A team of scientists from the Whitehead Institute for Biomedical Research and the Broad Institute at MIT and Harvard have systematically assessed the functions of more than 5,000 essential human genes using a new imaging-based screening method. Their analysis leverages CRISPR-Cas9 to knock out gene activity and provides a unique resource for understanding and visualizing gene function in a wide range of cellular processes with spatial and temporal resolution. The team’s findings cover more than 31 million individual cells and include quantitative data on hundreds of different parameters that help predict how genes work and work together. The new study appears in the November 7 online issue of the journal Cell.
“Throughout my career, I’ve wanted to see what happens in cells when the function of an essential gene is knocked out,” says MIT Professor Iain Cheeseman, lead author of the study and fellow at the Whitehead Institute. “Now we can do this, not just for one gene, but for every gene that matters to a human cell dividing in a box, and it’s extremely powerful. The resource we’ve created will not only benefit our own lab , but also to laboratories around the world.
Systematic disruption of essential gene function is not a new concept, but conventional methods have been limited by various factors, including cost, feasibility, and the ability to completely eliminate essential gene activity. Cheeseman, who is the Herman and Margaret Sokol Professor of Biology at MIT, and his colleagues collaborated with MIT Associate Professor Paul Blainey and his team at the Broad Institute to define and realize this ambitious common goal. Broad Institute researchers have pioneered a new genetic screening technology that combines two approaches: large-scale pooled genetic screens using CRISPR-Cas9 and cell imaging to reveal quantitative and qualitative differences. Moreover, the method is inexpensive compared to other methods and is performed using commercially available equipment.
“We are proud to show the incredible resolution of cellular processes accessible with low-cost imaging assays in partnership with Iain’s lab at the Whitehead Institute,” says Blainey, lead study author, associate professor in the Department of Engineering Biology at MIT, Fellow of the Koch Institute for Integrative Cancer Research at MIT, and Senior Fellow of the Institute at the Broad Institute.” And clearly that’s just the tip of the iceberg for our approach. The ability to link genetic perturbations based on even more detailed phenotypic readouts is imperative, and now accessible, for many areas of future research.
Cheeseman adds, “The ability to bioscreen clustered cells is a fundamental game-changer. You have two cells side by side and therefore your ability to make statistically significant calculations of whether they are the same or not is so much higher, and you can discern very small differences.
Cheeseman, Blainey, lead authors Luke Funk and Kuan-Chung Su and their colleagues assessed the functions of 5,072 essential genes in a human cell line. They analyzed four markers across the cells on their screen – DNA; DNA damage response, a key cellular pathway that detects and responds to damaged DNA; and two important structural proteins, actin and tubulin. In addition to their main screen, the scientists also carried out a smaller follow-up screen focused on some 200 genes involved in cell division (also called “mitosis”). The genes were identified in their initial screen as playing a clear role in mitosis but had not previously been associated with the process. This data, which is made available through a companion website, provides a resource for other scientists to study the functions of genes of interest to them.
“There’s a tremendous amount of information we’ve gathered about these cells. For example, for the cell nucleus, it is not only its brilliant color, but its size, its roundness, are the edges smooth or bumpy? said Cheeseman. “A computer can really extract a wealth of spatial information.”
From this rich and multidimensional data, the work of the scientists provides a kind of cellular biological “fingerprint” for each gene analyzed in the screen. Using sophisticated computational clustering strategies, researchers can compare these fingerprints to each other and establish potential regulatory relationships between genes. Because the team’s data confirms multiple relationships that are already known, they can be used to make confident predictions about genes whose functions and/or interactions with other genes are unknown.
There are a host of notable findings to emerge from the researchers’ screening data, including a surprising one related to ion channels. two genes, AQP7 and ATP1A1, have been identified for their roles in mitosis, particularly the proper segregation of chromosomes. These genes code for membrane-bound proteins that transport ions in and out of the cell. “In all the years I’ve worked on mitosis, I never would have imagined that ion channels were involved,” says Cheeseman.
He adds: “We are only scratching the surface of what can be discovered from our data. We hope that many more will not only benefit from this resource, but also be inspired by it.
This work was supported by grants from the U.S. National Institutes of Health as well as support from the Gordon and Betty Moore Foundation, a National Defense Science and Engineering Graduate Fellowship, and a Council Fellowship research in the natural sciences and engineering.
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