Assembly of Novel Gene Editing Particles to Understand Genome Surgery in Patient-Derived Cells

2016 - presentpresent

There is a fundamental gap in understanding how several components of engineered gene-editing nucleases achieve gene modification in human cells. Continued existence of this gap represents an important problem because, until it is filled, use of genome surgery tools will be limited, as it is not clear why various nucleases fail and why some succeed in producing desired gene edits. The long-term goal is to watch genome surgery in action to understand the bottlenecks in performing genome surgery on human cells in vitro with precisely controlled gene-editing particles, comprised of CRISPR-Cas9 components. Particles will be systematically assembled with various components and delivered in a controlled fashion to patient-derived cells and tissues. Live, in situ high content imaging and analysis within customized cell substrates will monitor genome surgery. These capabilities will explore large sequence variation of CRISPR-Cas9 components along with new assemblies of CRISPR-Cas9 components. The central hypothesis is that new assemblies of CRISPR-Cas9 particles can probe different biological processes of trafficking, DNA-double strand break formation and DNA repair involved in genome surgery. This hypothesis will be tested with respect to generating two types of gene edits involving non-homologous end joining (NHEJ) and homology-directed repair (HDR) pathways at several genomic loci within patient-derived stem cells and tissues. An overarching rationale for the proposed research programs is that robust gene editing techniques could enable the production of personalized drugs, cell therapies and gene therapies for future genomic and precision medicine.