Degree Type

Dissertation

Date of Award

2018

Degree Name

Doctor of Philosophy

Department

Genetics, Development and Cell Biology

Major

Molecular, Cellular and Developmental Biology

First Advisor

Drena Dobbs

Abstract

Designer nucleases permit the direction of DNA double-strand breaks and induction of DNA repair activities at virtually any genomic locus. The outcome of DNA repair is often non-random based on the structure of the double-strand DNA break and any homologies flanking the broken DNA ends. When an exogenous donor DNA molecule containing homology to a genomic DNA double-strand break site is supplied, DNA repair mechanisms can be hijacked to integrate the donor into the genome. However, to date rates of donor DNA integration are relatively low and recovery of precise gene targeting events is inefficient in vivo. Here, I demonstrate in zebrafish that liberating a donor cassette with CRISPR/Cas9 and the use of homologies as short as 12, 24, or 48 base pairs flanking double-stranded DNA donors can bias repair towards a Homology Directed Repair sub-pathway likely using strand annealing, herein called Homology-Mediated End Joining. This method, dubbed GeneWeld, drives precise integration in 50% of injected animals on average across 11 targeted loci and these events show low mosaicism in somatic tissue. GeneWeld events were recovered through the germline at an average rate of 50% across these loci. Southern blots demonstrate recovery of single copy, precise integration of donor cassettes at rates reasonable for average zebrafish labs, though some events are not precise. GeneWeld outperforms general Homologous Recombination (HR) in pig and human cells for integrating double-stranded DNA reporters. In addition, I apply an alternate, novel nuclease system from the CRISPR/Cas12a family, called CRISPR/Mad7, to GeneWeld in zebrafish and human cells, expanding genomic access and the genome editing toolbox. In the final part of this thesis, I describe genomic reporters that will be used to examine the genetic mechanisms of DNA repair using strand annealing in zebrafish. The work herein describes how short homologies can direct exogenous DNA integration effectively using the new method GeneWeld. GeneWeld increases accessibility and recovery of engineered genomes for functional genomics, agricultural engineering, therapeutic intervention, and addresses critical needs in the field of precision genome engineering.

Copyright Owner

Wesley Wierson

Language

en

File Format

application/pdf

File Size

159 pages

Share

COinS