Browsing by Subject "Gene Targeting"
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Item The impact of loss of function mutations of NHEJ genes on gene targeting and DNA DSB repair in human somatic cells.(2009-04) Fattah, Farjana JahanNon-homologous end-joining (NHEJ) is the predominant repair pathway for DNA double-strand breaks (DSBs) in human cells. The core NHEJ pathway is composed of seven factors: Ku70, Ku86, DNA-PKcs, Artemis, XRCC4, XLF and LIGIV. Mutation of any one of these NHEJ genes leads either to death, profound immune deficiencies, ionizing radiation sensitivity and/or cancer predisposition in human patients. We attempted to generate Ku70-null human somatic cells using a rAAV-based gene knockout strategy. Our data demonstrated that Ku70 is an essential gene in human somatic cells. More importantly, however, in Ku70+/- cells, the frequency of gene targeting was 5- to 10-fold higher than in wild type cells. RNA interference and short-hairpinned RNA strategies to deplete Ku70 phenocopied these results in wild-type cells and greatly accentuated them in Ku70+/- cell lines. Thus, Ku70 protein levels significantly influenced the frequency of rAAV-mediated gene targeting in human somatic cells. XLF is the newly identified core factor for NHEJ. To characterize XLF function in human cells, we knocked out XLF gene in HCT116 cells. XLF deficient cells are highly sensitive to ionizing radiation and DNA damaging agent, and have intrinsic DNA DSB repair defects. In V(D)J recombination assay, we find that XLF deficient cells have dramatic defect to form both V(D)J coding and signal joints. The phenotypes of XLF deficiency were rescued by a WT XLF cDNA over-expression. We conclude that, in humans, XLF is essential for C-NHEJ mediated repair of DNA-DSBs. Biochemical and genetic studies in mouse and hamster cells showed that DNA ends can also be joined via a backup pathway, especially when proteins responsible for NHEJ, are reduced or absent. In order to get insights in to backup NHEJ mechanism, we employed a reporter system based on the in vivo rejoining of cohesive and incompatible ends. We report here more than 10 to 20 fold reduction in NHEJ proficiency in DNA-PKcs, XLF and LIGIV null human cells, which is characterized by an increase in microhomology use. Strikingly, conditional knock-out of Ku86 did not result in defect in end-joining, while having an impact on repair fidelity.Item Quantification and Mechanistic Analysis of Plant Genome Editing Outcomes using Nanopore Sequencing(2020-08) Atkins, PaulPrecise genome modification via homologous recombination, or gene targeting (GT), allows crop genomes to be tailored to any application or environment. While GT’s potential is immense, it tends to be inefficient and technically challenging in plants. These problems are compounded by the slow and low-throughput nature of plant transformation, drastically hindering optimization. More insidiously, these issues result in dependence upon proxies and reporter readouts for estimating GT frequencies that vary between groups and delivery platform making it difficult to compare experimental outcomes. To enable widespread optimization of plant GT, a universal platform for directly measuring genome editing outcomes at the molecular level that accommodates plant-specific technical constraints is urgently needed. Here I develop such a platform, an amplicon-based analysis pipeline using Oxford Nanopore Sequencing (ONS). ONS has several valuable qualities for a plant GT optimization pipeline, namely its accessibility, speed, and read length, making it feasible for even the smallest labs to perform on-demand sequencing with their own equipment. These strengths are accompanied by a major shortcoming – sequencing error. I mitigate this problem using several approaches in a novel bioinformatics pipeline to minimize the effect of ONS error on estimates of targeted mutagenesis and virtually eliminating its effect on estimates of GT frequencies. Using this pipeline, I observed a significant impact of both geminiviral replicons (GVRs) and donor sequence divergence on gene targeting frequencies. Additionally, I was able to observe the conversion tracts of hundreds of gene targeting events, revealing their deposition by multiple DNA repair pathways and the prevalence of extremely short tracts, which will inform future optimization efforts. This work establishes a universal pipeline for quantifying plant gene targeting events, facilitating future optimization and communication of results between disparate experimental systems within the plant community.