Understanding homology directed DNA repair in human cells through analysis of CRISPR editing.

The core of this will be to continue the use of human cell lines that we have established in a previous DTP to assay CRISPR-based editing by Cas9 or CasX, and Cascade-Cas3, when cells have been modified in HDR genes. For example, first focus will be on the genes encoding DDX52, HelQ, Mre11 and CtiP. But there are other candidates of interest too. You would begin by deleting each of these genes, using CRISPR, sequencing the genomes, and establishing measurement of the DNA repair phenotype (usually we would use a DNA ICL agent). Each would then be used for measuring editing efficiency/efficacy for the deleted cells in a standard reaction based on any one of a number of commercially available kits. This would also aim to combine deletions for measuring the editing to determine if the deletions are additive or epistatic. These reactions would be done in parallel to DNA repair phentypes to get a full picture of the relationships between the HDR processes and editing. At that point we would then use ectopic expression of various forms of the proteins, e.g. mutant forms, to determine which properties of the proteins are needed.Depending on how the above starting point goes we would extend this into the cell-free assays for measuring editing, By comparing the outcomes for proteins like CtiP and Mre11, which are well-developed in understanding for their roles in HDR and their epistasis or not with proteins that are poorly understood for HDR (e.g. DDX52 and HelQ) we would aim to understand more about the roles of the unknown in HDR. This has implications for understanding cancer treatments too. Therefore your PhD project would aim for you to become expert through training in CRISPR editing and HDR. You would therefore learn skills of cell biology, genetics and protein biochemistry.