The year is 2540 and people have become sterile. Human embryos are grown in factories, where they are manipulated and conditioned to develop predetermined conditions and complexions.
Reading Aldous Huxley’s A Brave New World and similar dystopian novels as a child sparked an interest in the possibilities—and caveats—of genome manipulation tools in the young Brian Brown, now director of the Genomics Institute at Icahn School of Medicine at Mount Sinai. “The type of thinking that fictional writers apply is also important for scientists: what are the limits of human knowledge and technology, and how can we develop things to go past them?” Brown said.
Transcending these limits is the driving force behind Brown’s career; the geneticist has engineered technologies that improve gene editing, sequencing, and gene therapy methods. Perturb-map, Brown’s latest invention described in Cell, adds another tool to the experimental belt of cancer researchers.1 With this gene editing technology, scientists can model tumor heterogeneity in vivo and study its consequences on other cell types, such as immune cells or interstitial cells. Therefore, Perturb-map holds great promise to reveal important aspects of cancer biology, including cancer cell evolution and patient responses to treatment.
The advent of CRISPR technology revolutionized scientists’ ability to rapidly modify large gene sets and study the consequences for cell function. Pooled CRISPR screens, in which scientists use a library of CRISPR-inducing vectors to manipulate different genes in a tissue’s various cells, have helped identify myriad genes with important functions in a variety of cancer-related processes, including immune cell activation and cancer signaling pathways.2
Scientists use multiplex imaging to identify mutant cancer cells and study a mutation’s intra- and extracellular effects.
Brian Brown, Director, Genomics Institute, Icahn School of Medicine at Mount Sinai.
While pooled CRISPR screens are powerful, Brown felt restricted by some of the technology’s limitations. To identify CRISPR-induced manipulations and connect them with changes in a cell’s behavior, scientists must dissociate the tissue and perform single cell sequencing along with other single cell phenotyping technologies, such as flow cytometry. This physical manipulation prevents scientists from studying gene functions that stretch beyond the cell membrane. “It’s not just that you wouldn’t find genes with obvious extracellular functions, like cytokines, but you also wouldn’t find the downstream functions of a gene that are related to an extracellular function,” Brown said.
To overcome this limitation, Brown developed Perturb-map—a CRISPR screening method that retains a tissue’s spatial information so that scientists can study a manipulation’s consequences for the edited cell and its neighbors. He created a system to visually identify individual CRISPR-edited cells through reporter gene expression. This way, scientists can combine multiplex immunohistochemistry with other phenotyping technologies, such as spatial transcriptomics, to identify and analyze mutant cells in their native environments.1,3 “The technology allows you to study different mutant clones within the same mouse. It is beautifully internally controlled in an otherwise very variable system,” said Benjamin Izar, assistant professor at Columbia University, who was not involved in the study.
To showcase the technology’s potential, Brown’s research group performed a spatial functional genomics screen in mice, knocking out any one of 35 different genes in individual lung cancer cells to generate small pockets of mutant clones. The researchers analyzed more than 200 mutant clones per targeted gene, assaying each perturbation’s consequences on cellular function, immune cell infiltration, vascular development, and more to uncover the corresponding gene’s intra- and extracellular functions.
When Brown analyzed immune cell infiltration in various mutant clones, he was struck by the differences between neighboring cells. “These cells have the same antigens, and they’re side by side. And yet, the T cells are penetrating [one mutant clone], but they cannot migrate into [an adjacent mutant] lesion, representing potential pockets of resistance,” Brown said. These findings show Perturb-map’s potential to reveal genetic determinants of patient responses to immunotherapy, a brave new world for Brown to explore next.
- M. Dhainaut et al., “Spatial CRISPR genomics identifies regulators of the tumor microenvironment,” Cell, 185(7):1223-39.e20, 2022.
- C. Bock et al., “High-content CRISPR-screening,” Nat Rev Methods Primers, 2(8):1-23, 2022.
- A. Wroblewska et al, “Protein barcodes enable high-dimensional single-cell CRISPR screens,” Cell, 175(4):1141-55.e16, 2018.