Characterization of Emergent Defects in Retinal Morphology and Gene Expression in dCas9-SPH Transgenic Animals

Mouse genetics plays a crucial role in biomedical research, particularly in the generation of transgenic animals. These genetically modified animal models are essential for mimicking human eye diseases, to study disease mechanisms and potential treatments. A novel tool in the development of these models is CRISPR-Cas9, which enables targeted gene modifications to generate disease models. In addition, mutant variant of Cas9, the dead cas9 (dCas9) are also capable of modulating gene expression without cutting the DNA sequences which further enables to study gene regulatory mechanisms. Using this approach, researchers developed a novel transgenic mouse model, the dCas9-SPH (SunTag-p65-HSF1), which utilizes a dCas9 fused to an activator protein to upregulate specific target genes via guide RNAs. Unexpectedly, the homozygous state of this transgenic animal model exhibited an abnormal morphology around the optic disc. This raised a potential opportunity to mimic a disease model related to the glaucoma since this model showed similar characteristics to the optic cupping which occurs in the late stages of glaucoma. Here, we described and characterized this phenotype. dCas9-SPH homozygous animals exhibited persistent optic cupping, despite the absence of increased intraocular pressure (IOP). Retinal ganglion cell (RGC) markers were analyzed, revealing no RGC degeneration. Comparisons with models exhibiting RGC degeneration confirmed this finding. The absence of IOP but presence of optic cupping suggests this model may be used as a hereditary optic cupping model. Notably, GFP leakage in the animal model was observed, indicating activator protein leakage. ddPCR analysis implicated Dpysl2 gene knockdown from insertional mutagenesis as the most likely cause of the phenotype. These findings highlight the potential of the homozygous dCas9-SPH animals to contribute to our understanding of genetic factors in optic nerve diseases.