When you're ready to study disease causing mutations in a clinical context or protein function in a native cell biology, DefiniGEN can advance your studies through efficient creation of an appropriate knock-in cell line.
Combining years of experience handling iPSCs with an understanding of how iPS cells respond to CRISPR-based genome editing, we deliver high success rates and a collaborative customer experience.
Our CRISPR cas9 knock-in cell line service can generate the full range of knock-ins including:
|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|
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.|
We pride ourselves on working with each client as a collaborative research partner!
What you will receive:
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.
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.
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.