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

其核心将是继续使用我们在之前的DTP中建立的人类细胞系，以测试当细胞已在HDR基因中修改时，由Cas9或CasX和Casade-Cas3进行的基于CRISPR的编辑。例如，第一个重点将放在编码DDX52、Helq、Mre11和CtiP的基因上。但也有其他令人感兴趣的候选人。首先，使用CRISPR删除这些基因中的每一个，对基因组进行测序，并建立DNA修复表型的测量(通常我们会使用DNA ICL试剂)。然后，根据市售的多种试剂盒中的任何一种，每个试剂盒将用于在标准反应中测量被删除细胞的编辑效率/功效。这也将旨在合并删除，以衡量编辑，以确定删除是相加的还是上位的。这些反应将与DNA修复表型并行进行，以全面了解HDR过程和编辑之间的关系。在这一点上，我们将使用各种形式的蛋白质的异位表达，例如突变形式，来确定需要哪些蛋白质的属性。根据以上起点的进展，我们将把这一点扩展到无细胞分析中，用于测量编辑，通过比较像CtiP和Mre11这样的蛋白质的结果，这些蛋白质在理解它们在HDR中的作用以及它们的上位性是否与那些对HDR知之甚少的蛋白质(例如DDX52和Helq)相比，我们的目的是更多地了解未知因素在HDR中的作用。这对理解癌症治疗也有意义。因此，你的博士项目的目标是通过培训成为CRISPR编辑和人类发展报告方面的专家。因此，你将学习细胞生物学、遗传学和蛋白质生物化学的技能。

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.