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In a groundbreaking scientific achievement, researchers have engineered the most complex human cell lines to date, revealing that our genomes may have a greater capacity to withstand substantial structural modifications than previously understood.
Collaborating scientists from the Wellcome Sanger Institute, Imperial College London, Harvard University, and other institutions employed CRISPR prime editing techniques to create diverse human genome variations in cell lines, each characterized by distinct structural alterations. By applying genome sequencing, they meticulously examined the genetic implications of these variations on cell viability.
Published on January 30 in Science, the study demonstrates that human genomes can endure significant structural modifications, such as substantial deletions of the genetic material, provided that essential genes remain functional. This research lays the groundwork for better understanding and predicting how structural variations contribute to various diseases.
Structural variation refers to alterations in the structure of an organism’s genome, encompassing deletions, duplications, and inversions in the genetic sequence. These modifications can range widely, sometimes impacting hundreds or even thousands of nucleotides, which are the fundamental units of DNA and RNA.
Such structural variants have been linked to developmental disorders and malignancies. Nevertheless, the intricate complexity and scale of these variations made it challenging to study their effects in mammalian genomes until now.
To address this hurdle, the team at the Sanger Institute focused on developing innovative methodologies to create and investigate structural variations. By integrating a combination of CRISPR prime editing techniques and human cell lines, they successfully generated thousands of structural variants in human genomes within a single experimental framework.
This process involved using prime editing to introduce specific recognition sequences into the genomes of human cell lines, which could be targeted by recombinase enzymes—allowing the researchers to ‘shuffle’ the genome. By embedding these recombinase handles within repetitive genomic regions, the scientists managed to insert nearly 1,700 recognition sites into each cell line, enabling the induction of over 100 large-scale genetic structural changes per cell. This marked a significant advancement, as previous methods lacked the capacity for such extensive rearrangements in mammalian genomes.
Subsequently, the team carefully monitored the effects of these structural variations on the human cell lines, employing genomic sequencing to capture ‘snapshots’ of the cells and their altered genomes over several weeks, tracking survival rates and cell death.
As anticipated, deletion of essential genes resulted in strong selective pressure, leading to cell death. Conversely, cell groups that experienced large-scale deletions, while maintaining essential genes, sustained viability.
The researchers also carried out RNA sequencing to assess gene activity or gene expression. Their findings indicated that large-scale deletions, particularly in non-coding regions of the genome, did not adversely affect the gene expression of the surrounding cellular environment.
This led the researchers to propose that human genomes exhibit remarkable tolerance to structural variations, even those that reposition numerous genes, provided that no essential genes are eliminated. They also raised questions regarding the potential dispensability of non-coding DNA, suggesting future research could better evaluate this hypothesis by engineering additional deletions across varied cell lines.
In a collaborative effort, another paper published concurrently in Science by researchers from the University of Washington explored similar ambitions of generating large-scale structural variants and assessing their impacts on the human genome through a different methodology. Their approach involved adding recombinase sites to transposons—mobile genetic elements—that were then randomly integrated into human cell lines and mouse embryonic stem cells.
This innovative approach highlighted the capacity to detect the effects of these structural variants using single-cell RNA sequencing. The implications of this work not only enhance the understanding of structural variant impacts but also improve the classification of such variants in human genomes, distinguishing between benign and clinically significant changes. Both studies underscored the surprising resilience of human genomes to sizeable structural changes, while emphasizing that the full scope of this tolerance is still an area for future exploration facilitated by ongoing advancements in genetic technologies.
The implications of this research extend to a deeper comprehension of structural variants and their roles in disease, potentially leading to predictive models regarding the detrimental impact of these variations on individual health. Moreover, the advanced engineering tools developed may enable scientists to create more refined cell lines optimized for various applications, such as drug resistance studies or the development of therapeutic solutions.
Dr. Jonas Koeppel, co-first author and now at the University of Washington, remarked on the significance of their findings by likening a structural variant in a genome to ripping out an entire page of a book, contrasting it with a simple typo that a single nucleotide variant would represent. He noted that while structural variants are known to play critical roles in a range of diseases, studying them has posed considerable challenges until now. Through collaborative efforts, they have successfully demonstrated the flexibility of human genomes to endure significant structural alterations.
Dr. Raphael Ferreira, also a co-first author and a researcher in the Church Lab at Harvard Medical School, emphasized the convergence of genome sequencing capabilities, advanced genome engineering techniques, and collaborative scientific endeavors as pivotal in achieving these findings.
Professor Tom Ellis, an author of the study with ties to both the Wellcome Sanger Institute and Imperial College London, expressed excitement over the possibilities unlocked by this research. He highlighted a decade of speculation surrounding the feasibility of engineering rearrangeable human genomes being overcome through current capabilities, anticipating new biological insights from these efforts.
Dr. Leopold Parts, co-lead author at the Wellcome Sanger Institute, concluded that these studies signify a paradigm shift in the generation and examination of structural variations in human genomes. While methods for creating individual variants have existed for years, their work demonstrates the feasibility and importance of addressing multiple variants simultaneously, paving new pathways for disease research and bioengineering opportunities.
Notes:
For more information on prime editing, read the Sanger blog: https://sangerinstitute.blog/2023/07/17/prime-editing-explainer/
The human cell lines evaluated in this study include HEK293T, designed for genome engineering, and HAP1, which possesses a singular genome copy, facilitating clearer detection of subtle structural changes. These cell lines merely serve as tools for understanding the intricacies of the human genome and do not develop into actual organs or tissues.
Recombinases are specialized enzymes that facilitate specific recombination events within DNA sequences.
It is important to note that these findings are based on experiments conducted in cultured human cells and may not necessarily replicate the conditions present 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
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