Presentation Title

Using CRISPR/Cas9 to recreate a mutation in itx-1 to determine its role in spermatogenesis in C. elegans

Start Date

November 2016

End Date

November 2016

Location

Surge 171

Type of Presentation

Oral Talk

Abstract

Genetics research has long focused on mutations for discovering gene function. We are interested in a mutation that affects spermatogenesis in the nematode Caenorhabditis elegans. The mutation, hc202, restores the presence of functional sperm in worms that would otherwise be sterile due to the sperm defects associated with another mutation in the spe-27 gene. Further genetic evidence suggests that the protein encoded by the gene affected by the hc202 mutation has a lock and key mechanism with the SPE-27 protein. Thus, it is very important to identify the gene affected by hc202. Genetic mapping and subsequent whole genome DNA sequencing showed that only one mutation exists that could be hc202. This mutation affects the itx-1 gene. The purpose of this experiment is to test the hypothesis that the itx-1 mutation is responsible for the phenotype found in the hc202 strain by recreating the itx-1 mutation anew to see if it mimics the hc202 phenotype. Using CRISPR/Cas9 gene editing, a point mutation identical to that in hc202 is being made in the itx-1 gene. For efficient identification of the worms carrying the recreated mutation, we also induced an HpaII restriction site and eliminated a SacI restriction site by creating silent mutations nearby the itx-1 mutation. If the new itx-1 mutation causes worms carrying it and the spe-27-(it132) mutation to have functional sperm, it will confirm the role of itx-1 in sperm activation, leading to further experiments to determine the specific function of this neurexin-like gene product, whose homologs are involved in cell-cell contacts.

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Nov 12th, 10:45 AM Nov 12th, 11:00 AM

Using CRISPR/Cas9 to recreate a mutation in itx-1 to determine its role in spermatogenesis in C. elegans

Surge 171

Genetics research has long focused on mutations for discovering gene function. We are interested in a mutation that affects spermatogenesis in the nematode Caenorhabditis elegans. The mutation, hc202, restores the presence of functional sperm in worms that would otherwise be sterile due to the sperm defects associated with another mutation in the spe-27 gene. Further genetic evidence suggests that the protein encoded by the gene affected by the hc202 mutation has a lock and key mechanism with the SPE-27 protein. Thus, it is very important to identify the gene affected by hc202. Genetic mapping and subsequent whole genome DNA sequencing showed that only one mutation exists that could be hc202. This mutation affects the itx-1 gene. The purpose of this experiment is to test the hypothesis that the itx-1 mutation is responsible for the phenotype found in the hc202 strain by recreating the itx-1 mutation anew to see if it mimics the hc202 phenotype. Using CRISPR/Cas9 gene editing, a point mutation identical to that in hc202 is being made in the itx-1 gene. For efficient identification of the worms carrying the recreated mutation, we also induced an HpaII restriction site and eliminated a SacI restriction site by creating silent mutations nearby the itx-1 mutation. If the new itx-1 mutation causes worms carrying it and the spe-27-(it132) mutation to have functional sperm, it will confirm the role of itx-1 in sperm activation, leading to further experiments to determine the specific function of this neurexin-like gene product, whose homologs are involved in cell-cell contacts.