Faithin Omaha eBook Collection

Download or read book Advances in CRISPR-Cas9 Genome Engineering Technologies in Saccharomyces Cerevisiae and Tests for Cis-regulatory Evolution written by Justin Daniel Smith and published by . This book was released on 2016 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: This thesis is organized into two primary areas of research: cis-regulatory evolution and genome engineering. It is now known that noncoding regions comprise the vast majority of genomic regions under selective constraint in mammals. Despite this, the ability to detect natural selection on noncoding regions has lagged behind the ability to detect selection on coding regions. In Chapter 1, we introduce a new test to detect selection on cis-regulatory elements, and demonstrate its utility on three mammalian transcriptional enhancers. In Chapter 2, we investigate the evolution of resistance to the mycotoxin citrinin by comparing two closely related species of budding yeast, Saccharomyces paradoxus and Saccharomyces cerevisiae. Applying a genome-wide test for selection on cis-regulation, we identified five genes involved in resistance in S paradoxus, four of which are necessary for resistance and increase resistance in S cerevisiae when over-expressed. In the second half of this dissertation, I discuss my work with genome engineering and CRISPR-Cas9. In Chapter 2, we build an improved CRISPR activator (CRISPRa) to simultaneously induce overexpression (in S cerevisiae) of the four genes identified to confirm their role in citrinin resistance. In Chapter 3, we build an inducible CRISPR interference (CRISPRi) system and and designed gRNA libraries to determine design rules for CRISPRi gRNA design in S cerevisiae. We determined that gRNAs targeted to a region with low nucleosome occupancy and high chromatin accessibility within a window of 0 to 200bp upstream of the transcription start site (TSS) are most likely to be effective. In Chapter 4 we confirm and refine these rules in a much larger library consisting of ~9000 unique strains. Additionally, we present a novel method for parsing complex oligonucleotide libraries into single, sequence verified DNA sequences using high throughput sequencing and yeast synthetic biology. We utilize this technology to create and characterize a collection of ~9000 individual inducible CRISPRi strains to the vast majority of essential and respiratory essential genes in S cerevisiae. In Chapter 5, we compare mismatch tolerance for Cas9 in vitro and in vivo (in S cerevisiae) and test a variety of truncated and full-length gRNAs (with 17, 18, and 20 nucleotides of complementarity sequence). We observed notable differences between in vitro and in vivo Cas9 cleavage specificity profiles, with in vivo cleavage being more sensitive/less tolerant to mismatches.