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Full-Text Articles in Life Sciences
Dual-Active Genome-Editing Reagents, Jason M. Wolfs
Dual-Active Genome-Editing Reagents, Jason M. Wolfs
Electronic Thesis and Dissertation Repository
Manipulation of complex genomes has many beneficial downstream applications in agriculture and human gene therapy. Precise genome-editing requires the introduction of a specific DNA double-stand break at a locus of interest, in turn inducing host DNA repair pathways to cause gene knockout through non-homologous end-joining or gene repair using homologous recombination and donor template. No matter the application, the field has depended on a few reagents to introduce precise double-strand breaks in host genomes. LAGLIDADG homing endonucleases or meganucleases harness the natural properties of these rare-cutting enzymes to target precise sequences in a complex genome. Other successful reagents are derived …
Isolation And Characterisation Of Relaxed Specificity I-Tevi Nuclease Domain Mutants, Alexander C. Roy
Isolation And Characterisation Of Relaxed Specificity I-Tevi Nuclease Domain Mutants, Alexander C. Roy
Electronic Thesis and Dissertation Repository
Engineering nucleases is important to the advancement of genetic engineering and gene therapy approaches. Engineering requires a knowledge of which residues are contributing to each function of the nuclease. The residues which contribute to cleavage specificity of the I-TevI nuclease domain (ND) are unknown. I suspect that some of these contributions derive from the ND, thus my null hypothesis is that mutation of the ND will not alter the substrates this enzyme can cut. I have mutagenised the I-TevI nuclease domain and using directed evolution I have isolated mutations which were characterised in vivo and in vitro. These mutations permit …
Examining The Nucleotide Preference Of The Linker Domain In Engineered Tev-Mtalens, Brendon C. Mcdowell
Examining The Nucleotide Preference Of The Linker Domain In Engineered Tev-Mtalens, Brendon C. Mcdowell
Electronic Thesis and Dissertation Repository
Tev-mTALENs are genome-editing nucleases which combine the nuclease and linker domains of I-TevI with the DNA-binding domain of a TAL effector. The linker domain interacts with a portion of the Tev-mTALEN target site called the DNA Spacer, facilitating DNA cleavage. Linker-DNA Spacer interactions are poorly understood but necessary for Tev-mTALEN activity. I examined the DNA Spacer sequence requirements of the linker by assaying Tev-mTALEN activity on targets with mutated DNA Spacer sequences. I also performed activity assays using Tev-mTALENs with mutations to the I-TevI linker domain. My results indicate that the linker DNA Spacer sequence requirements are highly cryptic. No …
Dna Hydrolysis And Genome Editing Applications Of Giy-Yig Family Homing Endonucleases, Benjamin P. Kleinstiver
Dna Hydrolysis And Genome Editing Applications Of Giy-Yig Family Homing Endonucleases, Benjamin P. Kleinstiver
Electronic Thesis and Dissertation Repository
The ability to manipulate complex genomes in a precise manner is essential for studying biological processes in model systems, engineering plant strains for agriculture, or advancing human cellular therapies to treat diseases. Genomic alterations are most efficient when a double-strand DNA break is introduced at the loci where the modification is desired. Different classes of naturally occurring DNA endonucleases, including homing endonucleases, have therefore been explored as candidates for genome modification studies as they target long stretches of DNA. Homing endonucleases are mobile genetic elements whose biological role is to introduce site-specific double-strand breaks into naïve genomes, ultimately resulting in …