The scientists used the Perturb-seq cell sequencing tool on each expressed gene in the human genome, linking each to its function in the cell. Genetic research has progressed rapidly in recent decades. For example, just a few months ago, scientists announced the first complete, non-empty human genome sequence. Now researchers are moving forward again, creating the first complete functional map of genes expressed in human cells. The work of the human genome was an ambitious initiative to sequence every piece of human DNA. The project brought together collaborators from research institutes around the world, including the MIT Whitehead Institute for Biomedical Research, and was completed in 2003. Now, more than two decades later, MIT professor Jonathan Weissman and his colleagues have gone to great lengths to present the first complete 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 function in the cell and is the culmination of many years of collaboration in the Perturb-seq cell sequencing method. The data is available for use by other scientists. “It’s a great resource in the way the human genome is a great resource, as 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 . Institute. “Instead of defining in advance which biology to study, you have this map of genotype-phenotype relationships and you can go in and check the database without having to experiment.” CRISPR, which means grouped short reciprocating repetitions at regular intervals, a genome-processing tool invented in 2009, made DNA processing easier than ever. It is easier, faster, less expensive and more expensive than previous genetic treatment methods. The screen allowed researchers to delve deeper into various biological questions. They used it to explore the cellular effects of genes with unknown functions, to investigate the mitochondrial response to stress, and to test for genes that cause the loss or acquisition of chromosomes, a phenotype that has proven difficult to study in the past. “I think this data set will allow for all kinds of analyzes that we haven’t even thought about yet from people from other parts of biology and suddenly they just have it available to use,” says the former Weissman Lab postdoctoral fellow. Norman, co-author of the work. Innovative Perturb-seq The project takes advantage of the Perturb-seq approach that makes it possible to monitor the impact of activating or deactivating genes with unprecedented depth. This method was first published in 2016 by a team of researchers, including Weissman and fellow MIT professor Aviv Regev, but could only be used on small sets of genes and at great cost. The huge Perturb-seq map was made possible by fundamental work by Joseph Replogle, an MD-PhD student in Weissman’s lab and co-author of this paper. Replogle, in collaboration with Norman, who now leads a lab at the Memorial Sloan Kettering Cancer Center. Britt Adamson, Assistant Professor in the Department of Molecular Biology at Princeton University. and a team at 10x Genomics, started building a new version of Perturb-seq that could be scaled up. The researchers published a paper proving the idea in Nature Biotechnology in 2020. The Perturb-seq method uses CRISPR-Cas9 genome processing to introduce genetic changes into cells, and then uses a single-cell RNA sequence to capture information about RNAs expressed as a result of a given genetic change. Because RNA controls all aspects of how cells behave, this method can help decode the many cellular effects of genetic modification. Since their initial work to prove the idea, Weissman, Regev, and others have used this sequencing method on a smaller scale. For example, researchers used Perturb-seq in 2021 to investigate how human and viral genes interact during an infection with HCMV, a common herpes virus. In the new study, Replogle and his colleagues, including Reuben Saunders, a graduate student in Weissman’s lab and co-author of the paper, upgraded the method across the entire genome. Using human blood cancer cell lines as well as non-cancerous cells derived from the retina, he performed Perturb-seq on more than 2.5 million cells and used the data to create a complete map linking genotypes to phenotypes. Deepening the data After completing the screen, the researchers decided to use their new data set and look at some biological questions. “The advantage of Perturb-seq is that it allows you to access a large body of data in an unbiased manner,” says Tom Norman. “No one knows exactly what the limits of what you can get from this kind of data. “Now the question is, what do you really do with it?” The first, most obvious application was the examination of genes with unknown functions. Because the screen also read phenotypes of many known genes, the researchers could use the data to compare unknown genes with known ones and look for similar transcriptional results, which could suggest that the gene products work together as part of a larger complex. The mutation of a gene called C7orf26 stood out. The researchers observed that the genes whose removal led to a similar phenotype were part of a complex of proteins called integrators that played a role in the formation of small nuclear RNAs. The Integrator complex is made up of many smaller subunits – previous studies had suggested 14 individual proteins – and the researchers were able to confirm that C7orf26 was a 15th component of the complex. They also discovered that the 15 subunits worked together in smaller units to perform specific functions within the Integrator complex. “Missing this picture of the thousands of feet high, it was not so clear that these different units were so functionally distinct,” says Saunders. Another advantage of Perturb-seq is that because the analysis focuses on individual cells, the researchers could use the data to examine more complex phenotypes that become muddy when studied in conjunction with data from other cells. “We often take all the cells in which the ‘X gene’ is down and count them on average to see how they changed,” says Weissman. “But sometimes when you break down a gene, different cells that lose the same gene behave differently and that behavior can be ignored by the average.” The researchers found that a subset of genes whose removal led to different results from cell to cell were responsible for the separation of chromosomes. Their removal caused the cells to lose a chromosome or to pick up an extra, a condition known as aneuploidy. “You could not predict the transcriptional response to the loss of this gene because it depended on the side effect of the chromosome you gained or lost,” says Weissman. “We realized that then we could reverse it and create this complex phenotype that seeks chromosome signatures that are acquired and lost. “In this way, we did the first test in the whole genome for factors that are needed for the proper separation of DNA.” “I think the aneuploidy study is the most interesting application of this data to date,” says Norman. “It captures a phenotype that you can only acquire by reading a single cell. “You can not chase it any other way.” The researchers also used their data set to study how mitochondria respond to stress. Mitochondria, which evolved from free bacteria, carry 13 genes in their genome. Within nuclear DNA, about 1,000 genes are somehow related to mitochondrial function. “People have long been interested in how nuclear and mitochondrial DNA are coordinated and regulated in different cell conditions, especially when a cell is stressed,” says Replogle. The researchers found that when they disrupted different genes associated with mitochondria, the nuclear genome responded similarly to many different genetic changes. However, mitochondrial genome responses were much more variable. “There is still an open question as to why mitochondria still have their own DNA,” said Replogle. “One big picture from our work is that one of the benefits of having a separate mitochondrial genome can be localized or very specific genetic regulation in response to different stressors.” “If you have one mitochondrion that has broken and another that has broken in a different way, those mitochondria could respond differently,” says Weissman. In the future, researchers hope to use Perturb-seq on different cell types in addition to the cancer cell line they started in. They also hope to continue exploring the map of their genetic functions and hope that others will do the same. “This is really the culmination of many years of work by writers and other contributors, and I’m very happy to see it continue to thrive and expand,” 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: 10.101 .κελλ .2022.05.013
