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Noah Goodson, Joseph A Brzezinski; Utilizing CRISPR to perturb photoreceptor/bipolar enhancer mediated cell fate decisions in the mouse retina. Invest. Ophthalmol. Vis. Sci. 2019;60(9):6043. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The mechanisms that set the numbers of photoreceptors and bipolar cell interneurons formed during development are poorly understood. Two transcription factors, Prdm1 and Vsx2, influence rod photoreceptor and bipolar cell fate decisions late in retinal development. Disruption of either factor changes the number of rods and bipolars formed. We tested the hypothesis that these transcription factors do not instruct fate choice directly, but instead work in a mutually inhibitory gene regulatory network to establish the proportion of rods and bipolars.
We probed how the Prdm1 and Vsx2 gene regulatory network affects fate choice by taking a CRISPR/Cas9 approach to perturb these genes individually and simultaneously. To minimize effects on retinal progenitor cells, we targeted key enhancers for Prdm1 and Vsx2. This was done by in vivo electroporation of mouse retinas at postnatal day (P) 0. We quantitatively examined cell fate at P4, P7, P14, and P28 (N=6 mice/condition) by immunohistochemistry.
CRISPR/Cas9 targeting of the Prdm1 and Vsx2 enhancers resulted in a strong decrease in the number of cells that expressed PRDM1 and VSX2 proteins in the retina. Perturbing Prdm1 or Vsx2 singly significantly (unpaired t-tests, P<0.001) altered the rod to bipolar ratio we observed. Conversely, simultaneous deletion of the Prdm1 and Vsx2 enhancers resulted in a rod to bipolar cell ratio that was similar to non-targeting control electroporated cells.
Our findings show that targeting key enhancers with CRISPR/Cas9 can be used to precisely perturb gene function singly or in combination. Our results suggest that Prdm1 and Vsx2 work in a mutually inhibitory network to set the numbers of rods and bipolar cells formed. While Vsx2 and Prdm1 regulate numbers, they are not necessary for photoreceptor or bipolar cell fate specification or formation. Future work will focus on uncovering the instructive factors driving rod and bipolar fate specification.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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