New tools, targets and approaches for gene, genome and metabolic engineering

Abstract

Genome engineering tools and DNA sequencing technologies have improved dramatically over the past decade. These technologies are transforming many areas of research ranging from evolutionary biology to human disease. My thesis research has been focused on developing and applying DNA sequence-guided approaches to genome engineering. The specific projects described in this dissertation and the associated manuscripts from this work have focused on characterizing the sequence of a new algal genome from Chrysochromulina tobin, an organism of considerable biotechnology interest for metabolic engineering and evolutionary studies. I describe the sequencing, assembly and annotation of the Chrysochromulina organellar and nuclear genomes, and transcript profiling over the light-dark cycle that entrains metabolism, lipid synthesis and degradation and cell division. Novel findings included the unique repeat structure of the organellar genomes and discovery of novel biochemical pathways never before seen in algae. I also worked on genome engineering application to human disease. This work included the engineering of DNA target site-specific nucleases to serve as a gene drive system designed to control fertility and thus the population size of Anopheles gambiae, the vector for transmission of malaria. This work was part of the first demonstration of homing as a gene drive system in metazoans. In addition, I developed the molecular tools and protocols to diagnose disease-causing mutations in Shwachman-Diamond syndrome (SDS) and to target the correction of these mutations in iPS cells derived from SDS patients. An alternative approach makes use of a complementing SDS gene into a human genome safe harbor site.