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Targeted Nutrient Metabolism and MicroRNA Gene Knockout Via CRISPR/Cas9 Genome Editing

Targeted Nutrient Metabolism and MicroRNA Gene Knockout Via CRISPR/Cas9 Genome Editing

Name:
Matthew Hart

Department:
Nutritional Sciences

Abstract:
Targeted Nutrient Metabolism and MicroRNA Gene Knockout Via CRISPR/Cas9 Genome Editing

Matthew D. Hart1, Tony Z. Tang1, Mariah R. Nacke1, Joanna L. Fiddler1, Stephen L. Clarke1, Brenda J. Smith1, Winyoo Chowanadisai1

1Department of Nutritional Sciences, Oklahoma State University, Stillwater, Oklahoma, 74078

Introduction: The CRISPR/Cas9 genome editing system has emerged as the most popular genome editing technique to date. With a single expression vector and a guide sequence specific to the target locus, gene knockouts can be achieved with relatively high efficiency. Gene knockout is a useful tool for studying gene function and a simple and effective technique such as the CRISPR/Cas9 system could be used to study whole metabolic or regulatory pathways when performed en masse.

Methods: Guide sequences specific for the miR-210, ZIP12, ZIP13, and ZNT8 genes in both humans and mice were designed using the guide design tool at http://crispr.mit.edu. Amino acid sequence alignments were performed in order to prioritize specific, conserved regions for CRISPR-mediated targeting. The expression vectors containing the Cas9 enzyme, a selection (puromycin) or screening (GFP) marker, and the guide RNA expression cassette for each gene were then transfected into HEK293 (human) and Neuro-2A (mouse) cells. Successful CRIPSR-mediated gene editing was detected by PCR and T7 endonuclease assay or DNA sequencing chromatogram analysis. Monoclonal cell lines containing genes deletions were manually isolated via dilution plating. We then chose ZIP12 to confirm cutting in all chromosomes by direct DNA sequencing of individual chromosomes through PCR and TOPO cloning. Sequences obtained from TOPO clones were then analyzed for predicted frameshift and deletion mutations in ZIP12 using the ExPASy Translate tool.

Results: Cutting was achieved in 4 genes in both human and mouse cell lines as confirmed by T7 endonuclease assays. Sequencing of TOPO clones of ZIP12 mutants revealed cutting in all chromosomes, the majority of which led to the introduction of premature stop codons or large deletions. This suggests that large scale genome editing is feasible in nutrient metabolism and regulatory genes with relative ease and efficiency.