Project Details
Projekt
Development of 3Cs fixed-pair for pooled DNA excisions with single-cell transcriptomic read outs.
Subject Area
General Genetics and Functional Genome Biology
Image and Language Processing, Computer Graphics and Visualisation, Human Computer Interaction, Ubiquitous and Wearable Computing
Image and Language Processing, Computer Graphics and Visualisation, Human Computer Interaction, Ubiquitous and Wearable Computing
Term
since 2022
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 495039689
After more than two decades of gene editing in human cells, understanding the complex genetics of coding and noncoding alterations remains a major challenge in biology and medicine. This is in large part due to technological constraints, stochastic outcomes of gene edits, and the lack of unbiased phenotypic readouts. Whilst recent efforts to map gene dependencies in human cells produced unprecedented biological insights, similarly comprehensive approaches targeting gene combinations or noncoding sequence elements lack feasibility so far. This is mainly due to (i) the large size of the human genome and (ii) limitations inherent to genome perturbation methods. Exploiting our recent 3Cs technology, we here propose to establish the experimental and computational framework for CRISPR screening with predefined gRNA combinations for the investigation of coding and noncoding sequences. We will develop EXCIsion SEquencing (EXCI-SEq), a CRISPR-based DNA excision screening technology that is coupled to single-cell transcriptomic profiling, both of which are key areas of expertise in the two applicant laboratories. We will harness DNA excision phenotypes for biological discovery at multiple levels. First, the exploitation of gRNA design parameters required to excise predefined genetic regions will inform about the biological constraints of incorrect end-bridging during DNA repair. These constraints will be used to implement the multiCRISPR2 software to facilitate the user-friendly design of paired gRNAs. Second, we will excise transcriptional start sites to provide new insights on whether DNA excision-mediated gene inactivation circumvents expression of artificially truncated proteins. Applying a single-cell barcoding strategy, we will be able to distinguish InDel and DNA excision mutagenesis and extrapolate the confounding degree that truncated proteins have on our knowledge on gene dependencies in human cells. Third, the coupling of DNA excisions to single-cell transcriptomics will give new insights into the transcriptional targets of noncoding sequence elements. By applying EXCI-SEq to clinically-relevant miRNAs, we will identify their transcriptional targets to ultimately predict selective vulnerabilities of noncoding sequences. Finally, we will provide a cloud-based software as a service platform to analyse and explore EXCI-SEq data. By separating this process into three phases, we will be able to provide quality control, data analysis, and data visualization and exploration through a web-based frontend. Our proposed project represents the first joined experimental and computational effort merging DNA excision screening with single-cell transcriptomic profiling in human cells to date. It promises to produce innovative technologies and multifaceted biological insights into function and associated vulnerabilities of coding and noncoding alterations.
DFG Programme
Research Grants
