A Medium-Throughput Single Cell CRISPR-Cas9 Assay to Assess Gene Essentiality.

BACKGROUND: Target selection for oncology is a crucial step in the successful development of therapeutics. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 editing of specific loci offers an alternative method to RNA interference and small molecule inhibitors for determining whether a cell line is dependent on a specific gene product for proliferation or survival. In our initial studies using CRISPR-Cas9 to verify the dependence on EZH2 activity for proliferation of a SMARCB1/SNF5/INI1 mutant malignant rhabdoid tumor (MRT) cell line, we noted that the initial reduction in proliferation was lost over time. We hypothesized that in the few cells that retain proliferative capacity, at least one allele of EZH2 remains functional. To verify this, we developed an assay to analyze 10s-100s of clonal cell populations for target gene disruption using restriction digest and fluorescent fragment length analyses. RESULTS: Our results clearly show that in cell lines in which EZH2 is essential for proliferation, at least one potentially functional allele of EZH2 is retained in the clones that survive. CONCLUSION: This assay clearly indicates whether or not a specific gene is essential for survival and/or proliferation in a given cell line. Such data can aid the development of more robust therapeutics by increasing confidence in target selection.

Extracellular matrix regulation of metabolism and implications for tumorigenesis.

Attachment to extracellular matrix (ECM) is required for the survival and proliferation of normal epithelial cells. Epithelial tumor cells, however, often acquire "anchorage independence," a property that may contribute to their ability to invade and grow in foreign environments. Although apoptosis is the most rapid and effective mechanism that causes the death of matrix-detached cells, it has become apparent that detachment from matrix alters other aspects of cell physiology prior to commitment to cell death and that some of these alterations can lead to cell death under conditions where apoptosis is suppressed. This report provides an overview of death processes that contribute to the death of matrix-detached normal cells and describes mechanisms that confer anchorage independence, with a focus on ECM regulation of cell metabolism. Loss of matrix attachment leads to metabolic stress characterized by reduced nutrient uptake, decreased ATP production, and increased levels of reactive oxygen species (ROS). The decrease in ATP levels is prevented by either constitutive activation of the PI3K/Akt pathway or exogenous antioxidants. Additionally, decreased Erk signaling in matrix-detached cells causes a disproportionate decrease in flux through pyruvate dehydrogenase (PDH), leading to decreased entry of glucose carbons into the citric acid cycle. Interestingly, forced overexpression of a PDH inhibitor suppresses de novo lipogenesis and proliferation, highlighting the importance of mitochondrial metabolism in supplying intermediates for biosynthetic processes required for proliferation. Thus, ECM attachment is a key regulator of cellular metabolism, and alterations in metabolism owing to changes or loss of ECM engagement during tumorigenesis may serve important tumor-suppressive functions.