Source 1primary paper2019Frontiers in Physiology
Toolkit/duplex CRISPR/Cas9 technology
duplex CRISPR/Cas9 technology
Taxonomy: Technique Branch / Method. Workflows sit above the mechanism and technique branches rather than replacing them.
Summary
Duplex CRISPR/Cas9 technology is a genome-editing method that uses two guide RNAs to target intronic sequences flanking an exon, enabling excision of the intervening exon by Cas9-mediated cleavage. In human U-2 OS osteosarcoma cells, it was applied to generate CRY1 knockout, CRY2 knockout, and CRY1/CRY2 double knockout cell models.
Usefulness & Problems
Why this is useful
This method is useful for producing defined exon deletions rather than relying on small indels at a single cut site. In the cited study, it enabled construction of human cellular circadian clock models whose phenotypes resembled those from classical knockout mouse-derived cells.
Source:
Here, we used duplex CRISPR/Cas9 technology to generate three cellular models for studying human circadian clocks: CRY1 knockout cells, CRY2 knockout cells as well as CRY1/CRY2 double knockout cells.
Problem solved
It addresses the problem of generating human knockout cell models for CRY genes by removing whole exons through paired intronic targeting. The demonstrated application was creation of CRY1-null, CRY2-null, and double-null U-2 OS cell lines for circadian biology studies.
Problem links
Need controllable genome or transcript editing
DerivedDuplex CRISPR/Cas9 technology is a genome-editing method that uses two guide RNAs to excise whole exons by targeting exon-flanking intronic regions. In U-2 OS human osteosarcoma cells, it was used to generate CRY1 knockout, CRY2 knockout, and CRY1/CRY2 double knockout cellular models.
Taxonomy & Function
Primary hierarchy
Technique Branch
Method: A concrete method used to build, optimize, or evolve an engineered system.
Mechanisms
crispr/cas9-mediated double-strand dna cleavagecrispr/cas9-mediated double-strand dna cleavagedual-guide exon excision via deletion between two cut sitesdual-guide exon excision via deletion between two cut sitesgenome editinggenome editingTechniques
No technique tags yet.
Target processes
editingImplementation Constraints
cofactor dependency: cofactor requirement unknownencoding mode: genetically encodedimplementation constraint: context specific validationimplementation constraint: multi component delivery burdenoperating role: builderswitch architecture: multi component
The method uses two guide RNAs designed against intronic regions flanking the exon intended for deletion, together with CRISPR/Cas9. The provided evidence supports implementation in human U-2 OS cells, but does not specify Cas9 format, vector system, selection strategy, or validation workflow.
The supplied evidence is limited to one 2019 study in a single human osteosarcoma cell line and focuses on CRY1 and CRY2. No broader evidence is provided here on editing efficiency metrics, off-target effects, delivery modality, or performance in other genes, cell types, or organisms.
Validation
Cell-freeBacteriaMammalianMouseHumanTherapeuticIndep. Replication
Supporting Sources
Ranked Claims
The circadian phenotypes of the human CRY knockout cell models were similar to phenotypes of cells derived from classical knockout mouse models.
similar to circadian phenotypes of cells derived from classical knockout mouse models
The circadian phenotypes of the human CRY knockout cell models were similar to phenotypes of cells derived from classical knockout mouse models.
similar to circadian phenotypes of cells derived from classical knockout mouse models
The circadian phenotypes of the human CRY knockout cell models were similar to phenotypes of cells derived from classical knockout mouse models.
similar to circadian phenotypes of cells derived from classical knockout mouse models
The circadian phenotypes of the human CRY knockout cell models were similar to phenotypes of cells derived from classical knockout mouse models.
similar to circadian phenotypes of cells derived from classical knockout mouse models
The circadian phenotypes of the human CRY knockout cell models were similar to phenotypes of cells derived from classical knockout mouse models.
similar to circadian phenotypes of cells derived from classical knockout mouse models
The circadian phenotypes of the human CRY knockout cell models were similar to phenotypes of cells derived from classical knockout mouse models.
similar to circadian phenotypes of cells derived from classical knockout mouse models
The circadian phenotypes of the human CRY knockout cell models were similar to phenotypes of cells derived from classical knockout mouse models.
similar to circadian phenotypes of cells derived from classical knockout mouse models
The circadian phenotypes of the human CRY knockout cell models were similar to phenotypes of cells derived from classical knockout mouse models.
similar to circadian phenotypes of cells derived from classical knockout mouse models
The circadian phenotypes of the human CRY knockout cell models were similar to phenotypes of cells derived from classical knockout mouse models.
similar to circadian phenotypes of cells derived from classical knockout mouse models
The circadian phenotypes of the human CRY knockout cell models were similar to phenotypes of cells derived from classical knockout mouse models.
similar to circadian phenotypes of cells derived from classical knockout mouse models
Duplex CRISPR/Cas9 technology efficiently removed whole exons of CRY genes using two guide RNAs targeting exon-flanking intron regions in human osteosarcoma U-2 OS cells.
Duplex CRISPR/Cas9 technology efficiently removed whole exons of CRY genes by using two guide RNAs targeting exon-flanking intron regions of human osteosarcoma cells (U-2 OS).
Duplex CRISPR/Cas9 technology efficiently removed whole exons of CRY genes using two guide RNAs targeting exon-flanking intron regions in human osteosarcoma U-2 OS cells.
Duplex CRISPR/Cas9 technology efficiently removed whole exons of CRY genes by using two guide RNAs targeting exon-flanking intron regions of human osteosarcoma cells (U-2 OS).
Duplex CRISPR/Cas9 technology efficiently removed whole exons of CRY genes using two guide RNAs targeting exon-flanking intron regions in human osteosarcoma U-2 OS cells.
Duplex CRISPR/Cas9 technology efficiently removed whole exons of CRY genes by using two guide RNAs targeting exon-flanking intron regions of human osteosarcoma cells (U-2 OS).
Duplex CRISPR/Cas9 technology efficiently removed whole exons of CRY genes using two guide RNAs targeting exon-flanking intron regions in human osteosarcoma U-2 OS cells.
Duplex CRISPR/Cas9 technology efficiently removed whole exons of CRY genes by using two guide RNAs targeting exon-flanking intron regions of human osteosarcoma cells (U-2 OS).
Duplex CRISPR/Cas9 technology efficiently removed whole exons of CRY genes using two guide RNAs targeting exon-flanking intron regions in human osteosarcoma U-2 OS cells.
Duplex CRISPR/Cas9 technology efficiently removed whole exons of CRY genes by using two guide RNAs targeting exon-flanking intron regions of human osteosarcoma cells (U-2 OS).
Duplex CRISPR/Cas9 technology efficiently removed whole exons of CRY genes using two guide RNAs targeting exon-flanking intron regions in human osteosarcoma U-2 OS cells.
Duplex CRISPR/Cas9 technology efficiently removed whole exons of CRY genes by using two guide RNAs targeting exon-flanking intron regions of human osteosarcoma cells (U-2 OS).
Duplex CRISPR/Cas9 technology efficiently removed whole exons of CRY genes using two guide RNAs targeting exon-flanking intron regions in human osteosarcoma U-2 OS cells.
Duplex CRISPR/Cas9 technology efficiently removed whole exons of CRY genes by using two guide RNAs targeting exon-flanking intron regions of human osteosarcoma cells (U-2 OS).
Duplex CRISPR/Cas9 technology efficiently removed whole exons of CRY genes using two guide RNAs targeting exon-flanking intron regions in human osteosarcoma U-2 OS cells.
Duplex CRISPR/Cas9 technology efficiently removed whole exons of CRY genes by using two guide RNAs targeting exon-flanking intron regions of human osteosarcoma cells (U-2 OS).
Duplex CRISPR/Cas9 technology efficiently removed whole exons of CRY genes using two guide RNAs targeting exon-flanking intron regions in human osteosarcoma U-2 OS cells.
Duplex CRISPR/Cas9 technology efficiently removed whole exons of CRY genes by using two guide RNAs targeting exon-flanking intron regions of human osteosarcoma cells (U-2 OS).
Duplex CRISPR/Cas9 technology efficiently removed whole exons of CRY genes using two guide RNAs targeting exon-flanking intron regions in human osteosarcoma U-2 OS cells.
Duplex CRISPR/Cas9 technology efficiently removed whole exons of CRY genes by using two guide RNAs targeting exon-flanking intron regions of human osteosarcoma cells (U-2 OS).
Duplex CRISPR/Cas9 technology efficiently removed whole exons of CRY genes using two guide RNAs targeting exon-flanking intron regions in human osteosarcoma U-2 OS cells.
Duplex CRISPR/Cas9 technology efficiently removed whole exons of CRY genes by using two guide RNAs targeting exon-flanking intron regions of human osteosarcoma cells (U-2 OS).
Duplex CRISPR/Cas9 technology efficiently removed whole exons of CRY genes using two guide RNAs targeting exon-flanking intron regions in human osteosarcoma U-2 OS cells.
Duplex CRISPR/Cas9 technology efficiently removed whole exons of CRY genes by using two guide RNAs targeting exon-flanking intron regions of human osteosarcoma cells (U-2 OS).
Duplex CRISPR/Cas9 technology efficiently removed whole exons of CRY genes using two guide RNAs targeting exon-flanking intron regions in human osteosarcoma U-2 OS cells.
Duplex CRISPR/Cas9 technology efficiently removed whole exons of CRY genes by using two guide RNAs targeting exon-flanking intron regions of human osteosarcoma cells (U-2 OS).
Duplex CRISPR/Cas9 technology efficiently removed whole exons of CRY genes using two guide RNAs targeting exon-flanking intron regions in human osteosarcoma U-2 OS cells.
Duplex CRISPR/Cas9 technology efficiently removed whole exons of CRY genes by using two guide RNAs targeting exon-flanking intron regions of human osteosarcoma cells (U-2 OS).
Duplex CRISPR/Cas9 technology efficiently removed whole exons of CRY genes using two guide RNAs targeting exon-flanking intron regions in human osteosarcoma U-2 OS cells.
Duplex CRISPR/Cas9 technology efficiently removed whole exons of CRY genes by using two guide RNAs targeting exon-flanking intron regions of human osteosarcoma cells (U-2 OS).
Duplex CRISPR/Cas9 technology efficiently removed whole exons of CRY genes using two guide RNAs targeting exon-flanking intron regions in human osteosarcoma U-2 OS cells.
Duplex CRISPR/Cas9 technology efficiently removed whole exons of CRY genes by using two guide RNAs targeting exon-flanking intron regions of human osteosarcoma cells (U-2 OS).
Duplex CRISPR/Cas9 technology efficiently removed whole exons of CRY genes using two guide RNAs targeting exon-flanking intron regions in human osteosarcoma U-2 OS cells.
Duplex CRISPR/Cas9 technology efficiently removed whole exons of CRY genes by using two guide RNAs targeting exon-flanking intron regions of human osteosarcoma cells (U-2 OS).
Duplex CRISPR/Cas9 technology was used to generate human CRY1 knockout, CRY2 knockout, and CRY1/CRY2 double knockout cellular models in U-2 OS cells.
Here, we used duplex CRISPR/Cas9 technology to generate three cellular models for studying human circadian clocks: CRY1 knockout cells, CRY2 knockout cells as well as CRY1/CRY2 double knockout cells.
Duplex CRISPR/Cas9 technology was used to generate human CRY1 knockout, CRY2 knockout, and CRY1/CRY2 double knockout cellular models in U-2 OS cells.
Here, we used duplex CRISPR/Cas9 technology to generate three cellular models for studying human circadian clocks: CRY1 knockout cells, CRY2 knockout cells as well as CRY1/CRY2 double knockout cells.
Duplex CRISPR/Cas9 technology was used to generate human CRY1 knockout, CRY2 knockout, and CRY1/CRY2 double knockout cellular models in U-2 OS cells.
Here, we used duplex CRISPR/Cas9 technology to generate three cellular models for studying human circadian clocks: CRY1 knockout cells, CRY2 knockout cells as well as CRY1/CRY2 double knockout cells.
Duplex CRISPR/Cas9 technology was used to generate human CRY1 knockout, CRY2 knockout, and CRY1/CRY2 double knockout cellular models in U-2 OS cells.
Here, we used duplex CRISPR/Cas9 technology to generate three cellular models for studying human circadian clocks: CRY1 knockout cells, CRY2 knockout cells as well as CRY1/CRY2 double knockout cells.
Duplex CRISPR/Cas9 technology was used to generate human CRY1 knockout, CRY2 knockout, and CRY1/CRY2 double knockout cellular models in U-2 OS cells.
Here, we used duplex CRISPR/Cas9 technology to generate three cellular models for studying human circadian clocks: CRY1 knockout cells, CRY2 knockout cells as well as CRY1/CRY2 double knockout cells.
Duplex CRISPR/Cas9 technology was used to generate human CRY1 knockout, CRY2 knockout, and CRY1/CRY2 double knockout cellular models in U-2 OS cells.
Here, we used duplex CRISPR/Cas9 technology to generate three cellular models for studying human circadian clocks: CRY1 knockout cells, CRY2 knockout cells as well as CRY1/CRY2 double knockout cells.
Duplex CRISPR/Cas9 technology was used to generate human CRY1 knockout, CRY2 knockout, and CRY1/CRY2 double knockout cellular models in U-2 OS cells.
Here, we used duplex CRISPR/Cas9 technology to generate three cellular models for studying human circadian clocks: CRY1 knockout cells, CRY2 knockout cells as well as CRY1/CRY2 double knockout cells.
Duplex CRISPR/Cas9 technology was used to generate human CRY1 knockout, CRY2 knockout, and CRY1/CRY2 double knockout cellular models in U-2 OS cells.
Here, we used duplex CRISPR/Cas9 technology to generate three cellular models for studying human circadian clocks: CRY1 knockout cells, CRY2 knockout cells as well as CRY1/CRY2 double knockout cells.
Duplex CRISPR/Cas9 technology was used to generate human CRY1 knockout, CRY2 knockout, and CRY1/CRY2 double knockout cellular models in U-2 OS cells.
Here, we used duplex CRISPR/Cas9 technology to generate three cellular models for studying human circadian clocks: CRY1 knockout cells, CRY2 knockout cells as well as CRY1/CRY2 double knockout cells.
Duplex CRISPR/Cas9 technology was used to generate human CRY1 knockout, CRY2 knockout, and CRY1/CRY2 double knockout cellular models in U-2 OS cells.
Here, we used duplex CRISPR/Cas9 technology to generate three cellular models for studying human circadian clocks: CRY1 knockout cells, CRY2 knockout cells as well as CRY1/CRY2 double knockout cells.
Duplex CRISPR/Cas9 technology was used to generate human CRY1 knockout, CRY2 knockout, and CRY1/CRY2 double knockout cellular models in U-2 OS cells.
Here, we used duplex CRISPR/Cas9 technology to generate three cellular models for studying human circadian clocks: CRY1 knockout cells, CRY2 knockout cells as well as CRY1/CRY2 double knockout cells.
Duplex CRISPR/Cas9 technology was used to generate human CRY1 knockout, CRY2 knockout, and CRY1/CRY2 double knockout cellular models in U-2 OS cells.
Here, we used duplex CRISPR/Cas9 technology to generate three cellular models for studying human circadian clocks: CRY1 knockout cells, CRY2 knockout cells as well as CRY1/CRY2 double knockout cells.
Duplex CRISPR/Cas9 technology was used to generate human CRY1 knockout, CRY2 knockout, and CRY1/CRY2 double knockout cellular models in U-2 OS cells.
Here, we used duplex CRISPR/Cas9 technology to generate three cellular models for studying human circadian clocks: CRY1 knockout cells, CRY2 knockout cells as well as CRY1/CRY2 double knockout cells.
Duplex CRISPR/Cas9 technology was used to generate human CRY1 knockout, CRY2 knockout, and CRY1/CRY2 double knockout cellular models in U-2 OS cells.
Here, we used duplex CRISPR/Cas9 technology to generate three cellular models for studying human circadian clocks: CRY1 knockout cells, CRY2 knockout cells as well as CRY1/CRY2 double knockout cells.
Duplex CRISPR/Cas9 technology was used to generate human CRY1 knockout, CRY2 knockout, and CRY1/CRY2 double knockout cellular models in U-2 OS cells.
Here, we used duplex CRISPR/Cas9 technology to generate three cellular models for studying human circadian clocks: CRY1 knockout cells, CRY2 knockout cells as well as CRY1/CRY2 double knockout cells.
Duplex CRISPR/Cas9 technology was used to generate human CRY1 knockout, CRY2 knockout, and CRY1/CRY2 double knockout cellular models in U-2 OS cells.
Here, we used duplex CRISPR/Cas9 technology to generate three cellular models for studying human circadian clocks: CRY1 knockout cells, CRY2 knockout cells as well as CRY1/CRY2 double knockout cells.
Duplex CRISPR/Cas9 technology was used to generate human CRY1 knockout, CRY2 knockout, and CRY1/CRY2 double knockout cellular models in U-2 OS cells.
Here, we used duplex CRISPR/Cas9 technology to generate three cellular models for studying human circadian clocks: CRY1 knockout cells, CRY2 knockout cells as well as CRY1/CRY2 double knockout cells.
CRY1/CRY2 double knockout cells were arrhythmic.
were arrhythmic (for CRY1/CRY2 double knockout)
CRY1/CRY2 double knockout cells were arrhythmic.
were arrhythmic (for CRY1/CRY2 double knockout)
CRY1/CRY2 double knockout cells were arrhythmic.
were arrhythmic (for CRY1/CRY2 double knockout)
CRY1/CRY2 double knockout cells were arrhythmic.
were arrhythmic (for CRY1/CRY2 double knockout)
CRY1/CRY2 double knockout cells were arrhythmic.
were arrhythmic (for CRY1/CRY2 double knockout)
CRY1/CRY2 double knockout cells were arrhythmic.
were arrhythmic (for CRY1/CRY2 double knockout)
CRY1/CRY2 double knockout cells were arrhythmic.
were arrhythmic (for CRY1/CRY2 double knockout)
CRY1/CRY2 double knockout cells were arrhythmic.
were arrhythmic (for CRY1/CRY2 double knockout)
CRY1/CRY2 double knockout cells were arrhythmic.
were arrhythmic (for CRY1/CRY2 double knockout)
CRY1/CRY2 double knockout cells were arrhythmic.
were arrhythmic (for CRY1/CRY2 double knockout)
CRY1 knockout cells showed short-period, low-amplitude circadian rhythms.
showed short period, low-amplitude rhythms (for CRY1 knockout)
CRY1 knockout cells showed short-period, low-amplitude circadian rhythms.
showed short period, low-amplitude rhythms (for CRY1 knockout)
CRY1 knockout cells showed short-period, low-amplitude circadian rhythms.
showed short period, low-amplitude rhythms (for CRY1 knockout)
CRY1 knockout cells showed short-period, low-amplitude circadian rhythms.
showed short period, low-amplitude rhythms (for CRY1 knockout)
CRY1 knockout cells showed short-period, low-amplitude circadian rhythms.
showed short period, low-amplitude rhythms (for CRY1 knockout)
CRY1 knockout cells showed short-period, low-amplitude circadian rhythms.
showed short period, low-amplitude rhythms (for CRY1 knockout)
CRY1 knockout cells showed short-period, low-amplitude circadian rhythms.
showed short period, low-amplitude rhythms (for CRY1 knockout)
CRY1 knockout cells showed short-period, low-amplitude circadian rhythms.
showed short period, low-amplitude rhythms (for CRY1 knockout)
CRY1 knockout cells showed short-period, low-amplitude circadian rhythms.
showed short period, low-amplitude rhythms (for CRY1 knockout)
CRY1 knockout cells showed short-period, low-amplitude circadian rhythms.
showed short period, low-amplitude rhythms (for CRY1 knockout)
CRY2 knockout cells showed long-period circadian rhythms.
long period rhythms (for CRY2 knockout)
CRY2 knockout cells showed long-period circadian rhythms.
long period rhythms (for CRY2 knockout)
CRY2 knockout cells showed long-period circadian rhythms.
long period rhythms (for CRY2 knockout)
CRY2 knockout cells showed long-period circadian rhythms.
long period rhythms (for CRY2 knockout)
CRY2 knockout cells showed long-period circadian rhythms.
long period rhythms (for CRY2 knockout)
CRY2 knockout cells showed long-period circadian rhythms.
long period rhythms (for CRY2 knockout)
CRY2 knockout cells showed long-period circadian rhythms.
long period rhythms (for CRY2 knockout)
CRY2 knockout cells showed long-period circadian rhythms.
long period rhythms (for CRY2 knockout)
CRY2 knockout cells showed long-period circadian rhythms.
long period rhythms (for CRY2 knockout)
CRY2 knockout cells showed long-period circadian rhythms.
long period rhythms (for CRY2 knockout)
Resulting CRY knockout cell clones did not express CRY proteins.
Resulting cell clones did not express CRY proteins
Resulting CRY knockout cell clones did not express CRY proteins.
Resulting cell clones did not express CRY proteins
Resulting CRY knockout cell clones did not express CRY proteins.
Resulting cell clones did not express CRY proteins
Resulting CRY knockout cell clones did not express CRY proteins.
Resulting cell clones did not express CRY proteins
Resulting CRY knockout cell clones did not express CRY proteins.
Resulting cell clones did not express CRY proteins
Resulting CRY knockout cell clones did not express CRY proteins.
Resulting cell clones did not express CRY proteins
Resulting CRY knockout cell clones did not express CRY proteins.
Resulting cell clones did not express CRY proteins
Resulting CRY knockout cell clones did not express CRY proteins.
Resulting cell clones did not express CRY proteins
Resulting CRY knockout cell clones did not express CRY proteins.
Resulting cell clones did not express CRY proteins
Resulting CRY knockout cell clones did not express CRY proteins.
Resulting cell clones did not express CRY proteins
Approval Evidence
1 source2 linked approval claimsfirst-pass slug duplex-crispr-cas9-technology
Here, we used duplex CRISPR/Cas9 technology to generate three cellular models
Source:
editing outcomesupports
Duplex CRISPR/Cas9 technology efficiently removed whole exons of CRY genes using two guide RNAs targeting exon-flanking intron regions in human osteosarcoma U-2 OS cells.
Duplex CRISPR/Cas9 technology efficiently removed whole exons of CRY genes by using two guide RNAs targeting exon-flanking intron regions of human osteosarcoma cells (U-2 OS).
Source:
method capabilitysupports
Duplex CRISPR/Cas9 technology was used to generate human CRY1 knockout, CRY2 knockout, and CRY1/CRY2 double knockout cellular models in U-2 OS cells.
Here, we used duplex CRISPR/Cas9 technology to generate three cellular models for studying human circadian clocks: CRY1 knockout cells, CRY2 knockout cells as well as CRY1/CRY2 double knockout cells.
Source:
Comparisons
Source-backed strengths
The reported strength is efficient removal of whole CRY exons using two guide RNAs directed to exon-flanking intronic regions in U-2 OS cells. The resulting human knockout cell models showed circadian phenotypes similar to those observed in classical knockout mouse models, supporting functional relevance.
Source:
similar to circadian phenotypes of cells derived from classical knockout mouse models
Compared with CRISPR/Cas
duplex CRISPR/Cas9 technology and CRISPR/Cas address a similar problem space because they share editing.
Shared frame: same top-level item type; shared target processes: editing; shared mechanisms: genome editing
Compared with CRISPR/Cas9 genome editing technique
duplex CRISPR/Cas9 technology and CRISPR/Cas9 genome editing technique address a similar problem space because they share editing.
Shared frame: same top-level item type; shared target processes: editing; shared mechanisms: genome editing
Compared with CRISPR/Cas9 mediated genome editing
duplex CRISPR/Cas9 technology and CRISPR/Cas9 mediated genome editing address a similar problem space because they share editing.
Shared frame: same top-level item type; shared target processes: editing; shared mechanisms: genome editing
Ranked Citations
- 1.