Source 1primary paper2025MED
Toolkit/GFP activation assay
GFP activation assay
Taxonomy: Technique Branch / Method. Workflows sit above the mechanism and technique branches rather than replacing them.
Summary
In this study, we screened a panel of 11 CjCas9 orthologous using a GFP activation assay and identified seven active nucleases.
Usefulness & Problems
Why this is useful
The GFP activation assay was used to screen a panel of CjCas9 orthologs for activity. In the abstract, it served as the initial assay that identified seven active nucleases.; screening Cas9 ortholog activity
Source:
The GFP activation assay was used to screen a panel of CjCas9 orthologs for activity. In the abstract, it served as the initial assay that identified seven active nucleases.
Source:
screening Cas9 ortholog activity
Problem solved
It provides a way to triage multiple orthologs and find active candidates before deeper characterization.; provides an activity-based screen to identify active nucleases from an ortholog panel
Source:
It provides a way to triage multiple orthologs and find active candidates before deeper characterization.
Source:
provides an activity-based screen to identify active nucleases from an ortholog panel
Problem links
provides an activity-based screen to identify active nucleases from an ortholog panel
LiteratureIt provides a way to triage multiple orthologs and find active candidates before deeper characterization.
Source:
It provides a way to triage multiple orthologs and find active candidates before deeper characterization.
Published Workflows
Objective: Identify a compact active CjCas9 ortholog for in vivo genome editing and engineer it for higher activity and broader targeting scope.
Why it works: The workflow first screens multiple orthologs to find active nucleases, then prioritizes a compact candidate with a distinct PAM, then validates editing in mouse systems, and finally engineers the nuclease to improve activity and broaden PAM compatibility.
DNA cleavage by Cas9PAM recognitionortholog screeningGFP activation assaymutational engineeringAAV8-mediated in vivo delivery
Stages
- 1.Screen CjCas9 ortholog panel(broad_screen)
This stage identifies which orthologs are active before deeper prioritization and in vivo testing.
Selection: activity in a GFP activation assay
- 2.Prioritize Cj4Cas9 from active orthologs(hit_picking)
This stage narrows active orthologs to a particularly noteworthy candidate for downstream in vivo validation and engineering.
Selection: compact genome size and unique PAM preference
- 3.Validate editing in mouse zygotes(confirmatory_validation)
This stage confirms that the selected nuclease can perform genome editing in a mouse developmental context.
Selection: efficient Tyr disruption with albino phenotype in mouse zygotes
- 4.Validate AAV8-mediated editing in mouse liver(in_vivo_validation)
This stage tests whether the compact nuclease can function after AAV8 delivery in a therapeutically relevant in vivo setting.
Selection: efficient Pcsk9 editing in mouse liver with reduced serum cholesterol and LDL-C
- 5.Engineer Cj4Cas9 for improved activity and PAM scope(secondary_characterization)
This stage expands the utility of the selected nuclease after initial validation.
Selection: introduce L58Y/D900K mutations to increase activity and simplify PAM recognition
Taxonomy & Function
Primary hierarchy
Technique Branch
Method: A concrete measurement method used to characterize an engineered system.
Mechanisms
reporter activationTarget processes
recombinationselectionImplementation Constraints
cofactor dependency: cofactor requirement unknownencoding mode: genetically encodedimplementation constraint: context specific validationimplementation constraint: payload burdenoperating role: sensor
The abstract supports that the assay requires a GFP activation readout. It does not provide further protocol details.; requires a GFP activation readout
The abstract does not show that this assay alone establishes in vivo efficacy, therapeutic benefit, or full PAM characterization.; the abstract does not describe whether it measures in vivo therapeutic performance or off-target effects
Validation
Cell-freeBacteriaMammalianMouseHumanTherapeuticIndep. Replication
Supporting Sources
Ranked Claims
AAV8-delivered Cj4Cas9 efficiently edits Pcsk9 in mouse liver and reduces serum cholesterol and LDL-C levels.
Cj4Cas9 efficiently disrupts the Tyr gene in mouse zygotes and produces an albino phenotype.
The engineered Cj4Cas9 variant enCj4Cas9 containing L58Y/D900K has two-fold higher nuclease activity than wild-type Cj4Cas9 and recognizes an N3GG PAM.
nuclease activity increase versus wild type 2 foldPAM preference N3GG
Cj4Cas9 is a compact Cas9 ortholog of 985 amino acids with a 5'-NNNGRY-3' PAM preference.
PAM preference 5'-NNNGRY-3'protein length 985 amino acids
A GFP activation assay screen of 11 CjCas9 orthologs identified seven active nucleases.
active nucleases identified 7orthologs screened 11
Cj4Cas9 and its engineered variants are established for fundamental research and therapeutic applications.
Approval Evidence
1 source1 linked approval claimfirst-pass slug gfp-activation-assay
In this study, we screened a panel of 11 CjCas9 orthologous using a GFP activation assay and identified seven active nucleases.
Source:
screening resultsupports
A GFP activation assay screen of 11 CjCas9 orthologs identified seven active nucleases.
Source:
Comparisons
Source-stated alternatives
No alternative screening assay is named in the abstract.
Source:
No alternative screening assay is named in the abstract.
Source-backed strengths
enabled screening of 11 orthologs and identification of seven active nucleases
Source:
enabled screening of 11 orthologs and identification of seven active nucleases
Compared with haematoxylin-eosin stained histological sections
GFP activation assay and haematoxylin-eosin stained histological sections address a similar problem space because they share recombination, selection.
Shared frame: same top-level item type; shared target processes: recombination, selection
Compared with open-source microplate reader
GFP activation assay and open-source microplate reader address a similar problem space because they share recombination, selection.
Shared frame: same top-level item type; shared target processes: recombination, selection
Strengths here: looks easier to implement in practice.
Compared with touchscreen-equipped operant conditioning chambers
GFP activation assay and touchscreen-equipped operant conditioning chambers address a similar problem space because they share recombination, selection.
Shared frame: same top-level item type; shared target processes: recombination, selection
Ranked Citations
- 1.