We use a variety of genome engineering approaches, to systematically connect genotypes to phenotypes in human cancer cells. We have previously developed CRISPR-based, gene perturbation approaches and resources, that allow us to produce loss-of-function (LOF) and gain-of-function (GOF) phenotypes at genome-scale in a single reaction vessel (Figure 1). We will now begin to further characterise the identified candidate genes and genetic interactions in leukemias, in order to explore their value as biomarkers and therapeutic targets. In parallel, we will expand our drug target discovery strategies to triple negative breast cancer, a difficult to treat cancer and one of our longstanding research interests (1, 2).
Figure 1 - Systematic genotype-phenotype connection. Pools of tens of thousands of sgRNA templates are synthesized on a single microarray surface and cloned into lentiviral sgRNA expression vectors. Using low lentiviral titers for target cancer cell infection (MOI<0.3) ensures the genomic integration of no more than one sgRNA expression cassette per cell, hence only one gene in each cell is targeted for LOF or GOF. Cell pools are then challenged with a selective pressure of interest (here imatinib). Enrichment or depletion of perturbed cells is detected by sequencing the PCR-recovered sgRNA expression cassette pools, which serves as a surrogate read-out for the number of cells expressing the respective sgRNA. Hence, genetic perturbations can systematically be linked to functional phenotypes, like imatinib resistance.
In addition to the systematic perturbation of individual genes, we are going to continue with the development of combinatorial genome engineering. As we showed before, combining orthogonal CRISPR systems with one another, allows the simultaneous activation of one gene and deletion of a second gene in the same cell. We demonstrated, how this dual gene perturbation approach allows the reconstruction of directional genetic interaction networks (Figure 2). We are now expanding on this approach by combining additional CRISPR systems for genetic and epigenetic engineering, and by trying to achieve higher orders of combinatorial perturbations. Eventually, this should enable us to reversely engineer any combination of genetic and epigenetic cancer-associated alterations into cancer cell cultures, and to use these engineered models to better understand the functional dependencies between those alterations.
Figure 2 - Combinatorial genome engineering - proof-of-concept. (top panel) We combined a CRISPRa system from S. pyogenes with a Cas9 nuclease from S. aureus to facilitate GOF and LOF in the same cell. (bottom panel) We identified and validated many known and novel players of Ras signalling, and were able to connect them in a directional genetic interaction network based solely on our perturbation data, thereby illustrating the potential of combinatorial genome engineering for translational cancer research.
In addition to the research projects outlined above, we are always working on the development of novel CRISPR technologies, such as Cas variants with improved properties for human genome engineering. We are also going to implement CRISPR screens with single-cell transcriptome read-out - akin to CROP-seq or Perturb-seq - for transcriptional network studies in cancer cells.