Two landing pads expand the possibilities for engineering human stem cells

A new genome-engineering platform developed in the group of reNEW AI Richard Davis enables researchers to insert multiple large DNA constructs into human induced pluripotent stem cells (hiPSCs) with high precision and speed. The platform, called STRAIGHT-IN Dual, was published on April 30 in Nature Biomedical Engineering and has already been applied in a simultaneous publication in Science.

Genetically modifying hiPSCs is not a simple task – especially when researchers need to insert large DNA sequences or complex gene circuits. Conventional approaches can result in the DNA integrating at random sites in the genome, in multiple copies, or in genomic regions where the expression of the inserted genes is unreliable. This makes it difficult to determine which effects can truly be attributed to the genetic intervention.

The Davis group developed the STRAIGHT-IN platform to overcome these limitations. It uses a predefined “landing pad” in the genome, allowing researchers to insert DNA constructs at a precise location and as a single copy.

From one to two
“With STRAIGHT-IN Dual, we have added a second landing pad. This means that two different DNA constructs can now be simultaneously inserted into the same stem cell line while maintaining complete control over where each one is integrated. The process is also rapid – correctly engineered populations can be generated in just over a week, without the need for passaging, and with more than 95% of cells containing the desired genetic constructs,” explains Davis.

More complex and controlled
The addition of a second landing pad is not simply a way to insert more DNA. Its main advantage is that it allows different genetic functions to be separated, combined and compared in the same genetically uniform cell line. One landing pad can be used to control cell identity, while the other can be used to introduce a reporter, test a promoter, or add a second inducible gene circuit.

“This gives us much more flexibility,” says Davis. “Many biological systems are not controlled by a single gene or a single genetic switch. With STRAIGHT-IN Dual, we can start to build and study more complex genetic programs in a controlled way.”

Applications
In the Nature Biomedical Engineering paper, the team demonstrates several applications of the platform. In one example, they used the two landing pads to distribute transcription factors required to generate induced motor neurons. Splitting these factors across two defined genomic sites improved motor neuron production compared with placing all factors on a single construct, and removed the need for additional selection steps.

The researchers also used STRAIGHT-IN Dual to separate cell-fate programming from genetic readouts. This allowed them to convert stem cells into neurons or endothelial cells while simultaneously testing how different promoters behaved in those differentiated cell types. This is important because genetic elements that work well in stem cells may behave differently after the cells have differentiated.

A foundation for others
The platform has also already been used by researchers outside reNEW. In a simultaneous publication in Science, the group of Associate Professor Katie Galloway at Massachusetts Institute of Technology used STRAIGHT-IN Dual to study how the arrangement of genes and regulatory elements influences gene expression. Their work shows that this “gene syntax” can affect feedback between transcription and DNA supercoiling, thereby regulating neighbouring genes.

“This is exactly the kind of application we hoped the platform would enable,” says Davis. “It shows that STRAIGHT-IN Dual is not only useful for our own work but can also help other researchers answer precise questions about gene regulation and synthetic biology.”

Looking ahead, the team expects STRAIGHT-IN Dual to support a wide range of applications in stem cell research. These include building more advanced reporter lines, testing synthetic gene circuits, improving directed differentiation, and modelling complex genetic diseases. One future possibility is the study of compound heterozygous mutations, where two different variants affect the two copies of a gene. A dual landing-pad system could provide a controlled way to engineer and compare such allele-specific configurations in otherwise identical stem cell lines.

“We anticipate that many researchers will find uses for STRAIGHT-IN Dual that we have not yet imagined,” concludes Davis. “By making complex genome engineering in human stem cells faster, more precise and more efficient, we hope the platform will support new discoveries in disease modelling, synthetic biology and regenerative medicine.”