Knock-Out Cell Line Service

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.

Knock-in 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.

With our years of experience generating mature, primary-like cells from iPSCs and our proficiency at using CRISPR for targeted genome editing, we can build the cells that best reflect the biology you wish to model.

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-in cells, and several additional services are also available depending on your project needs.

We are an approved supplier on scientist.com

QC Testing and Additional Services

We offer an array of validation services to fully characterize your knock-in 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-in cells are free from mycoplasma contamination
qPCR Screening	Validation of your knock-in cells to confirm they carry the target deletion/indel on the coding sequence at the mRNA level
Additional Services	Depending on your project needs, we can perform any of the following additional validation assays on your knock-in cell lines
RNA-Seq	RNAseq provides insight regarding the impact of CRISPR gene knock-in 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 you will 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-in.

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How long does the entire gene-editing process take?

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.

How do you confirm that the cells have the correct modification?

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.

Which gene editing method do you use?

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.