Scientists have accomplished the intricate engineering of human cell lines, indicating that our genomes can withstand considerable structural alterations more than previously believed.
A team of researchers from the Wellcome Sanger Institute, Imperial College London, and Harvard University, among others, employed CRISPR prime editing to develop numerous iterations of human genomes in cell lines, each showcasing distinct structural modifications. Through genome sequencing, they assessed how these structural changes impacted the survival of cells.
The research, published today (30 January) in Science, demonstrates that as long as vital genes are preserved, our genomes can endure substantial structural modifications, including major deletions of genetic material. This study paves the way for exploring and predicting how structural variations contribute to diseases.
Structural variation refers to alterations in an organism’s genomic structure, such as deletions, duplications, and inversions of genetic sequences. These changes can significantly affect a large number of nucleotides—the fundamental components of DNA and RNA.
Such structural variants are linked to developmental disorders and cancer. Nonetheless, studying the consequences of structural variation in mammal genomes and understanding their role in diseases has proven challenging due to the difficulties in engineering these genetic alterations.
To address this issue, Sanger Institute researchers and their collaborators aimed to innovate methods for creating and examining structural variations.
In this recent research, the team combined CRISPR prime editing1 and human cell lines2—which are groups of human cells grown in a lab—to generate thousands of structural variants in human genomes within a single experiment.
Researchers achieved this by utilizing prime editing to introduce a recognition sequence into the human cell line genomes, which could then be targeted with recombinase3—an enzyme that enabled the rearrangement of the genome. By placing these recombinase sites into repetitive DNA sequences, they could use a single prime editor to add nearly 1,700 recognition sites to each cell line. This led to over 100 random, large-scale genetic structural changes per cell, marking the first time a mammalian genome has been ‘shuffled’ at this magnitude.
The researchers then assessed the effects of these structural variations on the human cell lines. With genomic sequencing, they captured ‘snapshots’ of the human cells and their ‘shuffled’ genomes over several weeks to observe which cells thrived and which perished.
Interestingly, they found that when essential genes were deleted, those cells were significantly disadvantaged and often died. In contrast, groups of cells that experienced large-scale deletions while avoiding key genes managed to survive.
Additionally, the team performed RNA sequencing on the human cell lines to evaluate gene activity—known as gene expression. This analysis revealed that large-scale deletions in non-coding regions of DNA had little impact on the gene expression in the rest of the cell.
The researchers concluded that human genomes are remarkably tolerant of structural variations, including those that alter the positions of numerous genes, granted that essential genes remain intact4. They also questioned whether much of the non-coding DNA in human genomes may be nonessential, though more research involving additional deletions in various cell lines is required.
In a related study published today in Science5, another group from the University of Washington shared a similar aim of generating large-scale structural variants and analyzing their effects on the human genome. They used a different strategy: adding recombinase sites to mobile genetic elements called transposons, which randomly integrated into the genomes of human cell lines and mouse embryonic stem cells.
Their approach demonstrated that the effects of these induced structural variants could be assessed using single-cell RNA sequencing. This progress hints at the potential for expansive screenings of structural variant impacts, which could enhance how we classify structural variants in human genomes as either harmless or clinically relevant. Both studies reached comparable conclusions about the human genome’s unexpected resilience to significant structural changes, although the full extent of this resilience requires further investigation using these new technologies.
In summary, this research showcases the most advanced engineering of human cell lines to date. For the first time, researchers can produce structural variants in human genomes on a large scale in a single experiment and investigate the numerous random modifications of our genomes.
This work has the potential to deepen our understanding of how structural variants contribute to disease, possibly enabling predictions regarding the harmful nature of such variants in individuals. The research also focuses on narrowing down the genomic areas for studying structural variations linked to disease, especially if non-coding DNA can be deemed unnecessary.
Furthermore, this innovative tool allows scientists to create new, optimized cell lines with distinct properties, such as improved growth rates, enhanced capabilities for studying drug resistance, or engineered sequences for therapeutic development.
Dr. Jonas Koeppel, co-first author, previously affiliated with the Wellcome Sanger Institute and now at the University of Washington, stated: “If we imagine the genome as a book, a single nucleotide variant would be akin to a typo, while a structural variant resembles a torn-out page. Although these structural variants are known to influence developmental disorders and cancer, experimental studies have been challenging. Through inventive and collaborative efforts, we’ve successfully engineered human cells in complex ways that no one has achieved before. Our genome shuffling on such a large scale reveals the flexibility of our genomic structure to endure substantial changes. These tools will facilitate future research into structural variations and their implications in disease.”
Dr. Raphael Ferreira, co-first author and postdoctoral researcher at the Church Lab within Harvard Medical School, remarked: “Our investigations were only feasible because the right mix of components came together at the right moment: extensive genome sequencing capabilities, advanced genome engineering techniques, and the utilization of recombinases. The collaborative nature of our international science community played a vital role, as our teams independently developed similar concepts and united to realize these groundbreaking studies.”
Professor Tom Ellis, a study author and Associate Faculty member at the Wellcome Sanger Institute, based in Imperial College London’s Department of Bioengineering, said: “A decade ago, it was believed that creating a rearrangeable human genome for scientific study would take many years and hundreds of millions in funding. This work demonstrates a pathway to achieve that goal now. It’s thrilling to consider what new biological insights we can gain from these rearrangeable genomes and where this research can lead next.”
Dr. Leopold Parts, co-lead author at the Wellcome Sanger Institute, stated: “These studies mark a significant advancement in the simultaneous production and analysis of structural variations in human genomes. While methods for creating individual variants have existed for decades, we have shown that it is now possible to interrogate and generate randomized human genomes at scale. This opens new avenues for studying disease-associated variations and offers bioengineering opportunities.”
Notes:
- For detailed information on prime editing, visit the Sanger blog: https://sangerinstitute.blog/2023/07/17/prime-editing-explainer/
- The human cell lines utilized in this study are HEK293T, which is tailored for genome engineering, and HAP1, which contains a single copy of the genome. This setup makes the subtle alterations caused by structural variants easier to observe. These cells do not possess the capability to develop into an organ or tissue; they are merely groups of isolated cells growing in a lab, serving as tools for understanding the human genome.
- Recombinases are enzymes that facilitate specific recombination events within DNA.
- The researchers caution that these findings stem from experiments conducted in cultured human cells and may not accurately reflect conditions in a living organism.
- Sudarshan Pinglay et al. (2025) ‘Multiplex generation and single cell analysis of structural variants in mammalian genomes.’ Science. DOI: 10.1126.science.ado5978
