Targeted gene integration for high-throughput applications

TTargeted genome editing has come a long way in recent decades, especially with the advent of endonuclease-based gene editing systems such as CRISPR-Cas9. However, targeted insertion of larger payloads remains problematic, hindering what researchers are able to do in terms of capacity and throughput. Richard Davis“We are pleased to be working with the stem cell research team at the Leiden University Medical Center (LUMC) and Associate Researcher at the Novo Nordisk Foundation Centre for Stem Cell Medicine (reNEW),” said Dr. New StrategiesThe technique, called “Serine and Tyrosine Recombinase-Assisted Gene Integration for High-Throughput Interrogation (STRAIGHT-IN),” provides scientists with a powerful new tool for research and clinical applications.¹

Precision genome editing Dependent Donor DNA is integrated into the target region using homology-directed repair (HDR).² However, the efficiency of HDR targeting Decrease It increases significantly with increasing insertion size, so it remains challenging to insert multi-kilobase payloads.³ Site-specific recombination can address this issue, and Davis and his team at LUMC built STRAIGHT-IN by taking advantage of the strengths of two major classes of site-specific recombinases (SSRs): “Serine recombinases can introduce constructs quickly, but they integrate the entire vector (backbone, plasmid, everything),” Davis says. “Our experience shows that these [unnecessary] If the sequences were retained, they would result in silencing that would prevent expression in the future. Therefore, we used tyrosine recombinase to remove these auxiliary sequences that are not needed in the final cell line.”

According to the findings of Davis and his team: Published in Cell Report Methodshowed that STRAIGHT-IN facilitates targeted integration or replacement of multi-kilobase genomic fragments while leaving only traces (<300 base pairs) of plasmid backbone DNA.¹ This ability is important for creating a more physiologically relevant system. [endogenous] “You need to remove introns and regulatory elements and preserve the whole genome context of the gene,” Davis explained. Davis also noted that in the past, inserting large payloads often required manipulations of the cell’s genome that could change the phenotype, which was particularly problematic for stem cell researchers. “These additional modifications could include knocking out or overexpressing certain genes, which could then affect their phenotype and differentiation capacity,” Davis said.

The researchers designed STRAIGHT-IN with human induced pluripotent stem cells (hiPSCs) in mind. These cells are harder to genetically modify than immortalized cell lines, hindering their potential for disease research. [hiPSC] It makes the model more useful.” David Ragaespada“For example, it’s difficult to mimic in mice what goes on in human cancer genomes because mouse chromosomes are different. With hiPSC models, we have the potential to model gene amplification events and enhancer hijacking events that are more relevant to human cancers,” said Dr. John F. Kennedy, a geneticist at the University of Minnesota Medical School who was not involved in the study.

In addition to functionality, Davis and his team wanted to improve the speed and throughput of model generation. They developed a procedure to replace 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 manipulations. Now, without having to develop an entirely new procedure each time, they can insert either the original gene of interest or a variant, and the sequence will be placed within its endogenous genomic context. “Typically, Cas9 targeting takes three to six months,” Davis explains. “But if you have a system where someone can rapidly reintroduce a gene with a different variant, it becomes much quicker, even taking into account the time needed to establish it.” The approach also allows for multiplexing, which Davis and his colleagues demonstrated by introducing plasmids with 12 different mutations into a cell population and recovering 11 of them in a single transfection.

STRAIGHT-IN’s potential is perhaps best illustrated by its high ceiling: Davis and others in the field are already working on the immediate challenges, such as finding better SSRs and delivering them to differentiated cells. Davis’ team has already Introduced STRAIGHT-IN v2 eliminates the need for a clonal isolation step by achieving 100 percent efficiency.^Four Finally, Larga-Espada noted that Davis and his team have made available to other scientists both the vectors and cell lines that already have the SSR sites engineered into them. [bring in] And once scaled up, it’s feasible for many labs, and we’re exploring that ourselves.”