Targeted Gene Integration for High-Throughput Applications

Targeted genomic editing made great strides in recent decades, especially thanks to the advent of endonuclease-based gene-editing systems such as CRISPR-Cas9. However, targeted insertion of larger payloads remains problematic, hampering what researchers can do in terms of capability and throughput. Richard Davis, a stem cell researcher at Leiden University Medical Center (LUMC) and associate investigator at The Novo Nordisk Foundation Center for Stem Cell Medicine (reNEW), believes that a new strategy, termed serine and tyrosine recombinase-assisted integration of genes for high-throughput investigation (STRAIGHT-IN), can give scientists a powerful new tool for research and clinical applications.¹

Precision genome editing relies on homology-directed repair (HDR) to incorporate donor DNA into targeted regions.² However, HDR targeting efficiency decreases significantly as insert size increases, so inserting multi-kilobase payloads remains difficult.³ Site-specific recombination can address this issue, and Davis and his team from LUMC used the strengths of two major site-specific recombinase (SSR) classes to build STRAIGHT-IN. “Serine recombinases can rapidly introduce constructs, but the whole vector—backbone, plasmid, everything—will integrate,” Davis said. “We knew from experience that if these [unnecessary] sequences were retained, there will be silencing down the road preventing expression. So, we used tyrosine recombinases to excise these auxiliary sequences which were not required in the final cell line.”

Davis and his team’s findings, published in Cell Reports Methods, showed that STRAIGHT-IN facilitated the targeted integration or substitution of multi-kilobase genomic fragments while leaving only traces (under 300 basepairs) of plasmid backbone DNA.¹ This capability is important for creating more physiologically relevant systems. “We wanted to keep all of the [endogenous] introns and regulatory elements; to retain the entire genomic context of the gene,” Davis explained. Davis also noted that in the past, inserting large payloads often required potentially phenotype-altering manipulations of cellular genomes. This was particularly problematic for stem cell researchers. “These additional modifications could involve knocking out or overexpressing certain genes, which would then affect their phenotype and their ability to differentiate,” said Davis.

The researchers designed STRAIGHT-IN with human induced pluripotent stem cells (hiPSCs) in mind, as the cells’ potential for disease research had been hampered because they were more difficult to genetically modify compared to immortalized cell lines. “STRAIGHT-IN technology is going to play a role in making [hiPSC] models more useful,” said David Largaespada, a geneticist at the University of Minnesota Medical School who was not associated with this study. “For example, we have trouble mimicking what happens in human cancer genomes in mice because murine chromosomes are different. With hiPSC models, we could potentially model things more relevant to human cancer like gene amplification events, enhancer hijacking events, and so on.”

In addition to capability, Davis and his team wanted to improve model generation speed and throughput. They developed a procedure where they replaced the gene of interest with a landing pad cassette containing SSR recognition and attachment sites, creating an “acceptor cell line” that serves as a template for future manipulation. Now, either the original gene of interest or a variant could be inserted without the need to develop a wholly new procedure each time, and the sequence would be placed within its endogenous genomic context. “Typically, Cas9 targeting requires three to six months,” Davis explained. “But having a system where someone can rapidly reintroduce the gene with different variants is far more rapid, even with the time necessary to establish it.” This approach also allowed multiplexing, which Davis and his colleagues demonstrated by introducing plasmids for twelve different mutations to a cell population, and recovering eleven of these from just a single transfection.

STRAIGHT-IN’s potential is perhaps best displayed by its high ceiling. Davis and others in the field are already tackling the immediate hurdles such as looking at potentially better SSRs and working on delivery into differentiated cells. Davis’s team has already introduced STRAIGHT-IN v2, which removed the need for a clonal isolation step by achieving 100 percent efficiency.⁴ Finally, Largaespada noted that Davis and his team have made both vectors and cell lines with already-integrated SSR sites available to other scientists: “I think it will be quite easy to [bring in] and to scale—it would be quite doable for many labs, and we’re considering it ourselves.”