Arrayed CRISPR/Cas9 Screening for the Functional Validation of Cancer Genetic Dependencies.

CRISPR/Cas9 screening has revolutionized functional genomics in biomedical research and is a widely used approach for the identification of genetic dependencies in cancer cells. Here, we present an efficient and versatile protocol for the cloning of guide RNAs (gRNA) into lentiviral vectors, the production of lentiviral supernatants, and the transduction of target cells in a 96-well format. To assess the effect of gene knockouts on cellular fitness, we describe a competition-based cell proliferation assay using flow cytometry, enabling the screening of many genes at the same time in a fast and reproducible manner. This readout can be extended to any parameter that is accessible to flow-based measurements, such as protein expression and stability, differentiation, cell death, and others. In summary, this protocol allows to functionally assess the effect of a set of 50-300 gene knockouts on various cellular parameters within eight weeks. This protocol was validated in: Leukemia (2021), DOI: 10.1038/s41375-021-01169-6 Graphical abstract.

Identification of gene targets of mutant C/EBPα reveals a critical role for MSI2 in CEBPA-mutated AML.

Mutations in the gene encoding the transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα) occur in 10-15% of acute myeloid leukemia (AML). Frameshifts in the CEBPA N-terminus resulting in exclusive expression of a truncated p30 isoform represent the most prevalent type of CEBPA mutations in AML. C/EBPα p30 interacts with the epigenetic machinery, but it is incompletely understood how p30-induced changes cause leukemogenesis. We hypothesized that critical effector genes in CEBPA-mutated AML are dependent on p30-mediated dysregulation of the epigenome. We mapped p30-associated regulatory elements (REs) by ATAC-seq and ChIP-seq in a myeloid progenitor cell model for p30-driven AML that enables inducible RNAi-mediated knockdown of p30. Concomitant p30-dependent changes in gene expression were measured by RNA-seq. Integrative analysis identified 117 p30-dependent REs associated with 33 strongly down-regulated genes upon p30-knockdown. CRISPR/Cas9-mediated mutational disruption of these genes revealed the RNA-binding protein MSI2 as a critical p30-target. MSI2 knockout in p30-driven murine AML cells and in the CEBPA-mutated human AML cell line KO-52 caused proliferation arrest and terminal myeloid differentiation, and delayed leukemia onset in vivo. In summary, this work presents a comprehensive dataset of p30-dependent effects on epigenetic regulation and gene expression and identifies MSI2 as an effector of the C/EBPα p30 oncoprotein.