Source 1primary paper2025MED
Toolkit/katA::mCherry replacement construct
katA::mCherry replacement construct
Also known as: katA locus replaced with mcherry
Taxonomy: Mechanism Branch / Architecture. Workflows sit above the mechanism and technique branches rather than replacing them.
Summary
For targeted gene insertion, the katA locus was replaced with mcherry, and successful integration was verified by PCR and increased mCherry fluorescence relative to the wild type.
Usefulness & Problems
Why this is useful
This construct pattern replaces the katA locus with mcherry as a targeted insertion example. It provides a fluorescently trackable integration outcome in B. methanolicus.; targeted gene insertion; reporter-based verification of locus replacement
Source:
This construct pattern replaces the katA locus with mcherry as a targeted insertion example. It provides a fluorescently trackable integration outcome in B. methanolicus.
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targeted gene insertion
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reporter-based verification of locus replacement
Problem solved
It shows that the platform can perform targeted gene insertion, not just deletions. The mCherry cargo also gives a convenient reporter signal for confirming successful replacement.; demonstrates targeted insertion at the katA locus with a fluorescent readout
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It shows that the platform can perform targeted gene insertion, not just deletions. The mCherry cargo also gives a convenient reporter signal for confirming successful replacement.
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demonstrates targeted insertion at the katA locus with a fluorescent readout
Problem links
demonstrates targeted insertion at the katA locus with a fluorescent readout
LiteratureIt shows that the platform can perform targeted gene insertion, not just deletions. The mCherry cargo also gives a convenient reporter signal for confirming successful replacement.
Source:
It shows that the platform can perform targeted gene insertion, not just deletions. The mCherry cargo also gives a convenient reporter signal for confirming successful replacement.
Published Workflows
Objective: Develop a CRISPR-Cas9 genome editing platform for Bacillus methanolicus MGA3 that supports precise deletions, gene replacements, targeted insertion, and template-free mutagenesis for metabolic engineering.
Why it works: The workflow couples Cas9 cleavage with native DNA repair pathways in B. methanolicus MGA3, allowing repair-template-guided precise edits or template-free indel formation depending on whether a repair template is provided.
Cas9-mediated double-strand break formationhomologous recombination for scarless deletions and gene replacementserror-prone end-joining repair for mutagenesis without a repair templateone-plasmid CRISPR-Cas9 engineeringhomology-directed repair editingtemplate-free Cas9 cuttinggenome sequencing validationPCR validationphenotypic complementation
Stages
- 1.Platform design and repair-mode definition(library_design)
This stage establishes the editing architecture and the two intended repair routes described in the abstract.
Selection: Design a one-plasmid Cas9 system that can exploit native DNA repair with or without a repair template.
- 2.Template-free mutagenesis assessment(functional_characterization)
The authors explicitly tested the no-template condition to determine whether native end-joining could support mutagenesis.
Selection: Assess whether Cas9 cutting without a repair template yields mutagenic repair outcomes.
- 3.Homology-directed deletion and replacement testing(functional_characterization)
This stage demonstrates the precise editing mode of the platform using repair templates.
Selection: Use homology-directed repair to generate scarless deletions and gene replacements at target loci.
- 4.Confirmatory validation of edited strains(confirmatory_validation)
The abstract explicitly reports orthogonal validation methods to verify both sequence changes and expected phenotypic consequences.
Selection: Confirm edits by genome sequencing, PCR, fluorescence, and expected phenotype with complementation.
Steps
- 1.Design a one-plasmid Cas9 editing system for B. methanolicus MGA3engineered genome editing system
Create a host-compatible CRISPR-Cas9 platform for genome editing in B. methanolicus MGA3.
The editing platform must be established before testing repair outcomes or validating edited loci.
- 2.Test Cas9 cutting without a repair templateediting system under no-template condition
Determine whether native end-joining can generate mutagenic repair outcomes in the absence of a repair template.
This directly evaluates the template-free editing mode described for the platform.
- 3.Use homology-directed repair to generate targeted deletions and replacementsediting system under repair-template condition
Demonstrate precise scarless deletions and gene replacements using repair templates.
After establishing that template-free cutting can yield indels, the authors also test the precise editing mode enabled by homologous recombination.
- 4.Confirm edits by orthogonal molecular and phenotypic assaysedited strains and reporter replacement construct
Verify that observed edits are correct at the sequence level and produce the expected functional consequences.
Confirmatory assays are performed after candidate edited strains are generated to reduce false-positive interpretation and establish functional validity.
Taxonomy & Function
Primary hierarchy
Mechanism Branch
Architecture: A reusable architecture pattern for arranging parts into an engineered system.
Mechanisms
crispr-cas9-mediated targeted genome cleavagefluorescent reporter expressionhomology-directed gene replacementTechniques
No technique tags yet.
Target processes
recombinationImplementation Constraints
cofactor dependency: cofactor requirement unknownencoding mode: genetically encodedimplementation constraint: context specific validationoperating role: sensor
It requires the CRISPR-Cas9 editing system, the katA-targeted replacement design, and PCR plus fluorescence measurement for verification. The abstract specifically mentions comparison to wild type for the fluorescence readout.; requires targeted replacement at the katA locus; verification used PCR and fluorescence comparison to wild type
The abstract does not show that this replacement strategy is general across many loci or cargos. It also does not provide quantitative fluorescence values.; the abstract only demonstrates this insertion at the katA locus
Validation
Cell-freeBacteriaMammalianMouseHumanTherapeuticIndep. Replication
Supporting Sources
Ranked Claims
Replacement of the katA locus with mcherry was successfully integrated and verified by PCR and increased mCherry fluorescence relative to wild type.
Approval Evidence
1 source1 linked approval claimfirst-pass slug kata-mcherry-replacement-construct
For targeted gene insertion, the katA locus was replaced with mcherry, and successful integration was verified by PCR and increased mCherry fluorescence relative to the wild type.
Source:
targeted insertionsupports
Replacement of the katA locus with mcherry was successfully integrated and verified by PCR and increased mCherry fluorescence relative to wild type.
Source:
Comparisons
Source-stated alternatives
The abstract also describes scarless deletions and gene replacements by homology-directed repair, as well as template-free mutagenesis after Cas9 cutting without a repair template.
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The abstract also describes scarless deletions and gene replacements by homology-directed repair, as well as template-free mutagenesis after Cas9 cutting without a repair template.
Source-backed strengths
integration was verified by PCR; integration was associated with increased mCherry fluorescence relative to wild type
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integration was verified by PCR
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integration was associated with increased mCherry fluorescence relative to wild type
Compared with CRISPR/Cas9
The abstract also describes scarless deletions and gene replacements by homology-directed repair, as well as template-free mutagenesis after Cas9 cutting without a repair template.
Shared frame: source-stated alternative in extracted literature
Strengths here: integration was verified by PCR; integration was associated with increased mCherry fluorescence relative to wild type.
Relative tradeoffs: the abstract only demonstrates this insertion at the katA locus.
Source:
The abstract also describes scarless deletions and gene replacements by homology-directed repair, as well as template-free mutagenesis after Cas9 cutting without a repair template.
Compared with CRISPR/Cas9 system
The abstract also describes scarless deletions and gene replacements by homology-directed repair, as well as template-free mutagenesis after Cas9 cutting without a repair template.
Shared frame: source-stated alternative in extracted literature
Strengths here: integration was verified by PCR; integration was associated with increased mCherry fluorescence relative to wild type.
Relative tradeoffs: the abstract only demonstrates this insertion at the katA locus.
Source:
The abstract also describes scarless deletions and gene replacements by homology-directed repair, as well as template-free mutagenesis after Cas9 cutting without a repair template.
Ranked Citations
- 1.