Genome Engineering to Create Dominant Alleles in Caenorhabditis elegans Using CRISPR-CAS9 Technology
Master's in Biomedical Sciences
Graduate School of Biomedical Sciences
School of Osteopathic Medicine
Eric Moss, PhD
Ronald Ellis, PhD
Susan Muller-Weeks, PhD
Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-Associated Protein 9, Caenorhabditis elegans
Animal Experimentation and Research | Genetics and Genomics | Investigative Techniques | Laboratory and Basic Science Research | Medicine and Health Sciences | Molecular Genetics
Many investigators have being using CRISPR-Cas9 as a method of genome engineering because it is easy, accurate and fast. This technique has been used to modify the genomes of a wide variety of organisms, including the nematode Caenorhabditis elegans (C. elegans). The short life cycle and ease of introducing exogenous plasmids make C. elegans an ideal system for advancing this technique. My thesis had two aims that focused on developing methods to create dominant alleles in C. elegans. Genetic modifications like precise deletion and insertions into a locus of chromosome are technically challenging. Additionally, although there are several ways of generating transgenic models that are useful for developmental studies, the efficiency of getting stable transgenic lines can be an issue. To address these problems, my first aim was using CRISPR-Cas9 specifically to generate ztf-16 mutants by deleting lin-4 and let-7 microRNAs regulatory regions in its 3’ UTR. I used microinjection to introduce the CRISPR plasmids into the gonad of a hermaphrodite C. elegans. In addition, I employed a co-CRISPR strategy to enhance the modification events and to visualize the morphology of the transgenic animals in the F1 progeny. PCR based screenings confirmed that two candidates have the ztf-16 3’UTR deletion in the F2 progeny. My second aim was to set up a method that allows locus-specific insertion of any desired sequences using a Cre/loxP system. The first step was to use the CRISPR-Cas9 method to insert mutant loxP (lox66) into chromosome IV and then inject a second loxP (lox71) to cause the single-copy insertion.
Sulaimani, Abrar, "Genome Engineering to Create Dominant Alleles in Caenorhabditis elegans Using CRISPR-CAS9 Technology" (2015). Graduate School of Biomedical Sciences Theses and Dissertations. 3.
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