light-activity dose-response curve.
For some of these variants, photosensitivity and expression levels could be changed independently, showing that the shape of the light-activity dose-response curve can be tuned and adjusted.
For some variants, photosensitivity and expression levels could be changed independently, enabling tuning of the light-activity dose-response curve.
For some of these variants, photosensitivity and expression levels could be changed independently, showing that the shape of the light-activity dose-response curve can be tuned and adjusted.
For some variants, photosensitivity and expression levels could be changed independently, enabling tuning of the light-activity dose-response curve.
For some of these variants, photosensitivity and expression levels could be changed independently, showing that the shape of the light-activity dose-response curve can be tuned and adjusted.
For some variants, photosensitivity and expression levels could be changed independently, enabling tuning of the light-activity dose-response curve.
For some of these variants, photosensitivity and expression levels could be changed independently, showing that the shape of the light-activity dose-response curve can be tuned and adjusted.
For some variants, photosensitivity and expression levels could be changed independently, enabling tuning of the light-activity dose-response curve.
For some of these variants, photosensitivity and expression levels could be changed independently, showing that the shape of the light-activity dose-response curve can be tuned and adjusted.
For some variants, photosensitivity and expression levels could be changed independently, enabling tuning of the light-activity dose-response curve.
For some of these variants, photosensitivity and expression levels could be changed independently, showing that the shape of the light-activity dose-response curve can be tuned and adjusted.
For some variants, photosensitivity and expression levels could be changed independently, enabling tuning of the light-activity dose-response curve.
For some of these variants, photosensitivity and expression levels could be changed independently, showing that the shape of the light-activity dose-response curve can be tuned and adjusted.
Mutations within the photosensory domains were identified that increase or decrease light sensitivity at sub-saturating light intensities, and some variants also improve dark-to-light fold change.
We identify a set of mutations located within the photosensory domains, which either increase or decrease the light sensitivity at sub-saturating light intensities, while also improving the dark-to-light fold change in certain variants.
Mutations within the photosensory domains were identified that increase or decrease light sensitivity at sub-saturating light intensities, and some variants also improve dark-to-light fold change.
We identify a set of mutations located within the photosensory domains, which either increase or decrease the light sensitivity at sub-saturating light intensities, while also improving the dark-to-light fold change in certain variants.
Mutations within the photosensory domains were identified that increase or decrease light sensitivity at sub-saturating light intensities, and some variants also improve dark-to-light fold change.
We identify a set of mutations located within the photosensory domains, which either increase or decrease the light sensitivity at sub-saturating light intensities, while also improving the dark-to-light fold change in certain variants.
Mutations within the photosensory domains were identified that increase or decrease light sensitivity at sub-saturating light intensities, and some variants also improve dark-to-light fold change.
We identify a set of mutations located within the photosensory domains, which either increase or decrease the light sensitivity at sub-saturating light intensities, while also improving the dark-to-light fold change in certain variants.
Mutations within the photosensory domains were identified that increase or decrease light sensitivity at sub-saturating light intensities, and some variants also improve dark-to-light fold change.
We identify a set of mutations located within the photosensory domains, which either increase or decrease the light sensitivity at sub-saturating light intensities, while also improving the dark-to-light fold change in certain variants.
Mutations within the photosensory domains were identified that increase or decrease light sensitivity at sub-saturating light intensities, and some variants also improve dark-to-light fold change.
We identify a set of mutations located within the photosensory domains, which either increase or decrease the light sensitivity at sub-saturating light intensities, while also improving the dark-to-light fold change in certain variants.
Mutations within the photosensory domains were identified that increase or decrease light sensitivity at sub-saturating light intensities, and some variants also improve dark-to-light fold change.
We identify a set of mutations located within the photosensory domains, which either increase or decrease the light sensitivity at sub-saturating light intensities, while also improving the dark-to-light fold change in certain variants.
A subset of Magnets variants can be transferred into mOptoT7 for gene expression regulation in mammalian cells.
We further show that a subset of these variants can be transferred into the mOptoT7 for gene expression regulation in mammalian cells.
A subset of Magnets variants can be transferred into mOptoT7 for gene expression regulation in mammalian cells.
We further show that a subset of these variants can be transferred into the mOptoT7 for gene expression regulation in mammalian cells.
A subset of Magnets variants can be transferred into mOptoT7 for gene expression regulation in mammalian cells.
We further show that a subset of these variants can be transferred into the mOptoT7 for gene expression regulation in mammalian cells.
A subset of Magnets variants can be transferred into mOptoT7 for gene expression regulation in mammalian cells.
We further show that a subset of these variants can be transferred into the mOptoT7 for gene expression regulation in mammalian cells.
A subset of Magnets variants can be transferred into mOptoT7 for gene expression regulation in mammalian cells.
We further show that a subset of these variants can be transferred into the mOptoT7 for gene expression regulation in mammalian cells.
A subset of Magnets variants can be transferred into mOptoT7 for gene expression regulation in mammalian cells.
We further show that a subset of these variants can be transferred into the mOptoT7 for gene expression regulation in mammalian cells.
A subset of Magnets variants can be transferred into mOptoT7 for gene expression regulation in mammalian cells.
We further show that a subset of these variants can be transferred into the mOptoT7 for gene expression regulation in mammalian cells.
Approval Evidence
2 sources6 linked approval claimsfirst-pass slug moptot7
transferred into the mOptoT7 for gene expression regulation in mammalian cells
Source:
Here we implement, characterize, and optimize a new optogenetic tool in mammalian cells based on a previously published system in bacteria called Opto-T7RNAPs. The tool is orthogonal to the cellular machinery for transcription and consists of a split T7 RNA polymerase coupled with the blue light-inducible magnets system (mammalian OptoT7-mOptoT7).
Source:
activation dynamic rangesupports
mOptoT7 reached almost 20-fold light activation over dark control after expression tuning.
reaching up to an almost 20-fold change activation over the dark control
Source:
application scopesupports
mOptoT7 was used to generate mRNA for protein expression, shRNA for protein inhibition, and Pepper aptamer for RNA visualization.
mOptoT7 is used here to generate mRNA for protein expression, shRNA for protein inhibition, and Pepper aptamer for RNA visualization
Source:
burden comparisonsupports
mOptoT7 can mitigate gene expression burden compared with another optogenetic construct.
we show that mOptoT7 can mitigate the gene expression burden when compared to another optogenetic construct
Source:
orthogonalitysupports
mOptoT7 is orthogonal to mammalian cellular transcriptional machinery.
The tool is orthogonal to the cellular machinery for transcription
Source:
performance improvementsupports
Transferred variants in mOptoT7 increased gene expression levels at low light intensities, which the authors state results in reduced potential phototoxicity in long-term experiments.
We demonstrate increased gene expression levels for low light intensities, resulting in reduced potential phototoxicity in long-term experiments.
Source:
transferabilitysupports
A subset of Magnets variants can be transferred into mOptoT7 for gene expression regulation in mammalian cells.
We further show that a subset of these variants can be transferred into the mOptoT7 for gene expression regulation in mammalian cells.
Source:
Comparisons
Source-backed strengths
After expression tuning, mOptoT7 achieved almost 20-fold light activation over dark control. The platform was demonstrated across multiple output modalities—protein-coding mRNA, shRNA, and Pepper aptamer—and transferred Magnets variants increased gene expression at low light intensities.
Source:
we develop and apply a simple, yet powerful, directed evolution and high-throughput screening strategy that allows us to alter the most fundamental property of the widely used nMag/pMag photodimerization system: its light sensitivity
Source:
We demonstrate increased gene expression levels for low light intensities, resulting in reduced potential phototoxicity in long-term experiments.
Ranked Citations
- 1.