Data for a new genetic function map is available for other scientists to use. “It’s a great resource in the way the human genome is a great resource, because you can go in and do research based on discoveries,” says Professor Jonathan Weissman.
The scientists used their Perturb-seq single-cell sequencing tool on all genes expressed in the human genome, each linking to their work in the cell.
Genetic research has advanced rapidly in recent decades. For example, just a few months ago, scientists announced the first complete sequencing of the human genome without gaps. Researchers are now moving forward again, creating the first complete functional map of genes that are expressed in human cells.
The Human Genome Project was an ambitious initiative to sequence every piece of human DNA. The project brought together collaborators from research institutions around the world, including the MIT Whitehead Institute for Biomedical Research, and was finally completed in 2003. Now, more than two decades later, the MIT professor Jonathan Weissman and his colleagues have gone beyond the sequence to present the first full sequence. functional map of genes expressed in human cells. The data from this project, published online on June 9, 2022 in the journal Cell, link each gene to its work in the cell and are the culmination of years of collaboration in the method of sequencing a single cell Perturb-seq.
The data is available for use by other scientists. “It’s a great resource in the way the human genome is a great resource, because you can go in and do discovery-based research,” says Weissman, who is also a member of the Whitehead Institute and a researcher at Howard Hughes Medical. High school. “Instead of defining in advance which biology you will look at, you have this map of genotype-phenotype relationships and you can go in and examine the database without having to do any experiments.”
CRISPR, meaning short grouped and regularly spaced palindromic repeats, a genome editing tool invented in 2009 made it easier than ever to edit DNA. It is easier, faster, less expensive, and more accurate than previous genetic editing methods.
The screen allowed researchers to delve into various biological issues. It was used to explore the cellular effects of genes with unknown functions, to investigate the response of mitochondria to stress, and to detect genes that cause chromosomes to be lost or gained, a phenotype that has been difficult to study in the United States. past. “I think this data set will allow for all sorts of analysis that we haven’t even thought about yet by people from other parts of biology, and suddenly they only have that available to take advantage of,” says the former postdoctoral fellow. of Weissman Lab Tom. Norman, lead co-author of the paper.
Perturb Perturb-seq
The project takes advantage of the Perturb-seq approach to track the impact of activating or deactivating genes with unprecedented depth. This method was first published in 2016 by a group of researchers including Weissman and MIT professor Aviv Regev, but it could only be used in small sets of genes and at great cost.
The great Perturb-seq map was made possible by the founding work of Joseph Replogle, a doctoral student in Weissman’s lab and co-author of this article. Replogle, in collaboration with Norman, who now runs a lab at the Memorial Sloan Kettering Cancer Center; Britt Adamson, assistant professor in the Department of Molecular Biology at Princeton University; and a group of 10x Genomics, proposed to create a new version of Perturb-seq that could be expanded. Researchers published a proof-of-concept paper in Nature Biotechnology in 2020.
The Perturb-seq method uses the editing of the CRISPR-Cas9 genome to introduce genetic changes into cells, and then uses unicellular RNA sequencing to capture information about RNAs that are expressed as a result of a particular genetic change. Because RNAs control all aspects of how cells behave, this method can help decode the many cellular effects of genetic changes.
Since their initial proof-of-concept paper, Weissman, Regev, and others have used this method of sequencing on smaller scales. For example, researchers used Perturb-seq in 2021 to explore how human and viral genes interact throughout an infection with HCMV, a common herpesvirus.
In the new study, Replogle and collaborators, including Reuben Saunders, a graduate student at Weissman’s lab and co-lead author of the paper, extended the method to the entire genome. Using human blood cancer cell lines and retinal-derived non-cancerous cells, he performed Perturb-seq on more than 2.5 million cells and used the data to build a complete map linking genotypes. to phenotypes.
Deepening in the data
Upon completing the screen, the researchers decided to use their new dataset and examine some biological questions. “The advantage of Perturb-seq is that it allows you to get a big data set in an unbiased way,” says Tom Norman. “No one really knows what the limits of what you can get out of this data set are. Now the question is, what are you really doing about it?
The first most obvious application was to examine genes with unknown functions. Because the screen also read phenotypes of many known genes, researchers could use the data to compare unknown genes with other known genes and look for similar transcriptional results, which might suggest that genetics worked together as part of a complex. bigger.
In particular, he highlighted the mutation of a gene called C7orf26. The researchers noted that genes whose elimination caused a similar phenotype were part of a protein complex called Integrator that played a role in the creation of small nuclear RNAs. The Integrator complex consists of many smaller subunits (previous studies had suggested 14 individual proteins) and the researchers were able to confirm that C7orf26 formed a 15th component of the complex.
They also found that the 15 subunits worked together in smaller modules to perform specific functions within the Integrator complex. “In the absence of this view of the thousand-foot-tall situation, it was not so clear that these different modules were so functionally different,” says Saunders.
Another advantage of Perturb-seq is that because the assay focuses on individual cells, researchers could use the data to examine more complex phenotypes that become muddy when studied in conjunction with data from other cells. lules. “We often take all the cells where the ‘X gene’ is knocked down and look at them together to see how they’ve changed,” says Weissman. “But sometimes, when you knock down a gene, different cells that are losing the same gene behave differently, and the average person can lose that behavior.”
The researchers found that a subset of genes whose deletion resulted in different cell-to-cell results was responsible for chromosomal segregation. Its removal was causing the cells to lose one chromosome or pick up one more, a condition known as aneuploidy. “You couldn’t predict what the transcriptional response to the loss of this gene was because it depended on the side effect of the chromosome you gained or lost,” says Weissman. “We realized that we could turn this around and create this compound phenotype in search of signed and lost chromosome signatures. In this way, we made the first genome-wide screen of the factors needed for the proper segregation of DNA. “
“I think the aneuploidy study is the most interesting application of this data to date,” Norman says. “It captures a phenotype that you can only get by reading a single cell. You can’t go after it any other way.”
The researchers also used their data set to study how mitochondria responded to stress. Mitochondria, which evolved from free-living bacteria, carry 13 genes in their genomes. Within nuclear DNA, about 1,000 genes are related in some way to mitochondrial function. “People have long been interested in how nuclear and mitochondrial DNA are coordinated and regulated under different cellular conditions, especially when a cell is stressed,” says Replogle.
The researchers found that when they disrupted different mitochondrial-related genes, the nuclear genome responded similarly to many different genetic changes. However, mitochondrial genome responses were much more variable.
“There’s still an open question as to why mitochondria still have their own DNA,” Replogle said. “A general idea of our work is that one of the benefits of having a separate mitochondrial genome could be to have localized or very specific genetic regulation in response to different stressors.”
“If you have one mitochondrion that is broken and another that is broken in a different way, those mitochondria might be responding differently,” Weissman says.
In the future, researchers hope to use Perturb-seq in different cell types in addition to the line of cancer cells in which they started. They also hope to continue exploring their gene function map and expect others to do the same. “This is really the culmination of many years of work by the authors and other contributors, and I’m very happy to see it continue to be successful and expanding,” says Norman.
Reference: “Mapping information-rich genotype-phenotype landscapes with genome-scale Perturb-seq” by Joseph M. Replogle, Reuben A. Saunders, Angela N. Pogson, Jeffrey A. Hussmann, Alexander Lenail, Alina Guna, Lauren Mascibroda, Eric J. Wagner, Karen Adelman, Gila Lithwick-Yanai, Nika Iremadze, Florian Oberstrass, Doron Lipson, Jessica L. Bonnar, Marco Jost, Thomas M. Norman and Jonathan S. Weissman, June 9, 2022, Cell.DOI: 10.1016 / j .cell.2022.05.013