HTS single cell CRISPR technology for experimental validation of population genetics and functional genomics discovery

Full Title

High throughput single cell CRISPR technology for experimental validation of population genetics and functional genomics discovery in endometriosis as a model disease.

Aims

This project will develop important, cutting-edge genomics capability that allows for the simultaneous functional screening of hundreds of genomics regions, including non-coding regions, in a single experiment. The technology uses pooled CRISPR perturbation of multiple genomic regions in tens of thousands of cells, followed by single-cell sequencing. The sequencing data allows us to detect cells containing the CRISPR construct, expression of the genes regulated by the genomic region and importantly, the downstream changes in the gene networks. Effectively, this approach allows us to validate the transcriptional regulation mechanisms of candidate gene regions and their susceptibility genes identified from genetics and genomics research. This will include the genomic regions implicated in disease risk through GWAS studies, as well as other regions identified in expression qualitative trait loci (eQTL), or those genes found differentially expressed in bulk-RNA sequencing experiments.

This new capability can likely be a “game-changer” to boost the population genetics and functional genomics research at UQ and in Australia. In this project the primary goal will be to identify causal risk variants and the non-coding regulatory elements they perturb and identify the endometriosis susceptibility genes underlying genetic risk associations.

Genomics-based innovative aspect 

This proposal is highly innovative and important in, for the first time, creating a platform for functionally validating very large-scale population genetics and genomics studies. The platform uniquely integrates the three cutting-edge research areas CRISPR, single-cell sequencing, and quantitative genetics. We will optimise this technology for primary cell and organoid culture, which is an important advancement from the recently published capture-perturb-seq protocol, which was applied for established cell lines. The ability to work with primary cell lines will open the potential for making a patient specific validation of gene regulation mechanisms underpinning the disease phenotype. For example, by comparing the transcriptomics of cells without gene expression perturbation with those targeted knock-down cells, we will see changes that are diverse from the background of healthy individuals.

Broad applicability of the technique

After successful completion of the project, the PCPS technology can be transferred to sequencing facility. It will provide the opportunity to create a high through put screening for functional follow up of the powerful GWAS data available for multiple traits and disease. It is anticipated the successful implementation of this technique would be adopted immediately by the groups interested in functional screening of multiple low penetrance genes.