Cardiovascular disease is a leading cause of mortality in both humans and canines, with one of the most 34 common forms being dilated cardiomyopathy (DCM). In the Doberman Pinscher, DCM is linked to two 35 known genetic mutations. One is a 16 base pair deletion located at the donor splice site of an intron in 36 the pyruvate dehydrogenase kinase 4 (PDK4) gene. This mutation results in an altered splice product, 37 which affects metabolic capacity of cardiomyocyte cells. The objective of this study was to determine the 38 efficacy of homology-independent targeted insertion (HITI), a CRISPR/Cas9 gene editing strategy, to 39 correct the PDK4 mutation. To do this, Cole is leading a team composed of one visiting professor from Egypt, one veterinary student, and two laboratory technicians in the project. His role is to conceptually guide, make decisions for, and teach other members of the team since he overtook the project from a postdoctoral associate.

Cole's team designed single guide RNA (sgRNA) using two approaches: 1) dropping 40 out the exon adjacent to the mutation and 2) targeting and cutting the unique mutant sequence to 41 enable the replacement of the deleted region using a HITI approach. The team tested five sgRNAs in vitro using 42 ribonucleoprotein (RNP) based cleavage assay. Four sgRNA candidates showed evidence of cutting in 43 vitro in the homozygous DNA template. To further evaluate the candidates, the group carried out transfection 44 using RNPs in vivo. Cleavage efficiency was assessed by Sanger sequencing and computational analysis. 45 For the two sgRNA candidates designed to exon drop, post-nucleofection analysis showed up to 100% 46 editing efficiency specific to the exon deletion.

These data show the promising potential of CRISPR/Cas9 technology for genetic correction in DCM. As we look to the future, the importance of CRISPR in therapeutic applications cannot be overstated. Its ability to precisely edit genetic mutations offers unprecedented opportunities to treat a variety of genetic disorders, including those affecting cardiovascular health. By continuing to refine these techniques, Cole's group hopes to not only mitigate the effects of diseases like DCM in dogs but also pave the way for similar treatments in humans, heralding a new era of personalized and highly effective medical interventions.