Source 1primary paper2026MED
Toolkit/nCas9ERT2
nCas9ERT2
Taxonomy: Mechanism Branch / Architecture. Workflows sit above the mechanism and technique branches rather than replacing them.
Summary
The nCas9ERT2 fusion protein, consisting of Cas9 and an estrogen receptor flanked by two nuclear localization signals, is usually located in the cytoplasm and efficiently translocated into nuclei upon 4-hydroxytamoxifen (4-OHT) treatment in cultured cells or embryos.
Usefulness & Problems
Why this is useful
nCas9ERT2 is an inducible Cas9 fusion that remains mainly cytoplasmic until 4-OHT treatment promotes nuclear translocation, enabling genome editing at chosen stages or in chosen tissues. The abstract presents it as a tool for temporospatial gene editing at the organismal level.; temporospatial gene editing; stage-specific gene editing; tissue-specific gene editing; 4-OHT-inducible control of Cas9 nuclear entry
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nCas9ERT2 is an inducible Cas9 fusion that remains mainly cytoplasmic until 4-OHT treatment promotes nuclear translocation, enabling genome editing at chosen stages or in chosen tissues. The abstract presents it as a tool for temporospatial gene editing at the organismal level.
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temporospatial gene editing
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stage-specific gene editing
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tissue-specific gene editing
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4-OHT-inducible control of Cas9 nuclear entry
Problem solved
It addresses the need for conditional genome editing when gene function depends on tissue and developmental stage. The abstract specifically highlights the difficulty of inducible editing in zebrafish early embryogenesis.; addresses the challenge of spatiotemporally inducible genome editing in zebrafish, particularly during early embryogenesis
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It addresses the need for conditional genome editing when gene function depends on tissue and developmental stage. The abstract specifically highlights the difficulty of inducible editing in zebrafish early embryogenesis.
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addresses the challenge of spatiotemporally inducible genome editing in zebrafish, particularly during early embryogenesis
Problem links
addresses the challenge of spatiotemporally inducible genome editing in zebrafish, particularly during early embryogenesis
LiteratureIt addresses the need for conditional genome editing when gene function depends on tissue and developmental stage. The abstract specifically highlights the difficulty of inducible editing in zebrafish early embryogenesis.
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It addresses the need for conditional genome editing when gene function depends on tissue and developmental stage. The abstract specifically highlights the difficulty of inducible editing in zebrafish early embryogenesis.
Published Workflows
Objective: Establish an inducible Cas9-based approach for stage- and tissue-specific genome editing in whole organisms, especially for contexts where spatiotemporal control is challenging.
Why it works: The abstract links inducibility to cytoplasmic sequestration of nCas9ERT2 followed by 4-OHT-induced nuclear entry, which is expected to restrict editing to chosen times and tissues where the system is present.
4-OHT-triggered nuclear translocation of a Cas9-estrogen receptor fusioninducible genome editingtransgenic stable expression
Stages
- 1.Inducible localization characterization(functional_characterization)
This stage establishes the mechanistic basis for inducible editing before organism-level proof-of-concept applications.
Selection: Demonstrate that nCas9ERT2 is cytoplasmic before induction and translocates to nuclei after 4-OHT treatment.
- 2.Zebrafish proof-of-concept editing(confirmatory_validation)
This stage tests whether inducible nuclear translocation translates into functional genome editing in a whole-organism context.
Selection: Show that 4-OHT induction can trigger mutation of genes in defined zebrafish germline-related cell populations from a stable transgenic line.
- 3.Cross-species embryo validation(confirmatory_validation)
This stage extends the proof of concept to another vertebrate system to support broader organism-level utility.
Selection: Test whether the inducible system functions beyond zebrafish by applying it in early mouse embryos.
Steps
- 1.Treat cultured cells or embryos with 4-OHT and assess nCas9ERT2 nuclear translocationengineered inducible Cas9 construct being characterized
Establish the inducible localization mechanism of nCas9ERT2.
The abstract presents nuclear translocation as the mechanistic basis for later conditional editing demonstrations.
- 2.Induce editing in zebrafish transgenic lines stably expressing nCas9ERT2 and gRNAsediting effector in stable transgenic context
Demonstrate functional gene mutation in defined zebrafish cell types and stages after induction.
After showing inducible nuclear entry, the workflow tests whether that mechanism yields actual genome editing in vivo.
- 3.Test the inducible system in early mouse embryosinducible editing system under cross-species validation
Assess whether the inducible editing approach generalizes to another vertebrate embryo system.
This follows zebrafish proof of concept to support broader organism-level applicability.
Taxonomy & Function
Primary hierarchy
Mechanism Branch
Architecture: A reusable architecture pattern for arranging parts into an engineered system.
Mechanisms
crispr-cas9 genome editingligand-dependent control of subcellular localizationsmall-molecule-induced nuclear translocationTechniques
No technique tags yet.
Target processes
editinglocalizationImplementation Constraints
cofactor dependency: cofactor requirement unknownencoding mode: genetically encodedimplementation constraint: context specific validationoperating role: regulator
The system requires the nCas9ERT2 construct, guide RNAs, and 4-OHT treatment. The proof-of-concept zebrafish experiments also used a transgenic line with stable expression of nCas9ERT2 and gRNAs.; requires expression of nCas9ERT2; requires gRNAs; requires 4-hydroxytamoxifen treatment
The abstract does not show that the system solves off-target editing, background leakiness, or all conditional genetics use cases. It also does not establish quantitative superiority over other conditional editing systems.; requires 4-OHT induction; abstract provides no quantitative data on leakiness, efficiency, or off-target effects
Validation
Cell-freeBacteriaMammalianMouseHumanTherapeuticIndep. Replication
Observations
successMouseapplication demomouse
Inferred from claim c4 during normalization. The inducible nCas9ERT2 system also works in early mouse embryos. Derived from claim c4. Quoted text: The system also works in early mouse embryos.
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Supporting Sources
Ranked Claims
In zebrafish, genes in primordial germ cells in embryos and germ cells in adult ovaries can be mutated by 4-OHT induction from a transgenic line stably expressing nCas9ERT2 and gRNAs.
As a proof of concept, we demonstrate that genes in primordial germ cells in embryos and germ cells in adult ovaries from a transgenic line with stable expression of nCas9ERT2 and gRNAs can be mutated by 4-OHT induction.
The inducible nCas9ERT2 approach enables gene editing in defined cell types and developmental stages.
Here, we establish inducible Cas9-based editing in defined cell types and stages.
The inducible nCas9ERT2 system also works in early mouse embryos.
The system also works in early mouse embryos.
nCas9ERT2 is a Cas9-estrogen receptor fusion flanked by two nuclear localization signals that is usually cytoplasmic and translocates to nuclei upon 4-OHT treatment.
The nCas9ERT2 fusion protein, consisting of Cas9 and an estrogen receptor flanked by two nuclear localization signals, is usually located in the cytoplasm and efficiently translocated into nuclei upon 4-hydroxytamoxifen (4-OHT) treatment in cultured cells or embryos.
The inducible nCas9ERT2 approach enables temporospatial gene editing at the organismal level and expands the tissue- and stage-specific gene-editing toolkit.
Thus, this inducible nCas9ERT2 approach enables temporospatial gene editing at the organismal level, expanding the tissue- and stage-specific gene-editing toolkit.
Approval Evidence
1 source5 linked approval claimsfirst-pass slug ncas9ert2
The nCas9ERT2 fusion protein, consisting of Cas9 and an estrogen receptor flanked by two nuclear localization signals, is usually located in the cytoplasm and efficiently translocated into nuclei upon 4-hydroxytamoxifen (4-OHT) treatment in cultured cells or embryos.
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applicationsupports
In zebrafish, genes in primordial germ cells in embryos and germ cells in adult ovaries can be mutated by 4-OHT induction from a transgenic line stably expressing nCas9ERT2 and gRNAs.
As a proof of concept, we demonstrate that genes in primordial germ cells in embryos and germ cells in adult ovaries from a transgenic line with stable expression of nCas9ERT2 and gRNAs can be mutated by 4-OHT induction.
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capabilitysupports
The inducible nCas9ERT2 approach enables gene editing in defined cell types and developmental stages.
Here, we establish inducible Cas9-based editing in defined cell types and stages.
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cross species applicationsupports
The inducible nCas9ERT2 system also works in early mouse embryos.
The system also works in early mouse embryos.
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mechanismsupports
nCas9ERT2 is a Cas9-estrogen receptor fusion flanked by two nuclear localization signals that is usually cytoplasmic and translocates to nuclei upon 4-OHT treatment.
The nCas9ERT2 fusion protein, consisting of Cas9 and an estrogen receptor flanked by two nuclear localization signals, is usually located in the cytoplasm and efficiently translocated into nuclei upon 4-hydroxytamoxifen (4-OHT) treatment in cultured cells or embryos.
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utilitysupports
The inducible nCas9ERT2 approach enables temporospatial gene editing at the organismal level and expands the tissue- and stage-specific gene-editing toolkit.
Thus, this inducible nCas9ERT2 approach enables temporospatial gene editing at the organismal level, expanding the tissue- and stage-specific gene-editing toolkit.
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Comparisons
Source-stated alternatives
The web research summary identifies related conditionality or inducible systems such as CreERT2, HIT, and CRISPR-Bac, plus prior zebrafish tissue-specific Cas9 systems. The abstract itself does not directly compare nCas9ERT2 against these alternatives.
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The web research summary identifies related conditionality or inducible systems such as CreERT2, HIT, and CRISPR-Bac, plus prior zebrafish tissue-specific Cas9 systems. The abstract itself does not directly compare nCas9ERT2 against these alternatives.
Source-backed strengths
supports inducible editing in defined cell types and stages; works in zebrafish embryos, adult ovaries, and early mouse embryos; uses 4-OHT-triggered nuclear translocation for temporal control
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supports inducible editing in defined cell types and stages
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works in zebrafish embryos, adult ovaries, and early mouse embryos
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uses 4-OHT-triggered nuclear translocation for temporal control
Compared with CRISPR/Cas9
The web research summary identifies related conditionality or inducible systems such as CreERT2, HIT, and CRISPR-Bac, plus prior zebrafish tissue-specific Cas9 systems. The abstract itself does not directly compare nCas9ERT2 against these alternatives.
Shared frame: source-stated alternative in extracted literature
Strengths here: supports inducible editing in defined cell types and stages; works in zebrafish embryos, adult ovaries, and early mouse embryos; uses 4-OHT-triggered nuclear translocation for temporal control.
Relative tradeoffs: requires 4-OHT induction; abstract provides no quantitative data on leakiness, efficiency, or off-target effects.
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The web research summary identifies related conditionality or inducible systems such as CreERT2, HIT, and CRISPR-Bac, plus prior zebrafish tissue-specific Cas9 systems. The abstract itself does not directly compare nCas9ERT2 against these alternatives.
Compared with CRISPR/Cas9 system
The web research summary identifies related conditionality or inducible systems such as CreERT2, HIT, and CRISPR-Bac, plus prior zebrafish tissue-specific Cas9 systems. The abstract itself does not directly compare nCas9ERT2 against these alternatives.
Shared frame: source-stated alternative in extracted literature
Strengths here: supports inducible editing in defined cell types and stages; works in zebrafish embryos, adult ovaries, and early mouse embryos; uses 4-OHT-triggered nuclear translocation for temporal control.
Relative tradeoffs: requires 4-OHT induction; abstract provides no quantitative data on leakiness, efficiency, or off-target effects.
Source:
The web research summary identifies related conditionality or inducible systems such as CreERT2, HIT, and CRISPR-Bac, plus prior zebrafish tissue-specific Cas9 systems. The abstract itself does not directly compare nCas9ERT2 against these alternatives.
Ranked Citations
- 1.