Discipline: Biological Sciences
Vanessa Hernandez - Dodge Community College
Co-Author(s): Katherine Hwang and Anna Zinovyeva, Kansas State University, Manhattan, Kansas
How genes are regulated is an important question in biology. Misregulation of genes causes a number of diseases and developmental disorders. These diseases include cancer, diabetes, neurological and autoimmune disorders. MicroRNAs (miRNA) are a class of small non-coding RNA that play an important role in regulating gene expression. Specifically, miRNAs negatively regulate gene expression at the post transcriptional level. During the miRNA biogenesis, a single miRNA strand is selected from a double stranded miRNA duplex. This strand becomes the dominant mature miRNA and dictates which genes get negatively regulated or repressed. Therefore, choosing the correct miRNA strand is important for regulating specific sets of gene targets. Not a lot is known about how the process of miRNA strand selection occurs in vivo. Our goal is to better understand which features of the miRNA duplex are important for miRNA strand selection in vivo. Caenorhabditis elegans is a great model system to help us understand the biogenesis of microRNAs and specifically how a single miRNA strand is selected. We are using CRISPR/Cas9 genome editing technology to build mutations into the C. elegans mir-58 gene. These mutations alter the features of the mir-58 duplex that are thought to be important for strand selection. To begin this project, we first focused on establishing a CRISPR method that uses a preloaded Cas9 protein/sgRNA complex. We also established a downstream PCR-restriction digest screening method. To implement this experiment, we injected 40 animals with the CRISPR/Cas9 components and screened their progeny for resulting mir-58 mutations using PCR followed by a restriction digest-based assay. Overall, we established methods for mir-58 CRISPR-induced mutation screening. Further work is required to generate mir-58 mutations of interest. Establishing stable mir-58 mutants using CRISPR technology will help to advance the goal of understanding strand selection in vivo.Not Submitted
Funder Acknowledgement(s): National Science Foundation, Kansas Louis Stokes Alliance for Minority Participation 1305059 (KS-LSAMP) K-State Johnson Cancer Research Center NIH K-INBRE P20 GM103418.
Faculty Advisor: Anna Zinovyeva, firstname.lastname@example.org
Role: We used a CRISPR/Cas9 method to edit genes. This allowed us to use this technique to introduce changes in the mir-58 locus. We injected a mir-58 sgRNA guide 1 into a Caenorhabditis elegans in order to produce F1 generations called dyp animals. The dyp-10 animals are the co-CRISPR marker and we screened for the desired mutation by PCR. We injected 40 animals with the CRISPR/Cas9 components and screened their progeny for resulting mir-58 mutations using PCR followed by a restriction digest-based assay. Overall, we established methods for mir-58 CRISPR-induced mutation screening.