Knock In Gene Editing Service - Definigen

GENE EDITING KNOCK-IN SERVICE

Expand your insight into cellular physiology and gene function with knock-in cell lines

Definigen test tube in lab

Overview

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:

  • Introduction of point mutations and SNPs
  • Integration of large cassettes
  • Addition of a tag or reporter gene

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.

DefiniGEN Vials in Dry Ice

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. 

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.
DefiniGEN scientist using microscope

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.

Frequently Asked Questions

Frequently Asked Questions

  • 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.