Human Cell Lines Reveal Genome's Tolerance to Structural Variation

Scientists have accomplished the most advanced engineering of human cell lines to date, demonstrating that genomes are more robust against significant structural changes than previously understood.

Researchers from the Wellcome Sanger Institute, Imperial College London, and Harvard University, and their collaborators utilized CRISPR prime editing to produce multiple versions of human genomes in cell lines, each containing distinct structural changes. Through genome sequencing, they analyzed the genetic effects of these structural variations on cell survival.

The study, published in Science, reveals that human genomes can endure substantial structural changes, including large deletions in the genetic code, as long as essential genes remain unaffected. This breakthrough paves the way for studying and predicting the role of structural variation in diseases.

Structural variation refers to alterations in the structure of an organism’s genome, such as deletions, duplications, and inversions of the genetic sequence. These changes can be extensive, sometimes impacting hundreds to thousands of nucleotides – the fundamental building blocks of DNA and RNA.

Structural variants are linked to developmental disorders and cancer. However, studying their effects on mammalian genomes and understanding their role in disease has been challenging due to the difficulty of engineering such genetic changes.

To address this, researchers from the Sanger Institute and their collaborators developed innovative methods for creating and studying structural variations.

In this new study, the team combined CRISPR prime editing and human cell lines – groups of human cells grown in a dish – to generate thousands of structural variants in human genomes within a single experiment.

To achieve this, the researchers used prime editing to insert recognition sequences into the genomes of human cell lines, which were then targeted with recombinase – an enzyme that allowed the team to “shuffle” the genome.

By embedding these recombinase handles into repetitive sequences, which consist of hundreds or thousands of identical sequences in the genome, they were able to integrate nearly 1,700 recombinase recognition sites into each cell line using a single prime editor.

This resulted in over 100 random large-scale genetic structural changes per cell. This marks the first time a mammalian genome has been “shuffled” on such a scale.

The team then examined the effects of these structural variations on the human cell lines. Using genomic sequencing, they captured “snapshots” of the human cells and their “shuffled” genomes over several weeks, observing which cells survived and which perished.

As anticipated, they discovered that structural variations deleting essential genes were strongly selected against, leading to cell death. However, cells with large-scale deletions in the genome that spared essential genes were able to survive.

The researchers also performed RNA sequencing on the human cell lines to measure gene activity or gene expression. They found that large-scale deletions in non-coding regions of the genome did not appear to affect the gene expression of the remaining genome.

The findings suggest that human genomes are highly tolerant of structural variations, including those that reposition hundreds of genes, as long as essential genes are not deleted. Furthermore, the researchers question whether much of the non-coding DNA in human genomes is dispensable, though further studies involving additional deletions in more cell lines are required to confirm this.

In a related study, also published today in Science, researchers from the University of Washington pursued a similar goal of generating large-scale structural variants and studying their effects on the human genome. This team employed a different method, adding recombinase sites to transposons – mobile genetic elements – which randomly integrated into the genomes of human cell lines and mouse embryonic stem cells.

Using their approach, they demonstrated that the effects of induced structural variants could be analyzed using single-cell RNA sequencing. This advancement enables large-scale screens of structural variant impacts, potentially improving the classification of structural variants in human genomes as either benign or clinically significant.

Both studies reached similar conclusions, showing that human genomes are surprisingly tolerant of substantial structural changes. The full extent of this tolerance remains to be explored in future research enabled by these technologies.

Overall, this research represents the most engineered human cell lines to date. For the first time, scientists can create structural variants in human genomes at large scales within a single experiment and analyze the many random versions of the genomes.

This work enhances the understanding of the role of structural variants in disease, potentially enabling predictions about their harmfulness in individuals. It also narrows the focus of genomic research on structural variations that contribute to disease, particularly if non-coding DNA can be excluded.

Additionally, this new tool allows scientists to develop streamlined cell lines with evolved properties, such as improved growth, resistance to drugs, or bioengineering capabilities for producing medicines.

If the genome was a book, you could think of a single nucleotide variant as a typo, whereas a structural variant is like ripping out a whole page. These structural variants are known to play roles in developmental diseases and cancer, but it has been difficult to study them experimentally. Through creative and collaborative thinking, we have been able to do complex engineering in human cells that no-one has done before.”

Dr. Jonas Koeppel, Study Co-First Author, University of Washington

Dr. Jonas Koeppel was previously associated with the Wellcome Sanger Institute.

Koeppel said, “By shuffling the genomes of human cell lines at large scale, we have shown that our genomes are flexible enough to tolerate significant structural changes. These tools will help focus future studies into structural variations and their roles in disease.”

Our studies were only made possible because the right mix of ingredients came together at the right time: the scale of genome sequencing, cutting-edge genome engineering, and the use of recombinases. And importantly, the open and collaborative nature of our science across global borders. Our teams independently had similar ideas and came together to make these pioneering studies happen.”

Dr. Raphael Ferreira, Study Co-First Author and Postdoctoral Researcher, Harvard Medical School

“Ten years ago, people thought it would take decades of work and hundreds of millions of dollars to engineer a rearrangeable human genome that scientists could use to study genome structure, but this work shows a way to make this possible right now. It is exciting to think about what new biology we can learn from rearrangeable genomes and where this might go next,” said, Professor Tom Ellis, Study Author and Associate Faculty at the Wellcome Sanger Institute, based at the Department of Bioengineering at Imperial College London

These studies represent a step change in the parallel creation and evaluation of structural variation in human genomes. The tools to create a single variant at a time had been available for decades, but we have demonstrated that interrogating variants and making randomized human genomes at scale is now doable. This gives new entry points both into the study of disease-associated variation, as well as opportunities for bioengineering.”

Dr. Leopold Parts, Study Co-Lead Author, Wellcome Sanger Institute