Simultaneous deletion of and enhancers in the retina alters photoreceptor and bipolar cell fate specification, yet differs from deleting both genes.

The transcription factor OTX2 is required for photoreceptor and bipolar cell formation in the retina. It directly activates the transcription factors and through cell type-specific enhancers. PRDM1 and VSX2 work in opposition, such that PRDM1 promotes photoreceptor fate and VSX2 bipolar cell fate. To determine how OTX2+ cell fates are regulated in mice, we deleted and or their cell type-specific enhancers simultaneously using a CRISPR/Cas9 retina electroporation strategy. Double gene or enhancer targeting effectively removed PRDM1 and VSX2 protein expression. However, double enhancer targeting favored bipolar fate outcomes, whereas double gene targeting favored photoreceptor fate. Both conditions generated excess amacrine cells. Combined, these fate changes suggest that photoreceptors are a default fate outcome in OTX2+ cells and that VSX2 must be present in a narrow temporal window to drive bipolar cell formation. and also appear to redundantly restrict the competence of OTX2+ cells, preventing amacrine cell formation. By taking a combinatorial deletion approach of both coding sequences and enhancers, our work provides new insights into the complex regulatory mechanisms that control cell fate choice.