Abstract

I will discuss our recent advances in levering single cell approaches for monitoring cell state and CRISPR approaches to effect targeted perturbations to enable principled and information-rich exploration of biological processes focusing on two applications: tumor evolution and systematic discovery of gene function. Tumor Evolution Cancer progression is characterized by rare, transient events, which are nonetheless highly consequential to disease etiology and mortality. Detailed cell phylogenies can recount the history and chronology of these critical events including tumor evolution and metastasis. We have applied our Cas9-based lineage tracer in two setting: (1) the study of metastatic spread in a lung cancer xenograft mouse model revealing the underlying rates, routes, and drivers of metastasis. (2) the study of tumor evolution in a mouse model of non-small cell lung cancer in which an oncogenic Kras mutation and homozygous loss of the P53 tumor suppressor gene is initiated sporadically in the adult mouse. Our Kras/P53 studies enabled us to track tumor evolution from single transformed cells to metastatic tumors at unprecedented resolution. We found that loss of the initial, stable alveolar-type2-like state was accompanied by transient increases in plasticity followed by clonal sweep of rare, stable subclones capable of metastasizing to distant sites. Finally, we showed that tumors develop through stereotypical evolutionary trajectories, and perturbing additional tumor suppressors accelerates tumor progression by creating novel evolutionary paths. Overall, our study supports a hierarchical model of tumor evolution, and more broadly enables the in-depth study of tumor progression in vivo. Principled Discovery of Gene Function A central goal of genetics is to define relationships between genotypes and phenotypes. High-content phenotypic screens such as Perturb-seq (CRISPR-based single-cell RNA-sequencing screens) enable massively parallel functional genomic mapping but, to date, have been used at limited scales. We have now developed and applied an approach to perform genome-scale Perturb-seq targeting all expressed genes with CRISPRi across >2.5 million human cells and present a framework to power biological discovery from the resulting genotype-phenotype map. We use transcriptional phenotypes to predict gene functions, uncovering new regulators of ribosome biogenesis, transcription, and mitochondrial respiration. In addition to assigning gene function, single-cell transcriptional phenotypes allow for in-depth dissection of complex cellular phenomena – from RNA processing to differentiation. Leveraging this, we systematically identified genetic drivers and consequences of aneuploidy and discovered an unanticipated layer of stress-specific regulation of the mitochondrial genome. More broadly, our studies establish the ability of information-rich genotype-phenotype map to reveal a multidimensional portrait of gene and cellular function.