Gene Editing
DefiniGEN is highly experienced in using CRISPR/Cas9 gene editing technology to manipulate the genome of iPSC to generate disease models
Why choose DefiniGEN?
Our expertise can help design the guide RNA’s (gRNA) in silico for maximum efficiency, then optimize the electroporation of the cell line in question to best establish ideal conditions for the gene edit. Our team then methodically sequence a number of clones to confirm the precise genetic manipulation has been successful.
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Collaborative partnershipWe offer a collaborative partnership with our clients to deliver models in a timely and efficient manner
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Precise targetingTo help you generate your disease models, we offer gene editing services to knock out a target gene or knock in a disease specific mutation
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High quality modelsLeverage our robust gene editing process that delivers physiologically relevant disease models to advance your discoveries
Edits available
| Service | Description | Applications |
Knockout |
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| Indel for gene disruption | Gene inactivation by introducing insertion or deletion | Study gene function via gene inactivation |
| Large deletions | Deletion from few base pairs to kilobase in the desired gene | |
| Multiple deletions | Editing with multiple guides in a single reaction | |
Knock-in |
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| Point mutations/SNPs | Introducing point mutation or correcting disease-causing mutation | Study disease causing mutations in a clinical context and protein function in a native cell biology setting |
| Large casettes integration | Introducing large casettes | |
| Tag Reporter | Integration of reporters or tags | |
Gene editing process
- Optimal sgRNA design – screening 3 sgRNA
- Optimal donor design – to maximize the efficiency of knock-ins/knockouts: selecting optimal homology arm length, amount of donor DNA, donor strand orientation and distance between cutting and insertion site
- Choice of donor template: ssDNA or dsDNA - Choice of donor type is often dictated by the size of the modifications to be introduced: ssDNA donors have been mostly used for applications requiring small edits, dsDNA are used for integration of gene-sized reporters. ssDNA donors are advantageous to achieve a higher specificity of integration, in a direct comparison with equivalent dsDNA sequences.
- Delivery method: via Electroporation
- Cell line: wide range of cells
- Genotyping/screening: clonal (homozygous/heterozygous modification) or pooled
Gene Editing Workflow
Gene-editing of iPSCs allows researchers to create isogenic cell models containing key disease driving mutations to achieve mechanistic understanding, without background genetic variability allowing determination of causative relationships between genotype and phenotype.
Deliverables
- Highly characterized/validated edited human cell lines (clonal or pool)
- Isogenic (parental) controls to remove any phenotypic effects driven by factors intrinsic to the particular mutation of interest