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Overcome CRISPR’s Limitations for Efficient Mammalian Cell Line Engineering

Scientist on February 27, 2025
in Challenges, Discovery & Innovation, Research Services

This blog post was written by Applied StemCell, founded in 2008 to provide industry and academic researchers with the ability to leverage the power of induced pluripotent stem cells (iPSC) and gene editing. Their services are available on the Scientist.com marketplace.

While CRISPR has revolutionized the life science industry, it’s not the right tool for every gene editing project. With a dependence on host DNA repair factors, low knock-in efficiency for DNA fragments above 3-5 kb and the risk of off-target events related to induction of double-strand breaks, CRISPR can be challenging and time-consuming to use for mammalian cell applications requiring large knock-ins and high safety. These applications include building mammalian cell lines for:

  • Expression/over-expression of proteins, especially large proteins
  • Co-expression of multiple proteins
  • Consistent protein expression from clone-to-clone for pool-based screening
  • Antibody display
  • Protein screening libraries
  • Cell therapy development

As a company dedicated to enabling allogeneic cell therapies, we knew we needed a better tool for safely and efficiently inserting DNA into mammalian cells, which is why we created TARGATT™ gene knock-in technology.

Figure 1. TARGATT™ integrase mediates recombination between attB and attP sites, resulting in insertion of the gene of interest into the genome.

TARGATT™ enables site-specific single copy insertion of 20+ kb of DNA

The heart of TARGATT™ gene knock-in technology is an optimized integrase that enables rapid, efficient and site-specific insertion of large DNA fragments into the genome — up to 20 kb in a single reaction and much larger insert sizes with nested reactions. Because integration is precise, single copy and targeted to a defined site, we can avoid the challenges encountered with random integration mechanisms such as position effects, gene silencing and gene instability due to the integration of multiple transgene copies.

The complete TARGATT™ platform consists of the TARGATT™ integrase and two DNA sequences (Figure 1) — the attB site, which is located on the donor plasmid containing your DNA payload, and the attP site, which is engineered into the genome at your location of choice (also referred to as the TARGATT™ landing pad).

TARGATT™ integrase binds the attP and attB sites, mediating a site-specific recombination event that results in the stable insertion of a single copy of the donor plasmid into the genome at the attP site.

You can also use a double attB and attP configuration to avoid inserting bacterial sequences from your cloning plasmid (Figure 2).

Figure 2. We use flanking attB sites paired with double attP sites to avoid insertion of bacterial plasmid sequences.

TARGATT™ works in both dividing and non-dividing cells because it does not require any host cell components, and the reaction is highly efficient and unidirectional.

We are also releasing an inducible TARGATT™ system in early 2025. To learn more about inducible TARGATT™ gene knock-in systems or how to make the most of TARGATT™ technology through our services and products — contact us.

The H11 safe harbor locus provides consistent and reliable gene expression after TARGATT™ knock-in

While you can engineer a TARGATT™ landing pad into any location, we recommend the H11 safe harbor genomic locus (22q12.2, an intergenic region of human chromosome 22 between the DRG1 and EIF4ENIF1 genes), which has consistently delivered reliable expression in our many TARGATT™ cell line engineering projects (Figure 3).

Figure 3. TARGATT™ delivers efficient integration with consistent expression. We inserted a construct expressing mCherry into the TARGATT™ landing pad at the H11 safe harbor site in HEK293 cells and used flow cytometry to assess mCherry expression in two pools of integrants. The high integration efficiency after drug selection can be seen in the preponderance of mCherry-expressing cells. The reproducibly tight clustering of the mCherry signal demonstrates the consistency of expression from the H11 locus.

TARGATT™ knock-in technology is only available through Applied StemCell. If you’re interested in using TARGATT™ in your own labs rather than through a service project with our team, you can start with iPSCs, HEK293 and/or CHO cell lines that have TARGATT™ landing pads pre-engineered into the H11 locus — contact us to learn more.

For cell therapy developers, we have GMP-grade iPSCs with the TARGATT™ landing pad available, or we can start with our GMP-grade iPSCs and engineer a TARGATT™ landing pad at the location of your choice, all under GMP-compliant conditions.

TARGATT™ knock-in is single copy

We can ensure that TARGATT™ knock-in occurs in single copy and at the landing pad by configuring the donor plasmid and landing pad with appropriate drug selection or FACS-based selection. However, we also have a very simple study that visually demonstrates single-copy insertion (Figure 4). After co-transfection of TARGATT™ donor plasmids with expressing either mCherry or GFP, we only find cells that are either red or green and not that are yellow in an overlay, indicating that each cell only stably integrated either the mCherry plasmid or the GFP plasmid and not both.

Figure 4. TARGATT™ knock-in is single copy. (A) We co-transfected GFP and mCherry TARGATT™ donor plasmids into CHO cells and imaged the resulting cells using the green channel (B), the red channel (C) and as an overlay (D). The lack of yellow cells in the overlay indicates the absence of cells expressing both GFP and mCherry, demonstrating that genomic insertion only occurs in single copy.

TARGATT™ is ideal for creating screening libraries in mammalian cells

One great application of TARGATT™ technology is creating screening libraries in mammalian cells. With high integration efficiency and large cargo insertion, you can create libraries of full-length antibodies or other large oligomeric proteins in CHO cells. You save time by assessing efficacy with native post-translational modifications and, for membrane proteins and mammalian display, on mammalian cell membranes.

TARGATT™ is great for cell therapy development

One of the drivers of TARGATT™ knock-in technology was to create a safe way to site-specifically insert large DNA fragments into the human genome to enable allogeneic cell therapy development efforts. With TARGATT™, you can efficiently insert chimeric antigen receptors (CARs) and create logic-gated CARS for highly targeted (pun intended) recognition.

Go bigger, stay on target and develop the cell lines that get the job done

At Applied StemCell, we are dedicated to accelerating advanced therapeutics. Developing enabling technology like the TARGATT™ knock-in system is just a small part of what we do. Saving you time with research use and GMP-grade services and products is where we excel. By combining our TARGATT™ technology with a service project and/or licensing TARGATT™ technology for product development, you’ll be able to move faster with an engineered cell line that delivers better biological relevance.

See how TARGATT™ can overcome CRISPR’s limitations and expand what you can achieve by contacting the team at Applied StemCell.

cell line engineering crispr targatt