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• 1 2020-05-06T00:09:38+00:00 Emilie Sandfeld 5 plain 2020-05-08T23:52:21+00:00

  
  Genomic Correction of Pompe Disease Knock-in Mouse Myoblasts via CRISPR-Cas9 Homology-directed Repair

  Overview: CRISPR/Cas9 technology, a 'molecular scissor' tool, is used to remove unwanted pieces of our DNA. For this research, the unwanted region is the mutated gene that causes Pompe disease. This mutation alters a human's ability to produce the enzyme acid alpha-glucosidase. Without it, our body cannot breakdown sugars naturally produced in the body. 
  
  Abstract: The goal of this study is to optimize CRISPR-Cas9 homology-directed repair (HDR) strategies to evaluate in vitro efficacy of genome correction in knock-in models of infantile-onset Pompe disease (PD). To accomplish this goal, we used a CRISPR-Cas9 knock-in system targeting the Gaa gene to introduce the known infantile-onset PD orthologue – Gaac.1826dupA (p.Y609*) into C2C12 mouse myoblasts. We confirmed the molecular and biochemical analogy of our Gaac.1826dupA knock-in myoblasts to PD by measuring GAA mRNA expression and enzymatic activity as well as glycogen accumulation. Next, for each Gaa knock-in line, we screened 6 CRISPR single-guide RNAs (sgRNA) and their respective single-stranded oligo DNA nucleotide donors (ssODNs) via nucleofection-mediated ribonucleoprotein (RNP) delivery. We then chose the top sgRNA candidates – with the highest levels of on-target nuclease activity and HDR template integration – to subclone into GFP-tagged CRISPR-Cas9 expression vectors (pX458). Lastly, we nucleofected CRISPR sgRNA-containing pX458 vectors with their respective ssODNs into knock-in myoblasts and used fluorescence activated cell sorting to isolate GFP-positive cells. Overall genome correction efficacy was determined by TIDER (Tracking of Insertion, DEletions and Recombination events) analysis. As determined by this study, the optimal genomic correction strategy will be used for future work to isolate, expand and characterize genome-corrected Gaa knock-in myoblasts prior to evaluating its therapeutic potential in an in vivo model system.
  
  
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