Clustered regularly interspaced short palindromic repeats (CRISPR/CRISPR-associated (Cas) protein system, CRISPR/Cas9, uses single-guide RNA to guide Cas9 to the target site for genome editing. In this study, the CRISPR/Cas9 system was used to knockout KED in tomato (Solanum lycopersicum). KED was first identified while screening the wounded tobacco (Nicotiana tabacum) leaves. We found that alignment of the protein sequence of SlKED (Solanum lycopersicum KED) and NtKED (Nicotiana tabacum KED) showed 55.1% identity. To investigate, we generated SlKED knockout tomato plants with a single base pair deletion, a five base pair deletion and a three base pair deletion with a single base pair insertion. We performed wounding assays and analyzed gene expression and found that the wounded SlKED knockout plant showed no gene induction. Furthermore, the biological assay results revealed that the tobacco hornworm (Manduca sexta) gained more mass when fed on the SlKED knockout plant. Our studies show that the KED gene plays a role in wound-induced mechanism and suggested it may involve in the plant defense system against biological stress and insect feeding. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2019. / FAU Electronic Theses and Dissertations Collection

The ability to generate multiplexed genomic modifications using CRISPR-based gene editing has fundamentally changed the scope of possible reverse genetic screening approaches that can be executed in human cells. A diversity of Cas effector proteins lies at the center of pooled CRISPR screens. Working in unison with targeting gRNAs, CRISPR-Cas effector complexes can produce a range of alterations at user specified genomic sites. The type of alteration, ranging from double-strand break (DSB) formation to precise single nucleotide substitutions, is dictated by the Cas protein. Initially, pooled CRISPR screens were conducted using the Cas9 endonuclease to generate loss of function mutations in single genes through the formation of DSBs. As CRISPR technologies matured, the discovery and engineering of novel Cas proteins has allowed for increasingly complex sets of genomic alterations to be studied in a high-throughput manner. In Chapter 1, I introduce a variety of CRISPR-based functional genomic technologies that have been used in high-throughput screening approaches. Here, I also describe discoveries that have been made in the human DNA damage response (DDR) using these approaches. In Chapter 2, I present my work using Cas12a to interrogate the genetic interaction landscape of the DDR. This work leverages the ability of Cas12a to generate several DSBs from a single gRNA array to investigate ~27,000 genetic interactions between 233 DDR genes. In these screens, novel synthetic lethal interactions were identified, with three sets of synthetic lethal interactions between gene complexes being highlighted. In Chapter 3, I present a published manuscript that demonstrates the utility of precision base editing screens. This study uses BE3-dependent base editing to induce mutational tiling of 86 human DDR genes and analyze the effects of these mutations in response to DNA damaging agents. In total, the work presented here highlights the utility of novel CRISPR screening platforms through the interrogation of the human DDR.