GENE EDITING KNOCK-OUT SERVICE
Build a deeper understanding of gene function with knock-out iPSCs
Overview
With CRISPR technology you can build isogenic knock-out cell lines that advance your insight into biological function. Combining CRISPR editing with iPSCs gives you the added advantages of a virtually unlimited supply of knock-out cells. But using CRISPR on iPSCs can be challenging. Unless you're DefiniGEN.
With DefiniGEN, you're choosing a team with an extensive track record of success using CRISPR technology to edit finicky iPSCs. Whether you take advantage of our well-characterized in-house iPSC lines or use your own cells, we have the experience and processes in place to quickly deliver the knock-out cell lines your studies need.
Our knock-out gene editing service can inactivate your gene of interest using a deletion or through insertion of a disruptive indel. We can also knock-out multiple genes in the same reaction:
- Large deletions of >2kb in the target gene
- Indel introduction for gene disruption
- Multiple deletions of different genes
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Rapid turnaround timeOur optimized protocols and standardized workflows ensure fast, efficient project completion.
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High success ratesOur deep expertise handling challenging iPSCs easily translates into successful CRISPR editing of iPSCs
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Functional ValidationWe validate successful knock-out cell line generation through a variety of assays including genotyping, phenotyping, and cell marker analysis.
Knock-out workflow
Because every cell line responds differently to CRISPR gene editing, we start each project with an evaluation of your iPS cell line's response to editing. Alternatively, you can accelerate turnaround times and maximize success by starting with one of our in-house cell lines that do not require as extensive an evaluation.
How it Works
- We design up to 3 single guide RNAs (sgRNAs) per target gene using software powered by the latest scoring algorithms.
- Depending on their cutting efficiency and predicted on-target and off-target scores, we select the best sgRNA for gene editing.
- Purified, high-fidelity Cas9 protein is delivered into your chosen cells together with the optimal sgRNA in the form of ribonucleoprotein (RNP) complexes.
- Once CRISPR editing is complete, we perform an array of standard QC and validation tests on the knock-out cells, and several additional services are also available depending on your needs.
QC Testing and Additional Services
We offer an array of validation services to fully characterize your knock-out cell lines.
| Service | Description |
| Morphology, Sterility, and Pluripotency | We confirm that your cells have the expected morphology, express pluripotency mRNAs, and are sterility tested. |
| Mycoplasma Testing | We confirm that your knock-out cells are free from mycoplasma contamination. |
| qPCR Screening | Validation of your knock-out cells to confirm they carry the target deletion/indel on the coding sequence at the mRNA level |
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Additional Services |
Depending on your project needs, we can perform any of the following additional validation assays on your knock-out cell lines. |
| RNA-Seq | RNAseq provides insight regarding the impact of CRISPR gene knock-out on a global transcriptomic scale. |
| iPSC Differentiation |
We can save you time and costs by differentiating your CRISPR-edited iPSC lines for you using our highly efficient differentiation platform. |
| Off-target analysis | Additional, off-target screening is available using in silico prediction, targeted sequencing, exome sequencing, or whole-genome sequencing. |
Technical Support From Start to Finish
We pride ourselves on working with each client as a collaborative research partner!
What will you receive:
- Bi-weekly calls and interim emails to update you on the project progress.
- Highly characterized/validated CRISPR-edited human cell lines (clonal or pool).
- Final report containing details of the sgRNA design and the results of all QC and validation assays.
- Isogenic (parental) controls to determine any phenotypic effects driven by factors intrinsic to the gene knock-out.
Frequently Asked Questions
Gene Editing
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The entire gene editing process takes between 8-10 weeks depending on the complexity of the project. We will advise you of the lead time when we generate your quote.
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We use Sanger sequencing to verify that the correct modifications have been made. Upon request, we can perform whole exome sequencing to scan for off-target edits.
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We most commonly use a CRISPR RNP-based gene editing approach. To guarantee rapid and highly efficient gene inactivation (gene knockout), purified CAS9 protein is delivered into cells along with sgRNA (ribonucleoprotein (RNP) complex) via electroporation or transfection.
For knock-in experiments where the goal is either to introduce a point mutation or to insert a reporter/tag, we co-deliver donor repair template (ssODN or dsDNA, respectively) and RNP into the cells to facilitate the double-strand break (DSB)-mediated homology directed repair (HDR). Although we favour the Cas9 RNP delivery approach, we also offer plasmid or viral-based delivery of CRISPR components into the cells.