Toolkit/CRISPR-dCas9

CRISPR-dCas9

Multi-Component Switch·Research·Since 2017

Taxonomy: Mechanism Branch / Architecture. Workflows sit above the mechanism and technique branches rather than replacing them.

Summary

CRISPR-dCas9 was used in an optogenetic LITE configuration to control endogenous PIM1 transcription in U87 glioblastoma cells in vitro. In this study, the system mediated light-inducible, reversible transcriptional induction or repression rather than genome editing.

Usefulness & Problems

Why this is useful

This implementation is useful for temporally controlling endogenous gene expression with light in cultured glioblastoma cells. The study specifically demonstrates rapid and reversible regulation of PIM1 transcription, enabling perturbation of transcriptional state without describing DNA cleavage.

Source:

Here we manipulate PIM1 through an optogenetic system using a combination of CRISPR-dCas9 technology and light-inducible heterodimerizing proteins CRY2 and CIB1.

Problem solved

The tool addresses the problem of manipulating endogenous PIM1 transcription in a light-dependent manner in U87 cells. It also addresses guide selection by testing guide RNA efficiency with a NanoLuc luciferase reporter assay before downstream cell-based experiments.

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

editingtranscription

Input: Light

Implementation Constraints

The reported implementation used an optogenetic CRISPR-dCas9 LITE system in U87 glioblastoma cells in vitro to manipulate endogenous PIM1 transcription. Guide RNAs were pre-screened for efficiency using a NanoLuc luciferase reporter assay, but the evidence does not provide additional construct, delivery, or cofactor details.

The evidence is limited to a single reported in vitro application in U87 glioblastoma cells targeting PIM1. The provided evidence does not specify construct architecture, light wavelength, repression reversibility, quantitative effect sizes, or any genome-editing activity.

Validation

Cell-freeBacteriaMammalianMouseHumanTherapeuticIndep. Replication

Observations

successMammalian Cell Lineapplication demoU87 cells

Inferred from claim c2 during normalization. In U87 cells, induction or repression of endogenous PIM1 transcription occurred within minutes of light exposure, could theoretically be graded with light dose, and induction was fully reversible after light retraction. Derived from claim c2. Section: abstract. Quoted text: Induction or repression of PIM1 endogenous transcription in U87 cells occurred within minutes of light exposure and the response could be theoretically graded with light dose, with the induction being fully reversible after light retraction.

Source:

light intensity5 mW/cm2light wavelength466 nmmaximum exposure duration24 hresponse time(within minutes)stimulation frequency0.016 Hz
successMammalian Cell Lineapplication demoU87 cells

Inferred from claim c2 during normalization. In U87 cells, induction or repression of endogenous PIM1 transcription occurred within minutes of light exposure, could theoretically be graded with light dose, and induction was fully reversible after light retraction. Derived from claim c2. Section: abstract. Quoted text: Induction or repression of PIM1 endogenous transcription in U87 cells occurred within minutes of light exposure and the response could be theoretically graded with light dose, with the induction being fully reversible after light retraction.

Source:

light intensity5 mW/cm2light wavelength466 nmmaximum exposure duration24 hresponse time(within minutes)stimulation frequency0.016 Hz
successMammalian Cell Lineapplication demoU87 cells

Inferred from claim c2 during normalization. In U87 cells, induction or repression of endogenous PIM1 transcription occurred within minutes of light exposure, could theoretically be graded with light dose, and induction was fully reversible after light retraction. Derived from claim c2. Section: abstract. Quoted text: Induction or repression of PIM1 endogenous transcription in U87 cells occurred within minutes of light exposure and the response could be theoretically graded with light dose, with the induction being fully reversible after light retraction.

Source:

light intensity5 mW/cm2light wavelength466 nmmaximum exposure duration24 hresponse time(within minutes)stimulation frequency0.016 Hz
successMammalian Cell Lineapplication demoU87 cells

Inferred from claim c2 during normalization. In U87 cells, induction or repression of endogenous PIM1 transcription occurred within minutes of light exposure, could theoretically be graded with light dose, and induction was fully reversible after light retraction. Derived from claim c2. Section: abstract. Quoted text: Induction or repression of PIM1 endogenous transcription in U87 cells occurred within minutes of light exposure and the response could be theoretically graded with light dose, with the induction being fully reversible after light retraction.

Source:

light intensity5 mW/cm2light wavelength466 nmmaximum exposure duration24 hresponse time(within minutes)stimulation frequency0.016 Hz
successMammalian Cell Lineapplication demoU87 cells

Inferred from claim c2 during normalization. In U87 cells, induction or repression of endogenous PIM1 transcription occurred within minutes of light exposure, could theoretically be graded with light dose, and induction was fully reversible after light retraction. Derived from claim c2. Section: abstract. Quoted text: Induction or repression of PIM1 endogenous transcription in U87 cells occurred within minutes of light exposure and the response could be theoretically graded with light dose, with the induction being fully reversible after light retraction.

Source:

light intensity5 mW/cm2light wavelength466 nmmaximum exposure duration24 hresponse time(within minutes)stimulation frequency0.016 Hz
successMammalian Cell Lineapplication demoU87 cells

Inferred from claim c2 during normalization. In U87 cells, induction or repression of endogenous PIM1 transcription occurred within minutes of light exposure, could theoretically be graded with light dose, and induction was fully reversible after light retraction. Derived from claim c2. Section: abstract. Quoted text: Induction or repression of PIM1 endogenous transcription in U87 cells occurred within minutes of light exposure and the response could be theoretically graded with light dose, with the induction being fully reversible after light retraction.

Source:

light intensity5 mW/cm2light wavelength466 nmmaximum exposure duration24 hresponse time(within minutes)stimulation frequency0.016 Hz
successMammalian Cell Lineapplication demoU87 cells

Inferred from claim c2 during normalization. In U87 cells, induction or repression of endogenous PIM1 transcription occurred within minutes of light exposure, could theoretically be graded with light dose, and induction was fully reversible after light retraction. Derived from claim c2. Section: abstract. Quoted text: Induction or repression of PIM1 endogenous transcription in U87 cells occurred within minutes of light exposure and the response could be theoretically graded with light dose, with the induction being fully reversible after light retraction.

Source:

light intensity5 mW/cm2light wavelength466 nmmaximum exposure duration24 hresponse time(within minutes)stimulation frequency0.016 Hz

Supporting Sources

Ranked Claims

Claim 1assay usesupports2017Source 1needs review

Guide RNAs were tested for efficiency using a NanoLuc luciferase reporter assay before downstream cell-based experiments.

Guide RNAs were tested for optimal efficiencies using the NanoLuc® luciferase reporter assay.
Section: abstract
Claim 2assay usesupports2017Source 1needs review

Guide RNAs were tested for efficiency using a NanoLuc luciferase reporter assay before downstream cell-based experiments.

Guide RNAs were tested for optimal efficiencies using the NanoLuc® luciferase reporter assay.
Section: abstract
Claim 3assay usesupports2017Source 1needs review

Guide RNAs were tested for efficiency using a NanoLuc luciferase reporter assay before downstream cell-based experiments.

Guide RNAs were tested for optimal efficiencies using the NanoLuc® luciferase reporter assay.
Section: abstract
Claim 4assay usesupports2017Source 1needs review

Guide RNAs were tested for efficiency using a NanoLuc luciferase reporter assay before downstream cell-based experiments.

Guide RNAs were tested for optimal efficiencies using the NanoLuc® luciferase reporter assay.
Section: abstract
Claim 5assay usesupports2017Source 1needs review

Guide RNAs were tested for efficiency using a NanoLuc luciferase reporter assay before downstream cell-based experiments.

Guide RNAs were tested for optimal efficiencies using the NanoLuc® luciferase reporter assay.
Section: abstract
Claim 6assay usesupports2017Source 1needs review

Guide RNAs were tested for efficiency using a NanoLuc luciferase reporter assay before downstream cell-based experiments.

Guide RNAs were tested for optimal efficiencies using the NanoLuc® luciferase reporter assay.
Section: abstract
Claim 7assay usesupports2017Source 1needs review

Guide RNAs were tested for efficiency using a NanoLuc luciferase reporter assay before downstream cell-based experiments.

Guide RNAs were tested for optimal efficiencies using the NanoLuc® luciferase reporter assay.
Section: abstract
Claim 8system performancesupports2017Source 1needs review

In U87 cells, induction or repression of endogenous PIM1 transcription occurred within minutes of light exposure, could theoretically be graded with light dose, and induction was fully reversible after light retraction.

Induction or repression of PIM1 endogenous transcription in U87 cells occurred within minutes of light exposure and the response could be theoretically graded with light dose, with the induction being fully reversible after light retraction.
Section: abstract
light intensity 5 mW/cm2light wavelength 466 nmmaximum exposure duration 24 hresponse time within minutesstimulation frequency 0.016 Hz
Claim 9system performancesupports2017Source 1needs review

In U87 cells, induction or repression of endogenous PIM1 transcription occurred within minutes of light exposure, could theoretically be graded with light dose, and induction was fully reversible after light retraction.

Induction or repression of PIM1 endogenous transcription in U87 cells occurred within minutes of light exposure and the response could be theoretically graded with light dose, with the induction being fully reversible after light retraction.
Section: abstract
light intensity 5 mW/cm2light wavelength 466 nmmaximum exposure duration 24 hresponse time within minutesstimulation frequency 0.016 Hz
Claim 10system performancesupports2017Source 1needs review

In U87 cells, induction or repression of endogenous PIM1 transcription occurred within minutes of light exposure, could theoretically be graded with light dose, and induction was fully reversible after light retraction.

Induction or repression of PIM1 endogenous transcription in U87 cells occurred within minutes of light exposure and the response could be theoretically graded with light dose, with the induction being fully reversible after light retraction.
Section: abstract
light intensity 5 mW/cm2light wavelength 466 nmmaximum exposure duration 24 hresponse time within minutesstimulation frequency 0.016 Hz
Claim 11system performancesupports2017Source 1needs review

In U87 cells, induction or repression of endogenous PIM1 transcription occurred within minutes of light exposure, could theoretically be graded with light dose, and induction was fully reversible after light retraction.

Induction or repression of PIM1 endogenous transcription in U87 cells occurred within minutes of light exposure and the response could be theoretically graded with light dose, with the induction being fully reversible after light retraction.
Section: abstract
light intensity 5 mW/cm2light wavelength 466 nmmaximum exposure duration 24 hresponse time within minutesstimulation frequency 0.016 Hz
Claim 12system performancesupports2017Source 1needs review

In U87 cells, induction or repression of endogenous PIM1 transcription occurred within minutes of light exposure, could theoretically be graded with light dose, and induction was fully reversible after light retraction.

Induction or repression of PIM1 endogenous transcription in U87 cells occurred within minutes of light exposure and the response could be theoretically graded with light dose, with the induction being fully reversible after light retraction.
Section: abstract
light intensity 5 mW/cm2light wavelength 466 nmmaximum exposure duration 24 hresponse time within minutesstimulation frequency 0.016 Hz
Claim 13system performancesupports2017Source 1needs review

In U87 cells, induction or repression of endogenous PIM1 transcription occurred within minutes of light exposure, could theoretically be graded with light dose, and induction was fully reversible after light retraction.

Induction or repression of PIM1 endogenous transcription in U87 cells occurred within minutes of light exposure and the response could be theoretically graded with light dose, with the induction being fully reversible after light retraction.
Section: abstract
light intensity 5 mW/cm2light wavelength 466 nmmaximum exposure duration 24 hresponse time within minutesstimulation frequency 0.016 Hz
Claim 14system performancesupports2017Source 1needs review

In U87 cells, induction or repression of endogenous PIM1 transcription occurred within minutes of light exposure, could theoretically be graded with light dose, and induction was fully reversible after light retraction.

Induction or repression of PIM1 endogenous transcription in U87 cells occurred within minutes of light exposure and the response could be theoretically graded with light dose, with the induction being fully reversible after light retraction.
Section: abstract
light intensity 5 mW/cm2light wavelength 466 nmmaximum exposure duration 24 hresponse time within minutesstimulation frequency 0.016 Hz
Claim 15tool applicationsupports2017Source 1needs review

The study used an optogenetic CRISPR-dCas9 LITE system to manipulate PIM1 transcription in U87 glioblastoma cells in vitro.

Here we manipulate PIM1 through an optogenetic system using a combination of CRISPR-dCas9 technology and light-inducible heterodimerizing proteins CRY2 and CIB1.
Section: abstract
Claim 16tool applicationsupports2017Source 1needs review

The study used an optogenetic CRISPR-dCas9 LITE system to manipulate PIM1 transcription in U87 glioblastoma cells in vitro.

Here we manipulate PIM1 through an optogenetic system using a combination of CRISPR-dCas9 technology and light-inducible heterodimerizing proteins CRY2 and CIB1.
Section: abstract
Claim 17tool applicationsupports2017Source 1needs review

The study used an optogenetic CRISPR-dCas9 LITE system to manipulate PIM1 transcription in U87 glioblastoma cells in vitro.

Here we manipulate PIM1 through an optogenetic system using a combination of CRISPR-dCas9 technology and light-inducible heterodimerizing proteins CRY2 and CIB1.
Section: abstract
Claim 18tool applicationsupports2017Source 1needs review

The study used an optogenetic CRISPR-dCas9 LITE system to manipulate PIM1 transcription in U87 glioblastoma cells in vitro.

Here we manipulate PIM1 through an optogenetic system using a combination of CRISPR-dCas9 technology and light-inducible heterodimerizing proteins CRY2 and CIB1.
Section: abstract
Claim 19tool applicationsupports2017Source 1needs review

The study used an optogenetic CRISPR-dCas9 LITE system to manipulate PIM1 transcription in U87 glioblastoma cells in vitro.

Here we manipulate PIM1 through an optogenetic system using a combination of CRISPR-dCas9 technology and light-inducible heterodimerizing proteins CRY2 and CIB1.
Section: abstract
Claim 20tool applicationsupports2017Source 1needs review

The study used an optogenetic CRISPR-dCas9 LITE system to manipulate PIM1 transcription in U87 glioblastoma cells in vitro.

Here we manipulate PIM1 through an optogenetic system using a combination of CRISPR-dCas9 technology and light-inducible heterodimerizing proteins CRY2 and CIB1.
Section: abstract
Claim 21tool applicationsupports2017Source 1needs review

The study used an optogenetic CRISPR-dCas9 LITE system to manipulate PIM1 transcription in U87 glioblastoma cells in vitro.

Here we manipulate PIM1 through an optogenetic system using a combination of CRISPR-dCas9 technology and light-inducible heterodimerizing proteins CRY2 and CIB1.
Section: abstract

Approval Evidence

1 source2 linked approval claimsfirst-pass slug crispr-dcas9
Here we manipulate PIM1 through an optogenetic system using a combination of CRISPR-dCas9 technology

Source:

system performancesupports

In U87 cells, induction or repression of endogenous PIM1 transcription occurred within minutes of light exposure, could theoretically be graded with light dose, and induction was fully reversible after light retraction.

Induction or repression of PIM1 endogenous transcription in U87 cells occurred within minutes of light exposure and the response could be theoretically graded with light dose, with the induction being fully reversible after light retraction.

Source:

tool applicationsupports

The study used an optogenetic CRISPR-dCas9 LITE system to manipulate PIM1 transcription in U87 glioblastoma cells in vitro.

Here we manipulate PIM1 through an optogenetic system using a combination of CRISPR-dCas9 technology and light-inducible heterodimerizing proteins CRY2 and CIB1.

Source:

Comparisons

Source-backed strengths

In U87 cells, endogenous PIM1 induction or repression occurred within minutes of light exposure and induction was reported to be fully reversible after light retraction. The study also states that the response could theoretically be graded with light dose, supporting tunable transcriptional control.

Ranked Citations

  1. 1.
    StructuralSource 1Neuro-Oncology2017Claim 1Claim 2Claim 3

    Extracted from this source document.