Targeted Perturb-seq enables genome-scale genetic screens in single cells

The transcriptome contains rich information on molecular, cellular and organismal phenotypes. However, experimental and statistical limitations constrain sensitivity and throughput of genetic screening with single-cell transcriptomics readout. To overcome these limitations, we introduce targeted Perturb-seq (TAP-seq), a sensitive, inexpensive and platform-independent method focusing single-cell RNA-seq coverage on genes of interest, thereby increasing the sensitivity and scale of genetic screens by orders of magnitude. TAP-seq permits routine analysis of thousands of CRISPR-mediated perturbations within a single experiment, detects weak effects and lowly expressed genes, and decreases sequencing requirements by up to 50-fold. We apply TAP-seq to generate perturbation-based enhancer–target gene maps for 1,778 enhancers within 2.5% of the human genome. We thereby show that enhancer–target association is jointly determined by three-dimensional contact frequency and epigenetic states, allowing accurate prediction of enhancer targets throughout the genome. In addition, we demonstrate that TAP-seq can identify cell subtypes with only 100 sequencing reads per cell

Identification of leukemic and pre-leukemic stem cells by clonal tracking from single-cell transcriptomics

Cancer stem cells drive disease progression and relapse in many types of cancer. Despite this, a thorough characterization of these cells remains elusive and with it the ability to eradicate cancer at its source. In acute myeloid leukemia (AML), leukemic stem cells (LSCs) underlie mortality but are difficult to isolate due to their low abundance and high similarity to healthy hematopoietic stem cells (HSCs). Here, we demonstrate that LSCs, HSCs, and pre-leukemic stem cells can be identified and molecularly profiled by combining single-cell transcriptomics with lineage tracing using both nuclear and mitochondrial somatic variants. While mutational status discriminates between healthy and cancerous cells, gene expression distinguishes stem cells and progenitor cell populations. Our approach enables the identification of LSC-specific gene expression programs and the characterization of differentiation blocks induced by leukemic mutations. Taken together, we demonstrate the power of single-cell multi-omic approaches in characterizing cancer stem cells.This project was financially supported by the Deutsche José Carreras Leukämie Stiftung grant DJCLS 20R/2017 (to L.V., S.H., L.M.S., and A.T.), the Emerson foundation grant 643577 (to L.V. and L.M.S.) and the German Bundesministerium für Bildung und Forschung (BMBF) through the Juniorverbund in der Systemmedizin “LeukoSyStem” (FKZ 01ZX1911D to L.V., S.H., and S.R). Contributions by S.R. were further supported by Emmy Noether Fellowship RA 3166/1-1 (DFG). Contributions by C.P. were supported by a Max-Eder Grant (German Cancer Aid 70111531). Contributions by D.N., J.C.J., W.K.H., and T.B. were supported by the Gutermuth Foundation, the H.W. & J. Hector fund, Baden-Württemberg, and the Dr. Rolf M. Schwiete Fund, Mannheim. D.N. is an endowed professor of the Deutsche José Carreras Leukämie Stiftung (DJCLS H 03/01

cAMP-mediated Induction of Cyclin E Sensitizes Growth-arrested Adipose Stem Cells to DNA Damage–induced Apoptosis

The differentiation capacity of mesenchymal stem cells has been extensively studied, but little is known on cell cycle–related events in the proliferation and differentiation phases of these cells. Here, we demonstrate that exposure to cAMP-increasing agents inhibits proliferation of adipose stem cells (ASCs). This antiproliferative effect is associated with both reduced cdk2 activity and pRB phosphorylation. Concomitantly, however, the level of cyclin E markedly increases upon cAMP induction, indicating that cyclin E may have cdk2-independent functions in these cells besides its role as a cdk2 activator. Indeed, we found indications of a cdk2-independent role of cyclin E in DNA damage–induced apoptosis. 8-CPT-cAMP sensitizes ASCs to γ-irradiation–induced apoptosis, an effect abolished by knockdown of cyclin E. Moreover, cAMP induces early activation of ERK, leading to reduced degradation of cyclin E. The cAMP-mediated up-regulation of cyclin E was blocked by knockdown of ERK or by an inhibitor of the ERK kinase MEK. We conclude that cAMP inhibits cdk2 activity and pRB phosphorylation, leading to reduced ASC proliferation. Concomitant with this growth inhibition, however, cyclin E levels are increased in a MEK/ERK-dependent manner. Our results suggest that cyclin E plays an important, cdk2-independent role in genotoxic stress–induced apoptosis in mesenchymal stem cells