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Across biomanufacturing, disease modeling, synthetic biology, and cell therapy development, researchers increasingly need to insert large DNA constructs into mammalian genomes. Yet despite significant advances in genome editing technologies over the past decade, integrating large DNA payloads into mammalian genomes efficiently, site-specifically, and safely has remained a challenge.

Cell line developers are engineering bigger

For cell therapy applications, simple chimeric antigen receptor (CAR) designs are no longer sufficient. Next-generation constructs incorporate logic gating, safety switches, multiple signaling domains, and regulatory elements. At the same time, many therapeutic strategies require expression of full-length genes to address loss-of-function mutations—no small task, given that the median human gene length is 24–27 kb.

This challenge extends to biomanufacturing and synthetic biology, where multi-gene constructs and synthetic promoters lead to large expression cassettes.^1,2

Why conventional knock-in approaches struggle

Most targeted genome editing strategies rely on homology-directed repair (HDR) following a CRISPR-induced DNA double-strand break. While this method works well for small edits, there is a risk of off-target events. In addition, low efficiency makes it difficult to generate inserts greater than 3–5 kb and, since HDR requires an active cell cycle and DNA repair, this method is not usable for non-dividing cells.

Random integration approaches, including lentiviral vectors and transposon systems, can improve insertion efficiency but at the cost of control. DNA integrates at unpredictable genomic locations, often resulting in multiple copies and highly variable expression, which extends timelines by requiring clone screening and selection.

Large serine recombinases provide a partial solution

Large serine recombinases enable efficient, site-specific integration of large DNA payloads without relying on cellular repair pathways. With optimization, these systems can accommodate multi-gene constructs and full-length genes with high efficiency.³

However, integration site still matters. Without a defined genomic target, expression of inserted constructs can vary due to chromatin context, leading to inconsistent performance across clones.

Targeted large knock-ins with TARGATT technology

TARGATT technology addresses the large knock-in challenge by combining efficient, site-specific integration using a large serine recombinase with a validated genomic target site.

In this approach, a TARGATT landing pad is established at the H11 safe harbor locus, enabling precise insertion of large DNA constructs into a location that supports robust, stable, and uniform gene expression. By directing integration to this single, well-characterized site, TARGATT technology enables consistent expression within cell populations—streamlining cell line development for biomanufacturing and therapeutic applications.

This strategy provides several key advantages:

• Efficient integration of large DNA payloads, including multi-gene constructs and multiple expression cassettes⁴
• Targeted insertion at the H11 safe harbor locus
• Single-copy, defined genomic integration
• Reduced clonal variability and screening burden
• Enables pool-based screening
• Reproducible expression across engineered cell lines

Because each construct is inserted into the same genomic location, developers can generate cell lines with greater predictability significantly reducing the time and effort required for clone screening and selection.

Toward more predictable cell engineering

From advanced CAR-T designs to multi-gene therapeutic constructs and synthetic biology circuits, many next-generation applications depend on stable integration of large genetic payloads.

By pairing efficient large DNA integration with the proven H11 safe harbor locus, TARGATT technology enables a more controlled and reproducible approach to mammalian cell engineering—helping researchers move beyond the traditional limitations of large knock-ins.

References

1. Joshua Mayne, et al. Billion-Scale Deciphering of Human Gene Regulatory Grammar. BioRxiv 2025.11.10.687627.

2. Leonid Gaidukov, et al. A multi-landing pad DNA integration platform for mammalian cell engineering. Nucleic Acids Res. 2018 May 4;46(8):4072-4086. doi: 10.1093/nar/gky216.

3. W. Marshal Stark. Making serine integrases work for us. Curr Opin Microbiol. 2017 Aug:38:130-136. doi: 10.1016/j.mib.2017.04.006.

4. Applied StemCell. Expanding the capabilities of targeted integration. Published December 18, 2025.

See how TARGATT technology can help you engineer bigger by visiting appliedstemcell.com/targatt-technology.

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