Source 1primary paper2015Nucleic Acids Research
Toolkit/split-Cas9D10A nickase version
split-Cas9D10A nickase version
Also known as: analogously designed split-Cas9D10A nickase version
Taxonomy: Mechanism Branch / Architecture. Workflows sit above the mechanism and technique branches rather than replacing them.
Summary
The split-Cas9D10A nickase version is an intein-mediated split form of Cas9 carrying the D10A nickase mutation, designed to reconstitute an active nickase from separate polypeptide components. In the cited 2015 study, the analogously designed split-Cas9D10A showed similar activity to full-length Cas9D10A.
Usefulness & Problems
Why this is useful
This tool is useful for deploying Cas9D10A nickase activity in a split, multi-component format while preserving activity similar to the unsplit nickase. The source context indicates that intein-mediated split Cas9 systems can be packaged, delivered, and reconstituted in cells via rAAV, which is relevant to delivery-constrained applications.
Problem solved
It addresses the problem of delivering a large Cas9-derived genome editing effector by dividing it into separate components that can be reassembled intracellularly. In the nickase context, it supports double-nick genome editing strategies associated with homologous directed recombination, as stated in the provided tool description.
Taxonomy & Function
Primary hierarchy
Mechanism Branch
Architecture: A composed arrangement of multiple parts that instantiates one or more mechanisms.
Mechanisms
double-nick genome editingenzymatic dna nickinghomologous directed recombinationprotein trans-splicingTechniques
Computational DesignTarget processes
recombinationImplementation Constraints
Implementation is based on intein-mediated splitting and reconstitution of Cas9D10A from separate components. The source literature indicates cellular delivery and reconstitution via rAAV for the split-Cas9 platform, but the provided evidence does not specify split site, intein identity, guide design, or construct architecture for the nickase version.
The supplied evidence is limited to a single study and a brief statement of similar activity, without quantitative editing rates, locus-specific performance, or off-target measurements for the nickase version. Independent replication and broad validation across cell types or organisms are not provided in the evidence set.
Validation
Cell-freeBacteriaMammalianMouseHumanTherapeuticIndep. Replication
Supporting Sources
Ranked Claims
The split-Cas9D10A nickase version showed similar activity to Cas9D10A.
An analogously designed split-Cas9D10A nickase version showed similar activity as Cas9D10A.
The split-Cas9D10A nickase version showed similar activity to Cas9D10A.
An analogously designed split-Cas9D10A nickase version showed similar activity as Cas9D10A.
The split-Cas9D10A nickase version showed similar activity to Cas9D10A.
An analogously designed split-Cas9D10A nickase version showed similar activity as Cas9D10A.
The split-Cas9D10A nickase version showed similar activity to Cas9D10A.
An analogously designed split-Cas9D10A nickase version showed similar activity as Cas9D10A.
The split-Cas9D10A nickase version showed similar activity to Cas9D10A.
An analogously designed split-Cas9D10A nickase version showed similar activity as Cas9D10A.
The split-Cas9D10A nickase version showed similar activity to Cas9D10A.
An analogously designed split-Cas9D10A nickase version showed similar activity as Cas9D10A.
The split-Cas9D10A nickase version showed similar activity to Cas9D10A.
An analogously designed split-Cas9D10A nickase version showed similar activity as Cas9D10A.
The split-intein split-Cas9 system has nuclease activity comparable to wild-type Cas9.
We demonstrated that the nuclease activity of our split-intein system is comparable to wild-type Cas9, shown by a genome-integrated surrogate reporter and by targeting three different endogenous genes.
The split-intein split-Cas9 system has nuclease activity comparable to wild-type Cas9.
We demonstrated that the nuclease activity of our split-intein system is comparable to wild-type Cas9, shown by a genome-integrated surrogate reporter and by targeting three different endogenous genes.
The split-intein split-Cas9 system has nuclease activity comparable to wild-type Cas9.
We demonstrated that the nuclease activity of our split-intein system is comparable to wild-type Cas9, shown by a genome-integrated surrogate reporter and by targeting three different endogenous genes.
The split-intein split-Cas9 system has nuclease activity comparable to wild-type Cas9.
We demonstrated that the nuclease activity of our split-intein system is comparable to wild-type Cas9, shown by a genome-integrated surrogate reporter and by targeting three different endogenous genes.
The split-intein split-Cas9 system has nuclease activity comparable to wild-type Cas9.
We demonstrated that the nuclease activity of our split-intein system is comparable to wild-type Cas9, shown by a genome-integrated surrogate reporter and by targeting three different endogenous genes.
The split-intein split-Cas9 system has nuclease activity comparable to wild-type Cas9.
We demonstrated that the nuclease activity of our split-intein system is comparable to wild-type Cas9, shown by a genome-integrated surrogate reporter and by targeting three different endogenous genes.
The split-intein split-Cas9 system has nuclease activity comparable to wild-type Cas9.
We demonstrated that the nuclease activity of our split-intein system is comparable to wild-type Cas9, shown by a genome-integrated surrogate reporter and by targeting three different endogenous genes.
Intein-mediated split-Cas9 can be packaged, delivered, and efficiently reconstitute nuclease activity in cells via rAAV.
Most importantly, we revealed for the first time that intein-mediated split-Cas9 can be packaged, delivered and its nuclease activity reconstituted efficiently, in cells via rAAV.
Intein-mediated split-Cas9 can be packaged, delivered, and efficiently reconstitute nuclease activity in cells via rAAV.
Most importantly, we revealed for the first time that intein-mediated split-Cas9 can be packaged, delivered and its nuclease activity reconstituted efficiently, in cells via rAAV.
Intein-mediated split-Cas9 can be packaged, delivered, and efficiently reconstitute nuclease activity in cells via rAAV.
Most importantly, we revealed for the first time that intein-mediated split-Cas9 can be packaged, delivered and its nuclease activity reconstituted efficiently, in cells via rAAV.
Intein-mediated split-Cas9 can be packaged, delivered, and efficiently reconstitute nuclease activity in cells via rAAV.
Most importantly, we revealed for the first time that intein-mediated split-Cas9 can be packaged, delivered and its nuclease activity reconstituted efficiently, in cells via rAAV.
Intein-mediated split-Cas9 can be packaged, delivered, and efficiently reconstitute nuclease activity in cells via rAAV.
Most importantly, we revealed for the first time that intein-mediated split-Cas9 can be packaged, delivered and its nuclease activity reconstituted efficiently, in cells via rAAV.
Intein-mediated split-Cas9 can be packaged, delivered, and efficiently reconstitute nuclease activity in cells via rAAV.
Most importantly, we revealed for the first time that intein-mediated split-Cas9 can be packaged, delivered and its nuclease activity reconstituted efficiently, in cells via rAAV.
Intein-mediated split-Cas9 can be packaged, delivered, and efficiently reconstitute nuclease activity in cells via rAAV.
Most importantly, we revealed for the first time that intein-mediated split-Cas9 can be packaged, delivered and its nuclease activity reconstituted efficiently, in cells via rAAV.
The double nick strategy increased homologous directed recombination.
Moreover, we showed that the double nick strategy increased the homologous directed recombination (HDR).
The double nick strategy increased homologous directed recombination.
Moreover, we showed that the double nick strategy increased the homologous directed recombination (HDR).
The double nick strategy increased homologous directed recombination.
Moreover, we showed that the double nick strategy increased the homologous directed recombination (HDR).
The double nick strategy increased homologous directed recombination.
Moreover, we showed that the double nick strategy increased the homologous directed recombination (HDR).
The double nick strategy increased homologous directed recombination.
Moreover, we showed that the double nick strategy increased the homologous directed recombination (HDR).
The double nick strategy increased homologous directed recombination.
Moreover, we showed that the double nick strategy increased the homologous directed recombination (HDR).
The double nick strategy increased homologous directed recombination.
Moreover, we showed that the double nick strategy increased the homologous directed recombination (HDR).
The authors developed an intein-mediated split-Cas9 system to bypass the Cas9 packaging limit for gene therapy delivery.
Therefore, we developed a split-Cas9 system, bypassing the packaging limit using split-inteins.
The authors developed an intein-mediated split-Cas9 system to bypass the Cas9 packaging limit for gene therapy delivery.
Therefore, we developed a split-Cas9 system, bypassing the packaging limit using split-inteins.
The authors developed an intein-mediated split-Cas9 system to bypass the Cas9 packaging limit for gene therapy delivery.
Therefore, we developed a split-Cas9 system, bypassing the packaging limit using split-inteins.
The authors developed an intein-mediated split-Cas9 system to bypass the Cas9 packaging limit for gene therapy delivery.
Therefore, we developed a split-Cas9 system, bypassing the packaging limit using split-inteins.
The authors developed an intein-mediated split-Cas9 system to bypass the Cas9 packaging limit for gene therapy delivery.
Therefore, we developed a split-Cas9 system, bypassing the packaging limit using split-inteins.
The authors developed an intein-mediated split-Cas9 system to bypass the Cas9 packaging limit for gene therapy delivery.
Therefore, we developed a split-Cas9 system, bypassing the packaging limit using split-inteins.
The authors developed an intein-mediated split-Cas9 system to bypass the Cas9 packaging limit for gene therapy delivery.
Therefore, we developed a split-Cas9 system, bypassing the packaging limit using split-inteins.
Co-expression of the two Cas9 halves fused to corresponding split-intein moieties reconstitutes full Cas9 protein by intein-mediated trans-splicing.
Each Cas9 half was fused to the corresponding split-intein moiety and, only upon co-expression, the intein-mediated trans-splicing occurs and the full Cas9 protein is reconstituted.
Co-expression of the two Cas9 halves fused to corresponding split-intein moieties reconstitutes full Cas9 protein by intein-mediated trans-splicing.
Each Cas9 half was fused to the corresponding split-intein moiety and, only upon co-expression, the intein-mediated trans-splicing occurs and the full Cas9 protein is reconstituted.
Co-expression of the two Cas9 halves fused to corresponding split-intein moieties reconstitutes full Cas9 protein by intein-mediated trans-splicing.
Each Cas9 half was fused to the corresponding split-intein moiety and, only upon co-expression, the intein-mediated trans-splicing occurs and the full Cas9 protein is reconstituted.
Co-expression of the two Cas9 halves fused to corresponding split-intein moieties reconstitutes full Cas9 protein by intein-mediated trans-splicing.
Each Cas9 half was fused to the corresponding split-intein moiety and, only upon co-expression, the intein-mediated trans-splicing occurs and the full Cas9 protein is reconstituted.
Co-expression of the two Cas9 halves fused to corresponding split-intein moieties reconstitutes full Cas9 protein by intein-mediated trans-splicing.
Each Cas9 half was fused to the corresponding split-intein moiety and, only upon co-expression, the intein-mediated trans-splicing occurs and the full Cas9 protein is reconstituted.
Co-expression of the two Cas9 halves fused to corresponding split-intein moieties reconstitutes full Cas9 protein by intein-mediated trans-splicing.
Each Cas9 half was fused to the corresponding split-intein moiety and, only upon co-expression, the intein-mediated trans-splicing occurs and the full Cas9 protein is reconstituted.
Co-expression of the two Cas9 halves fused to corresponding split-intein moieties reconstitutes full Cas9 protein by intein-mediated trans-splicing.
Each Cas9 half was fused to the corresponding split-intein moiety and, only upon co-expression, the intein-mediated trans-splicing occurs and the full Cas9 protein is reconstituted.
Approval Evidence
1 source2 linked approval claimsfirst-pass slug split-cas9d10a-nickase-version
An analogously designed split-Cas9D10A nickase version showed similar activity as Cas9D10A.
Source:
activity comparisonsupports
The split-Cas9D10A nickase version showed similar activity to Cas9D10A.
An analogously designed split-Cas9D10A nickase version showed similar activity as Cas9D10A.
Source:
editing strategy effectsupports
The double nick strategy increased homologous directed recombination.
Moreover, we showed that the double nick strategy increased the homologous directed recombination (HDR).
Source:
Comparisons
Source-backed strengths
The key reported strength is that the split-Cas9D10A nickase version showed similar activity to full-length Cas9D10A. More broadly, the parent split-intein Cas9 platform was reported to be packageable, deliverable, and efficiently reconstituted in cells via rAAV.
Source:
Therefore, we developed a split-Cas9 system, bypassing the packaging limit using split-inteins.
Ranked Citations
- 1.