CRISPR Cas9
For Cell Line Development
For Cell Line Development
Save Your Time, Money & Effort!
Leverage our extensive expertise in CRISPR/Cas9 genome editing technologies to generate genetically modified induced pluripotent stem cells with various modifications in a targeted gene of interest. As one of the earliest licensees of CRISPR/Cas9 technology, we have genetically engineered > 1800 unique cell lines and animal models for disease modeling, functional genomics, target identification, antibody validation, and validation for drug discovery and screening, and more. We offer affordable, comprehensive custom service with a fast turnaround time to meet the exact requirements of your projects. You can combine it with our downstream custom assay services for a seamless project workflow.
We can utilize CRISPR to create disease models and assist in developing your cell therapy products by precisely manipulating the genome. With CRISPR-edited disease models, you can accurately recapitulate disease phenotypes observed in human patients. Additionally, CRISPR-modified cells for cell therapy allow for the creation of improved allogeneic and autologous cell therapies customized to individual needs.
Do you have a project in mind? Take advantage of our expert consultation services. Get in touch with us today to speak with one of our experts who can help you design and execute your project.
Genetic modifications available through our CRISPR/Cas9 gene editing platform:
Gene knockout, point mutation knock-in, gene insertion in any locus, including safe harbor locus (large fragment insertion, reporter gene knock-in, gene replacement), conditional knockout/ knock-in models, conditional/ inducible gene expression models.
CRISPR applications:
Functional genomics, disease modeling, target identification and validation for drug discovery and screening, and many more.
Choosing the right genome editing technology:
Applied StemCell uses two complementary genome editing technologiesto generate advanced cell line and animal models very efficiently and effectively: CRISPR/Cas9 technology and our propriety site-specific gene integration technology, TARGATT™ for large fragment (up to 20 kb) knock-in into a safe harbor locus.
Frequently Asked Questions
What is CRISPR/Cas9 genome editing technology?
The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) Genome Editing technology is a versatile tool for efficient gene modification of nearly all types of cells and has gained popularity in just a few years due to the simplicity of design and delivery of its RNA-probes, high specificity and efficiency, ease of implementation, cost and turnaround time.
Developed from a naturally occurring genome editing system in bacteria adaptive immune response, CRISPR/Cas technology utilizes Cas endonuclease to cleave and introduce sequence-specific double stranded DNA breaks (DSB) as directed by non-coding guide RNAs (gRNAs). The gRNA or single guide RNA (sgRNA) is a synthetic RNA sequence made of crispr RNA (crRNA), a 17-20 nucleotide sequence complementary to the target DNA, and a trans-activating crRNA (tracrRNA), which serves as a binding scaffold for the Cas nuclease. After the gRNA guides the Cas nuclease to bind to the target DNA sequence, each Cas nuclease cuts 3-4 nucleotides upstream of a specific protospacer adjacent motif (PAM) sequence. To repair the DSB, the cell uses its own DNA repair machinery to add or delete or replace pieces of DNA sequence via homology direct repair (HDR) or non-homologous end joining repair (NHEJ) pathway.
CRISPR/Cas9 genome editing applications are varied, including functional genomics, disease modeling, target identification and validation for drug discovery and screening to name a few. Traditional methods of targeted genome editing such as homologous recombination and bacterial artificial chromosome for generation of animal and cellular genome have been labor intensive and time-consuming endeavors. The discovery and adaptation of site-specific nucleases, especially CRISPR, and recombinase-integrases for animal and human cells has revolutionized genome engineering and enabled the precise manipulation of the eukaryotic genome. This in turn has paved the way for better animal and cellular disease models, thereby improving our understanding of human diseases and the scope of gene-based therapies.
The CRISPR/Cas9 technology although very popular as a do-it-yourself genome editing tool, is still in its infancy and its outcome is still tainted by off-target mutagenesis in non-targeted sites after indiscriminate Cas9 cleavage and epigenetic factors influencing gene functions. Applied StemCell is one of the earliest commercial service providers for this technology, and we know how this system works. Enjoy stress-free research and let the experts at Applied StemCell engineer your cell line or animal model according to your specification.
What genome editing method do you use in your Cell Line service?
For knockout, point mutation, and DNA insertion (small and large DNA insertion): CRISPR/Cas9. For knock-in of DNA fragments in a genomic safe harbor locus, e.g. Rosa26, AAVS1, H11: TARGATT™.
Is there a size limit on DNA to be inserted into the genome (CRISPR Knock-in)?
For CRISPR/Cas9, we can insert fragments up to 9kb in cell lines with drug selection.
Have you encountered difficulties in genome editing in cell lines?
Typical challenges are:
How many guide RNAs do you typically design for a CRISPR Genome Editing Service?
We start with 2 gRNAs and validate them. If no active gRNA is identified, we do another 2 gRNAs. In most projects, two rounds of testing are sufficient.
Can you provide off-target analysis report?
Yes, upon customer’s request.
Are you currently providing gene editing service to biotech/pharmaceutical companies as well?
Yes.
Can we have a confidentiality disclosure agreement (CDA) before disclosing my project details?
Yes.
What is the efficiency of CRISPR-Cas9 technology as compared to TARGATT™ and random transgenesis for mouse model generation?
Efficiency of random transgenesis is dependent on the species and the strain of animal, if any. In mice, depending on the strain of mouse being used, the efficiency can vary between 3-30%. The CRISPR efficiency can be very high if the parameters are optimized for the strain of mouse and type of modification required (such as knockout, knock-in, and conditional knockout). The efficiency of TARGATT™ insertion at preselected safe harbor docking sites (attP sites) averages around 20-30%.
Which technology is suitable for inserting a gene of interest with GFP or Cre-ERT in a mouse model?
The choice of gene knock-in technology will depend on the promoter that the transgene will be expressed under. If the knock-in fragment is under control of an endogenous promoter, CRISPR/Cas9 methodology will be adopted. If the transgene expression cassette requires a specific promoter (Ex. CRE expressed under control of a tissue-specific promoter), TARGATT™ technology will be better suited for integrating the transgene.
What is the basis of your design algorithm for the sgRNAs?
There are a number of open source tools that have efficient gRNA design capabilities. We cannot share information regarding the design tool we use at Applied StemCell.