Source 1primary paper2015Molecular Therapy
Toolkit/LACE system
LACE system
Also known as: LACE, light-activated CRISPR/Cas9 effector
Taxonomy: Mechanism Branch / Architecture. Workflows sit above the mechanism and technique branches rather than replacing them.
Summary
LACE is a light-activated CRISPR/Cas9 effector system that induces transcription of endogenous genes in the presence of gene-specific guide RNAs and blue light. It functions as a multi-component optical switch for programmable gene activation and has also been used to spatially pattern reporter expression under photomask illumination.
Usefulness & Problems
Why this is useful
LACE provides optical control over CRISPR-based transcriptional activation, enabling gene induction only under blue-light exposure and in the presence of target-specific gRNAs. This is useful for experiments requiring inducible and spatially patterned gene expression with low dark-state activity.
Source:
Spatial patterning of gene expression was also achieved using an eGFP reporter. Cells illuminated though a photomask containing slits of varying width resulted in a corresponding pattern of eGFP-expressing cells.
Problem solved
LACE addresses the problem of controlling endogenous gene activation with external, noninvasive light input rather than constitutive CRISPR activator activity. It also helps solve the need for spatially restricted transcriptional activation, as shown by photomask-based patterning of an eGFP reporter.
Source:
Spatial patterning of gene expression was also achieved using an eGFP reporter. Cells illuminated though a photomask containing slits of varying width resulted in a corresponding pattern of eGFP-expressing cells.
Taxonomy & Function
Primary hierarchy
Mechanism Branch
Architecture: A composed arrangement of multiple parts that instantiates one or more mechanisms.
Mechanisms
HeterodimerizationTechniques
No technique tags yet.
Target processes
editingtranscriptionInput: Light
Implementation Constraints
The reported system requires blue light and gene-specific gRNAs to induce endogenous transcription. It is described as a multi-component light-activated CRISPR/Cas9 effector, implying coordinated delivery and expression of multiple components, but the supplied evidence does not specify construct architecture, cofactors, or host systems. Photomask illumination was used to spatially pattern eGFP reporter expression.
The available evidence supports transcriptional activation, but does not document genome editing activity despite the CRISPR/Cas9 framework. Performance was gene dependent, because ASCL1 activation was lower than that achieved by dCas9-VP64. Evidence here comes from a single 2015 study, so broader validation across cell types and targets is not established in the supplied record.
Validation
Cell-freeBacteriaMammalianMouseHumanTherapeuticIndep. Replication
Supporting Sources
Ranked Claims
LACE enabled spatial patterning of gene expression using an eGFP reporter and photomask illumination.
Spatial patterning of gene expression was also achieved using an eGFP reporter. Cells illuminated though a photomask containing slits of varying width resulted in a corresponding pattern of eGFP-expressing cells.
LACE enabled spatial patterning of gene expression using an eGFP reporter and photomask illumination.
Spatial patterning of gene expression was also achieved using an eGFP reporter. Cells illuminated though a photomask containing slits of varying width resulted in a corresponding pattern of eGFP-expressing cells.
LACE enabled spatial patterning of gene expression using an eGFP reporter and photomask illumination.
Spatial patterning of gene expression was also achieved using an eGFP reporter. Cells illuminated though a photomask containing slits of varying width resulted in a corresponding pattern of eGFP-expressing cells.
LACE enabled spatial patterning of gene expression using an eGFP reporter and photomask illumination.
Spatial patterning of gene expression was also achieved using an eGFP reporter. Cells illuminated though a photomask containing slits of varying width resulted in a corresponding pattern of eGFP-expressing cells.
LACE enabled spatial patterning of gene expression using an eGFP reporter and photomask illumination.
Spatial patterning of gene expression was also achieved using an eGFP reporter. Cells illuminated though a photomask containing slits of varying width resulted in a corresponding pattern of eGFP-expressing cells.
LACE enabled spatial patterning of gene expression using an eGFP reporter and photomask illumination.
Spatial patterning of gene expression was also achieved using an eGFP reporter. Cells illuminated though a photomask containing slits of varying width resulted in a corresponding pattern of eGFP-expressing cells.
LACE enabled spatial patterning of gene expression using an eGFP reporter and photomask illumination.
Spatial patterning of gene expression was also achieved using an eGFP reporter. Cells illuminated though a photomask containing slits of varying width resulted in a corresponding pattern of eGFP-expressing cells.
LACE enabled spatial patterning of gene expression using an eGFP reporter and photomask illumination.
Spatial patterning of gene expression was also achieved using an eGFP reporter. Cells illuminated though a photomask containing slits of varying width resulted in a corresponding pattern of eGFP-expressing cells.
In the dark, LACE-targeted cells maintained target gene expression levels that were not significantly different from mock-transfected cells.
In all instances, transfected cells incubated in the dark maintained levels of the targeted gene that did not significantly differ from mock-transfected cells.
In the dark, LACE-targeted cells maintained target gene expression levels that were not significantly different from mock-transfected cells.
In all instances, transfected cells incubated in the dark maintained levels of the targeted gene that did not significantly differ from mock-transfected cells.
In the dark, LACE-targeted cells maintained target gene expression levels that were not significantly different from mock-transfected cells.
In all instances, transfected cells incubated in the dark maintained levels of the targeted gene that did not significantly differ from mock-transfected cells.
In the dark, LACE-targeted cells maintained target gene expression levels that were not significantly different from mock-transfected cells.
In all instances, transfected cells incubated in the dark maintained levels of the targeted gene that did not significantly differ from mock-transfected cells.
In the dark, LACE-targeted cells maintained target gene expression levels that were not significantly different from mock-transfected cells.
In all instances, transfected cells incubated in the dark maintained levels of the targeted gene that did not significantly differ from mock-transfected cells.
In the dark, LACE-targeted cells maintained target gene expression levels that were not significantly different from mock-transfected cells.
In all instances, transfected cells incubated in the dark maintained levels of the targeted gene that did not significantly differ from mock-transfected cells.
In the dark, LACE-targeted cells maintained target gene expression levels that were not significantly different from mock-transfected cells.
In all instances, transfected cells incubated in the dark maintained levels of the targeted gene that did not significantly differ from mock-transfected cells.
In the dark, LACE-targeted cells maintained target gene expression levels that were not significantly different from mock-transfected cells.
In all instances, transfected cells incubated in the dark maintained levels of the targeted gene that did not significantly differ from mock-transfected cells.
For IL1RN and HBG1/2 activation, LACE achieved activation levels equivalent to dCas9-VP64, whereas for ASCL1 activation it was lower than dCas9-VP64.
Illuminated cells in which IL1RN or HBG1/2 was targeted demonstrated significantly greater mRNA levels in the light compared to the dark (p<0.0001 and 0.005, respectively), as well as equivalent activation levels to dCas9-VP64 (p=0.17 and 0.35, respectively). Significant light-dependent activation was also observed when the ASCL1 locus was targeted with the LACE system (p<0.0001). However, in this case mRNA levels were not activated to the same extent as cells that received dCas9-VP64 and the same four ASCL1-targeting gRNAs.
HBG1/2 versus dCas9-VP64 p-value 0.35IL1RN versus dCas9-VP64 p-value 0.17
For IL1RN and HBG1/2 activation, LACE achieved activation levels equivalent to dCas9-VP64, whereas for ASCL1 activation it was lower than dCas9-VP64.
Illuminated cells in which IL1RN or HBG1/2 was targeted demonstrated significantly greater mRNA levels in the light compared to the dark (p<0.0001 and 0.005, respectively), as well as equivalent activation levels to dCas9-VP64 (p=0.17 and 0.35, respectively). Significant light-dependent activation was also observed when the ASCL1 locus was targeted with the LACE system (p<0.0001). However, in this case mRNA levels were not activated to the same extent as cells that received dCas9-VP64 and the same four ASCL1-targeting gRNAs.
HBG1/2 versus dCas9-VP64 p-value 0.35IL1RN versus dCas9-VP64 p-value 0.17
For IL1RN and HBG1/2 activation, LACE achieved activation levels equivalent to dCas9-VP64, whereas for ASCL1 activation it was lower than dCas9-VP64.
Illuminated cells in which IL1RN or HBG1/2 was targeted demonstrated significantly greater mRNA levels in the light compared to the dark (p<0.0001 and 0.005, respectively), as well as equivalent activation levels to dCas9-VP64 (p=0.17 and 0.35, respectively). Significant light-dependent activation was also observed when the ASCL1 locus was targeted with the LACE system (p<0.0001). However, in this case mRNA levels were not activated to the same extent as cells that received dCas9-VP64 and the same four ASCL1-targeting gRNAs.
HBG1/2 versus dCas9-VP64 p-value 0.35IL1RN versus dCas9-VP64 p-value 0.17
For IL1RN and HBG1/2 activation, LACE achieved activation levels equivalent to dCas9-VP64, whereas for ASCL1 activation it was lower than dCas9-VP64.
Illuminated cells in which IL1RN or HBG1/2 was targeted demonstrated significantly greater mRNA levels in the light compared to the dark (p<0.0001 and 0.005, respectively), as well as equivalent activation levels to dCas9-VP64 (p=0.17 and 0.35, respectively). Significant light-dependent activation was also observed when the ASCL1 locus was targeted with the LACE system (p<0.0001). However, in this case mRNA levels were not activated to the same extent as cells that received dCas9-VP64 and the same four ASCL1-targeting gRNAs.
HBG1/2 versus dCas9-VP64 p-value 0.35IL1RN versus dCas9-VP64 p-value 0.17
For IL1RN and HBG1/2 activation, LACE achieved activation levels equivalent to dCas9-VP64, whereas for ASCL1 activation it was lower than dCas9-VP64.
Illuminated cells in which IL1RN or HBG1/2 was targeted demonstrated significantly greater mRNA levels in the light compared to the dark (p<0.0001 and 0.005, respectively), as well as equivalent activation levels to dCas9-VP64 (p=0.17 and 0.35, respectively). Significant light-dependent activation was also observed when the ASCL1 locus was targeted with the LACE system (p<0.0001). However, in this case mRNA levels were not activated to the same extent as cells that received dCas9-VP64 and the same four ASCL1-targeting gRNAs.
HBG1/2 versus dCas9-VP64 p-value 0.35IL1RN versus dCas9-VP64 p-value 0.17
For IL1RN and HBG1/2 activation, LACE achieved activation levels equivalent to dCas9-VP64, whereas for ASCL1 activation it was lower than dCas9-VP64.
Illuminated cells in which IL1RN or HBG1/2 was targeted demonstrated significantly greater mRNA levels in the light compared to the dark (p<0.0001 and 0.005, respectively), as well as equivalent activation levels to dCas9-VP64 (p=0.17 and 0.35, respectively). Significant light-dependent activation was also observed when the ASCL1 locus was targeted with the LACE system (p<0.0001). However, in this case mRNA levels were not activated to the same extent as cells that received dCas9-VP64 and the same four ASCL1-targeting gRNAs.
HBG1/2 versus dCas9-VP64 p-value 0.35IL1RN versus dCas9-VP64 p-value 0.17
For IL1RN and HBG1/2 activation, LACE achieved activation levels equivalent to dCas9-VP64, whereas for ASCL1 activation it was lower than dCas9-VP64.
Illuminated cells in which IL1RN or HBG1/2 was targeted demonstrated significantly greater mRNA levels in the light compared to the dark (p<0.0001 and 0.005, respectively), as well as equivalent activation levels to dCas9-VP64 (p=0.17 and 0.35, respectively). Significant light-dependent activation was also observed when the ASCL1 locus was targeted with the LACE system (p<0.0001). However, in this case mRNA levels were not activated to the same extent as cells that received dCas9-VP64 and the same four ASCL1-targeting gRNAs.
HBG1/2 versus dCas9-VP64 p-value 0.35IL1RN versus dCas9-VP64 p-value 0.17
For IL1RN and HBG1/2 activation, LACE achieved activation levels equivalent to dCas9-VP64, whereas for ASCL1 activation it was lower than dCas9-VP64.
Illuminated cells in which IL1RN or HBG1/2 was targeted demonstrated significantly greater mRNA levels in the light compared to the dark (p<0.0001 and 0.005, respectively), as well as equivalent activation levels to dCas9-VP64 (p=0.17 and 0.35, respectively). Significant light-dependent activation was also observed when the ASCL1 locus was targeted with the LACE system (p<0.0001). However, in this case mRNA levels were not activated to the same extent as cells that received dCas9-VP64 and the same four ASCL1-targeting gRNAs.
HBG1/2 versus dCas9-VP64 p-value 0.35IL1RN versus dCas9-VP64 p-value 0.17
Endogenous gene expression controlled by LACE is reversible and repeatable by modulating blue light exposure duration.
Endogenous gene expression could be controlled in a reversible and repeatable fashion by modulating the duration of blue light exposure.
Endogenous gene expression controlled by LACE is reversible and repeatable by modulating blue light exposure duration.
Endogenous gene expression could be controlled in a reversible and repeatable fashion by modulating the duration of blue light exposure.
Endogenous gene expression controlled by LACE is reversible and repeatable by modulating blue light exposure duration.
Endogenous gene expression could be controlled in a reversible and repeatable fashion by modulating the duration of blue light exposure.
Endogenous gene expression controlled by LACE is reversible and repeatable by modulating blue light exposure duration.
Endogenous gene expression could be controlled in a reversible and repeatable fashion by modulating the duration of blue light exposure.
Endogenous gene expression controlled by LACE is reversible and repeatable by modulating blue light exposure duration.
Endogenous gene expression could be controlled in a reversible and repeatable fashion by modulating the duration of blue light exposure.
Endogenous gene expression controlled by LACE is reversible and repeatable by modulating blue light exposure duration.
Endogenous gene expression could be controlled in a reversible and repeatable fashion by modulating the duration of blue light exposure.
Endogenous gene expression controlled by LACE is reversible and repeatable by modulating blue light exposure duration.
Endogenous gene expression could be controlled in a reversible and repeatable fashion by modulating the duration of blue light exposure.
Endogenous gene expression controlled by LACE is reversible and repeatable by modulating blue light exposure duration.
Endogenous gene expression could be controlled in a reversible and repeatable fashion by modulating the duration of blue light exposure.
Fusing CIBN to both the N- and C-termini of dCas9 produced 10- to 100-fold greater gene activation than fusing CIBN to only one terminus.
Importantly, the fusion of CIBN to both N- and C-termini of dCas9 yielded 10- to 100-fold greater gene activation than when CIBN was fused to only one terminus.
gene activation increase for dual-terminus CIBN fusion 10- to 100-fold greater
Fusing CIBN to both the N- and C-termini of dCas9 produced 10- to 100-fold greater gene activation than fusing CIBN to only one terminus.
Importantly, the fusion of CIBN to both N- and C-termini of dCas9 yielded 10- to 100-fold greater gene activation than when CIBN was fused to only one terminus.
gene activation increase for dual-terminus CIBN fusion 10- to 100-fold greater
Fusing CIBN to both the N- and C-termini of dCas9 produced 10- to 100-fold greater gene activation than fusing CIBN to only one terminus.
Importantly, the fusion of CIBN to both N- and C-termini of dCas9 yielded 10- to 100-fold greater gene activation than when CIBN was fused to only one terminus.
gene activation increase for dual-terminus CIBN fusion 10- to 100-fold greater
Fusing CIBN to both the N- and C-termini of dCas9 produced 10- to 100-fold greater gene activation than fusing CIBN to only one terminus.
Importantly, the fusion of CIBN to both N- and C-termini of dCas9 yielded 10- to 100-fold greater gene activation than when CIBN was fused to only one terminus.
gene activation increase for dual-terminus CIBN fusion 10- to 100-fold greater
Fusing CIBN to both the N- and C-termini of dCas9 produced 10- to 100-fold greater gene activation than fusing CIBN to only one terminus.
Importantly, the fusion of CIBN to both N- and C-termini of dCas9 yielded 10- to 100-fold greater gene activation than when CIBN was fused to only one terminus.
gene activation increase for dual-terminus CIBN fusion 10- to 100-fold greater
Fusing CIBN to both the N- and C-termini of dCas9 produced 10- to 100-fold greater gene activation than fusing CIBN to only one terminus.
Importantly, the fusion of CIBN to both N- and C-termini of dCas9 yielded 10- to 100-fold greater gene activation than when CIBN was fused to only one terminus.
gene activation increase for dual-terminus CIBN fusion 10- to 100-fold greater
Fusing CIBN to both the N- and C-termini of dCas9 produced 10- to 100-fold greater gene activation than fusing CIBN to only one terminus.
Importantly, the fusion of CIBN to both N- and C-termini of dCas9 yielded 10- to 100-fold greater gene activation than when CIBN was fused to only one terminus.
gene activation increase for dual-terminus CIBN fusion 10- to 100-fold greater
Fusing CIBN to both the N- and C-termini of dCas9 produced 10- to 100-fold greater gene activation than fusing CIBN to only one terminus.
Importantly, the fusion of CIBN to both N- and C-termini of dCas9 yielded 10- to 100-fold greater gene activation than when CIBN was fused to only one terminus.
gene activation increase for dual-terminus CIBN fusion 10- to 100-fold greater
LACE uses CRY2 and CIB1 heterodimerization to recruit VP64 to dCas9-targeted genomic loci under blue light.
This system is based on the plant proteins CRY2 and CIB1 from Arabidopsis thaliana that heterodimerize in response to blue light. The full-length CRY2 was fused to the N-terminus of the transcriptional activator VP64 (CRY2FL-VP64), and an N-terminal fragment of CIB1 was fused to the N- and C-terminus of the catalytically inactive form of Cas9 (CIBN-dCas9-CIBN). When these fusion proteins are expressed with a gRNA, CIBN-dCas9-CIBN localizes to the gRNA target. In the presence of blue light, CRY2FL binds to CIBN, which translocates CRY2FL-VP64 to the gene target and activates transcription.
LACE uses CRY2 and CIB1 heterodimerization to recruit VP64 to dCas9-targeted genomic loci under blue light.
This system is based on the plant proteins CRY2 and CIB1 from Arabidopsis thaliana that heterodimerize in response to blue light. The full-length CRY2 was fused to the N-terminus of the transcriptional activator VP64 (CRY2FL-VP64), and an N-terminal fragment of CIB1 was fused to the N- and C-terminus of the catalytically inactive form of Cas9 (CIBN-dCas9-CIBN). When these fusion proteins are expressed with a gRNA, CIBN-dCas9-CIBN localizes to the gRNA target. In the presence of blue light, CRY2FL binds to CIBN, which translocates CRY2FL-VP64 to the gene target and activates transcription.
LACE uses CRY2 and CIB1 heterodimerization to recruit VP64 to dCas9-targeted genomic loci under blue light.
This system is based on the plant proteins CRY2 and CIB1 from Arabidopsis thaliana that heterodimerize in response to blue light. The full-length CRY2 was fused to the N-terminus of the transcriptional activator VP64 (CRY2FL-VP64), and an N-terminal fragment of CIB1 was fused to the N- and C-terminus of the catalytically inactive form of Cas9 (CIBN-dCas9-CIBN). When these fusion proteins are expressed with a gRNA, CIBN-dCas9-CIBN localizes to the gRNA target. In the presence of blue light, CRY2FL binds to CIBN, which translocates CRY2FL-VP64 to the gene target and activates transcription.
LACE uses CRY2 and CIB1 heterodimerization to recruit VP64 to dCas9-targeted genomic loci under blue light.
This system is based on the plant proteins CRY2 and CIB1 from Arabidopsis thaliana that heterodimerize in response to blue light. The full-length CRY2 was fused to the N-terminus of the transcriptional activator VP64 (CRY2FL-VP64), and an N-terminal fragment of CIB1 was fused to the N- and C-terminus of the catalytically inactive form of Cas9 (CIBN-dCas9-CIBN). When these fusion proteins are expressed with a gRNA, CIBN-dCas9-CIBN localizes to the gRNA target. In the presence of blue light, CRY2FL binds to CIBN, which translocates CRY2FL-VP64 to the gene target and activates transcription.
LACE uses CRY2 and CIB1 heterodimerization to recruit VP64 to dCas9-targeted genomic loci under blue light.
This system is based on the plant proteins CRY2 and CIB1 from Arabidopsis thaliana that heterodimerize in response to blue light. The full-length CRY2 was fused to the N-terminus of the transcriptional activator VP64 (CRY2FL-VP64), and an N-terminal fragment of CIB1 was fused to the N- and C-terminus of the catalytically inactive form of Cas9 (CIBN-dCas9-CIBN). When these fusion proteins are expressed with a gRNA, CIBN-dCas9-CIBN localizes to the gRNA target. In the presence of blue light, CRY2FL binds to CIBN, which translocates CRY2FL-VP64 to the gene target and activates transcription.
LACE uses CRY2 and CIB1 heterodimerization to recruit VP64 to dCas9-targeted genomic loci under blue light.
This system is based on the plant proteins CRY2 and CIB1 from Arabidopsis thaliana that heterodimerize in response to blue light. The full-length CRY2 was fused to the N-terminus of the transcriptional activator VP64 (CRY2FL-VP64), and an N-terminal fragment of CIB1 was fused to the N- and C-terminus of the catalytically inactive form of Cas9 (CIBN-dCas9-CIBN). When these fusion proteins are expressed with a gRNA, CIBN-dCas9-CIBN localizes to the gRNA target. In the presence of blue light, CRY2FL binds to CIBN, which translocates CRY2FL-VP64 to the gene target and activates transcription.
LACE uses CRY2 and CIB1 heterodimerization to recruit VP64 to dCas9-targeted genomic loci under blue light.
This system is based on the plant proteins CRY2 and CIB1 from Arabidopsis thaliana that heterodimerize in response to blue light. The full-length CRY2 was fused to the N-terminus of the transcriptional activator VP64 (CRY2FL-VP64), and an N-terminal fragment of CIB1 was fused to the N- and C-terminus of the catalytically inactive form of Cas9 (CIBN-dCas9-CIBN). When these fusion proteins are expressed with a gRNA, CIBN-dCas9-CIBN localizes to the gRNA target. In the presence of blue light, CRY2FL binds to CIBN, which translocates CRY2FL-VP64 to the gene target and activates transcription.
LACE uses CRY2 and CIB1 heterodimerization to recruit VP64 to dCas9-targeted genomic loci under blue light.
This system is based on the plant proteins CRY2 and CIB1 from Arabidopsis thaliana that heterodimerize in response to blue light. The full-length CRY2 was fused to the N-terminus of the transcriptional activator VP64 (CRY2FL-VP64), and an N-terminal fragment of CIB1 was fused to the N- and C-terminus of the catalytically inactive form of Cas9 (CIBN-dCas9-CIBN). When these fusion proteins are expressed with a gRNA, CIBN-dCas9-CIBN localizes to the gRNA target. In the presence of blue light, CRY2FL binds to CIBN, which translocates CRY2FL-VP64 to the gene target and activates transcription.
The LACE system induces transcription of endogenous genes in the presence of gene-specific gRNAs and blue light.
We engineered a light-activated CRISPR/Cas9 effector (LACE) system that induces transcription of endogenous genes in the presence of gene-specific guide RNAs (gRNAs) and blue light.
The LACE system induces transcription of endogenous genes in the presence of gene-specific gRNAs and blue light.
We engineered a light-activated CRISPR/Cas9 effector (LACE) system that induces transcription of endogenous genes in the presence of gene-specific guide RNAs (gRNAs) and blue light.
The LACE system induces transcription of endogenous genes in the presence of gene-specific gRNAs and blue light.
We engineered a light-activated CRISPR/Cas9 effector (LACE) system that induces transcription of endogenous genes in the presence of gene-specific guide RNAs (gRNAs) and blue light.
The LACE system induces transcription of endogenous genes in the presence of gene-specific gRNAs and blue light.
We engineered a light-activated CRISPR/Cas9 effector (LACE) system that induces transcription of endogenous genes in the presence of gene-specific guide RNAs (gRNAs) and blue light.
The LACE system induces transcription of endogenous genes in the presence of gene-specific gRNAs and blue light.
We engineered a light-activated CRISPR/Cas9 effector (LACE) system that induces transcription of endogenous genes in the presence of gene-specific guide RNAs (gRNAs) and blue light.
The LACE system induces transcription of endogenous genes in the presence of gene-specific gRNAs and blue light.
We engineered a light-activated CRISPR/Cas9 effector (LACE) system that induces transcription of endogenous genes in the presence of gene-specific guide RNAs (gRNAs) and blue light.
The LACE system induces transcription of endogenous genes in the presence of gene-specific gRNAs and blue light.
We engineered a light-activated CRISPR/Cas9 effector (LACE) system that induces transcription of endogenous genes in the presence of gene-specific guide RNAs (gRNAs) and blue light.
The LACE system induces transcription of endogenous genes in the presence of gene-specific gRNAs and blue light.
We engineered a light-activated CRISPR/Cas9 effector (LACE) system that induces transcription of endogenous genes in the presence of gene-specific guide RNAs (gRNAs) and blue light.
LACE mediates light-dependent activation of endogenous IL1RN, HBG, and ASCL1 loci.
Light-dependent activation of the IL1RN, HBG, or ASCL1 genes was achieved by delivery of the LACE system and four gene-specific gRNAs per promoter region. Illuminated cells in which IL1RN or HBG1/2 was targeted demonstrated significantly greater mRNA levels in the light compared to the dark (p<0.0001 and 0.005, respectively)... Significant light-dependent activation was also observed when the ASCL1 locus was targeted with the LACE system (p<0.0001).
ASCL1 light-dependent activation p-value 0.0001HBG1/2 light versus dark p-value 0.005IL1RN light versus dark p-value 0.0001
LACE mediates light-dependent activation of endogenous IL1RN, HBG, and ASCL1 loci.
Light-dependent activation of the IL1RN, HBG, or ASCL1 genes was achieved by delivery of the LACE system and four gene-specific gRNAs per promoter region. Illuminated cells in which IL1RN or HBG1/2 was targeted demonstrated significantly greater mRNA levels in the light compared to the dark (p<0.0001 and 0.005, respectively)... Significant light-dependent activation was also observed when the ASCL1 locus was targeted with the LACE system (p<0.0001).
ASCL1 light-dependent activation p-value 0.0001HBG1/2 light versus dark p-value 0.005IL1RN light versus dark p-value 0.0001
LACE mediates light-dependent activation of endogenous IL1RN, HBG, and ASCL1 loci.
Light-dependent activation of the IL1RN, HBG, or ASCL1 genes was achieved by delivery of the LACE system and four gene-specific gRNAs per promoter region. Illuminated cells in which IL1RN or HBG1/2 was targeted demonstrated significantly greater mRNA levels in the light compared to the dark (p<0.0001 and 0.005, respectively)... Significant light-dependent activation was also observed when the ASCL1 locus was targeted with the LACE system (p<0.0001).
ASCL1 light-dependent activation p-value 0.0001HBG1/2 light versus dark p-value 0.005IL1RN light versus dark p-value 0.0001
LACE mediates light-dependent activation of endogenous IL1RN, HBG, and ASCL1 loci.
Light-dependent activation of the IL1RN, HBG, or ASCL1 genes was achieved by delivery of the LACE system and four gene-specific gRNAs per promoter region. Illuminated cells in which IL1RN or HBG1/2 was targeted demonstrated significantly greater mRNA levels in the light compared to the dark (p<0.0001 and 0.005, respectively)... Significant light-dependent activation was also observed when the ASCL1 locus was targeted with the LACE system (p<0.0001).
ASCL1 light-dependent activation p-value 0.0001HBG1/2 light versus dark p-value 0.005IL1RN light versus dark p-value 0.0001
LACE mediates light-dependent activation of endogenous IL1RN, HBG, and ASCL1 loci.
Light-dependent activation of the IL1RN, HBG, or ASCL1 genes was achieved by delivery of the LACE system and four gene-specific gRNAs per promoter region. Illuminated cells in which IL1RN or HBG1/2 was targeted demonstrated significantly greater mRNA levels in the light compared to the dark (p<0.0001 and 0.005, respectively)... Significant light-dependent activation was also observed when the ASCL1 locus was targeted with the LACE system (p<0.0001).
ASCL1 light-dependent activation p-value 0.0001HBG1/2 light versus dark p-value 0.005IL1RN light versus dark p-value 0.0001
LACE mediates light-dependent activation of endogenous IL1RN, HBG, and ASCL1 loci.
Light-dependent activation of the IL1RN, HBG, or ASCL1 genes was achieved by delivery of the LACE system and four gene-specific gRNAs per promoter region. Illuminated cells in which IL1RN or HBG1/2 was targeted demonstrated significantly greater mRNA levels in the light compared to the dark (p<0.0001 and 0.005, respectively)... Significant light-dependent activation was also observed when the ASCL1 locus was targeted with the LACE system (p<0.0001).
ASCL1 light-dependent activation p-value 0.0001HBG1/2 light versus dark p-value 0.005IL1RN light versus dark p-value 0.0001
LACE mediates light-dependent activation of endogenous IL1RN, HBG, and ASCL1 loci.
Light-dependent activation of the IL1RN, HBG, or ASCL1 genes was achieved by delivery of the LACE system and four gene-specific gRNAs per promoter region. Illuminated cells in which IL1RN or HBG1/2 was targeted demonstrated significantly greater mRNA levels in the light compared to the dark (p<0.0001 and 0.005, respectively)... Significant light-dependent activation was also observed when the ASCL1 locus was targeted with the LACE system (p<0.0001).
ASCL1 light-dependent activation p-value 0.0001HBG1/2 light versus dark p-value 0.005IL1RN light versus dark p-value 0.0001
LACE mediates light-dependent activation of endogenous IL1RN, HBG, and ASCL1 loci.
Light-dependent activation of the IL1RN, HBG, or ASCL1 genes was achieved by delivery of the LACE system and four gene-specific gRNAs per promoter region. Illuminated cells in which IL1RN or HBG1/2 was targeted demonstrated significantly greater mRNA levels in the light compared to the dark (p<0.0001 and 0.005, respectively)... Significant light-dependent activation was also observed when the ASCL1 locus was targeted with the LACE system (p<0.0001).
ASCL1 light-dependent activation p-value 0.0001HBG1/2 light versus dark p-value 0.005IL1RN light versus dark p-value 0.0001
The LACE system can be retargeted to new endogenous loci by changing gRNA specificity without re-engineering the light-inducible proteins.
Unlike other optogenetic systems, the LACE system can be targeted to new endogenous loci by solely manipulating the specificity of the gRNA without having to re-engineer the light-inducible proteins.
The LACE system can be retargeted to new endogenous loci by changing gRNA specificity without re-engineering the light-inducible proteins.
Unlike other optogenetic systems, the LACE system can be targeted to new endogenous loci by solely manipulating the specificity of the gRNA without having to re-engineer the light-inducible proteins.
The LACE system can be retargeted to new endogenous loci by changing gRNA specificity without re-engineering the light-inducible proteins.
Unlike other optogenetic systems, the LACE system can be targeted to new endogenous loci by solely manipulating the specificity of the gRNA without having to re-engineer the light-inducible proteins.
The LACE system can be retargeted to new endogenous loci by changing gRNA specificity without re-engineering the light-inducible proteins.
Unlike other optogenetic systems, the LACE system can be targeted to new endogenous loci by solely manipulating the specificity of the gRNA without having to re-engineer the light-inducible proteins.
The LACE system can be retargeted to new endogenous loci by changing gRNA specificity without re-engineering the light-inducible proteins.
Unlike other optogenetic systems, the LACE system can be targeted to new endogenous loci by solely manipulating the specificity of the gRNA without having to re-engineer the light-inducible proteins.
The LACE system can be retargeted to new endogenous loci by changing gRNA specificity without re-engineering the light-inducible proteins.
Unlike other optogenetic systems, the LACE system can be targeted to new endogenous loci by solely manipulating the specificity of the gRNA without having to re-engineer the light-inducible proteins.
The LACE system can be retargeted to new endogenous loci by changing gRNA specificity without re-engineering the light-inducible proteins.
Unlike other optogenetic systems, the LACE system can be targeted to new endogenous loci by solely manipulating the specificity of the gRNA without having to re-engineer the light-inducible proteins.
The LACE system can be retargeted to new endogenous loci by changing gRNA specificity without re-engineering the light-inducible proteins.
Unlike other optogenetic systems, the LACE system can be targeted to new endogenous loci by solely manipulating the specificity of the gRNA without having to re-engineer the light-inducible proteins.
Approval Evidence
1 source9 linked approval claimsfirst-pass slug lace-system
We engineered a light-activated CRISPR/Cas9 effector (LACE) system that induces transcription of endogenous genes in the presence of gene-specific guide RNAs (gRNAs) and blue light.
Source:
applicationsupports
LACE enabled spatial patterning of gene expression using an eGFP reporter and photomask illumination.
Spatial patterning of gene expression was also achieved using an eGFP reporter. Cells illuminated though a photomask containing slits of varying width resulted in a corresponding pattern of eGFP-expressing cells.
Source:
background activitysupports
In the dark, LACE-targeted cells maintained target gene expression levels that were not significantly different from mock-transfected cells.
In all instances, transfected cells incubated in the dark maintained levels of the targeted gene that did not significantly differ from mock-transfected cells.
Source:
comparisonmixed
For IL1RN and HBG1/2 activation, LACE achieved activation levels equivalent to dCas9-VP64, whereas for ASCL1 activation it was lower than dCas9-VP64.
Illuminated cells in which IL1RN or HBG1/2 was targeted demonstrated significantly greater mRNA levels in the light compared to the dark (p<0.0001 and 0.005, respectively), as well as equivalent activation levels to dCas9-VP64 (p=0.17 and 0.35, respectively). Significant light-dependent activation was also observed when the ASCL1 locus was targeted with the LACE system (p<0.0001). However, in this case mRNA levels were not activated to the same extent as cells that received dCas9-VP64 and the same four ASCL1-targeting gRNAs.
Source:
control propertysupports
Endogenous gene expression controlled by LACE is reversible and repeatable by modulating blue light exposure duration.
Endogenous gene expression could be controlled in a reversible and repeatable fashion by modulating the duration of blue light exposure.
Source:
design optimizationsupports
Fusing CIBN to both the N- and C-termini of dCas9 produced 10- to 100-fold greater gene activation than fusing CIBN to only one terminus.
Importantly, the fusion of CIBN to both N- and C-termini of dCas9 yielded 10- to 100-fold greater gene activation than when CIBN was fused to only one terminus.
Source:
mechanismsupports
LACE uses CRY2 and CIB1 heterodimerization to recruit VP64 to dCas9-targeted genomic loci under blue light.
This system is based on the plant proteins CRY2 and CIB1 from Arabidopsis thaliana that heterodimerize in response to blue light. The full-length CRY2 was fused to the N-terminus of the transcriptional activator VP64 (CRY2FL-VP64), and an N-terminal fragment of CIB1 was fused to the N- and C-terminus of the catalytically inactive form of Cas9 (CIBN-dCas9-CIBN). When these fusion proteins are expressed with a gRNA, CIBN-dCas9-CIBN localizes to the gRNA target. In the presence of blue light, CRY2FL binds to CIBN, which translocates CRY2FL-VP64 to the gene target and activates transcription.
Source:
mechanismsupports
The LACE system induces transcription of endogenous genes in the presence of gene-specific gRNAs and blue light.
We engineered a light-activated CRISPR/Cas9 effector (LACE) system that induces transcription of endogenous genes in the presence of gene-specific guide RNAs (gRNAs) and blue light.
Source:
performancesupports
LACE mediates light-dependent activation of endogenous IL1RN, HBG, and ASCL1 loci.
Light-dependent activation of the IL1RN, HBG, or ASCL1 genes was achieved by delivery of the LACE system and four gene-specific gRNAs per promoter region. Illuminated cells in which IL1RN or HBG1/2 was targeted demonstrated significantly greater mRNA levels in the light compared to the dark (p<0.0001 and 0.005, respectively)... Significant light-dependent activation was also observed when the ASCL1 locus was targeted with the LACE system (p<0.0001).
Source:
programmabilitysupports
The LACE system can be retargeted to new endogenous loci by changing gRNA specificity without re-engineering the light-inducible proteins.
Unlike other optogenetic systems, the LACE system can be targeted to new endogenous loci by solely manipulating the specificity of the gRNA without having to re-engineer the light-inducible proteins.
Source:
Comparisons
Source-backed strengths
In the dark, LACE-targeted cells maintained target gene expression levels that were not significantly different from mock-transfected cells, indicating low background activity under the reported conditions. For IL1RN and HBG1/2 activation, LACE reached activation levels equivalent to dCas9-VP64, although performance was target dependent. The system was also demonstrated for spatial patterning of reporter expression under photomask illumination.
Source:
Light-dependent activation of the IL1RN, HBG, or ASCL1 genes was achieved by delivery of the LACE system and four gene-specific gRNAs per promoter region. Illuminated cells in which IL1RN or HBG1/2 was targeted demonstrated significantly greater mRNA levels in the light compared to the dark (p<0.0001 and 0.005, respectively)... Significant light-dependent activation was also observed when the ASCL1 locus was targeted with the LACE system (p<0.0001).
Ranked Citations
- 1.