The SOXB1 group of transcription factors (SOX1, SOX2 & SOX3) are expressed within the neural progenitor/stem cells of the CNS during early embryogenesis, and are essential for proper development of the embryo. We have previously reported ChIP-Seq data showing the binding of SOX3 within mouse neural progenitor cells at predicted enhancers within the genome, and discovered a region within the intermediate filament gene, Nes, that contains 6 putative SOXB1 binding sites. This region is within a known enhancer commonly used to direct expression within neural progenitor cells in transgenic animals and cell lines. Whilst this region has been used extensively in vitro, it has not previously been removed in vivo; making it difficult to quantify the contribution of the enhancer to overall nestin expression. Using CRISPR we have generated independent mouse models that lack the binding sites, and by using qPCR, in situ hybridisation and immunohistochemisty to assess both RNA and protein expression we show decreased levels of nestin within embryos from 9.5 to 15.5dpc, indicating the enhancer is active during these developmental stages. Curiously, ectopic expression of nestin is also seen within the vasculature of mice lacking the enhancer, a tissue that does not express SOXB1’s, suggesting the enhancer may have additional SOXB1 independent repressor functions. This work shows the application of CRISPR in the functional assessment of putative enhancers, whilst also providing greater insight into how the SOXB1 transcription factors control target gene expression, furthering our understanding of SOX proteins in embryonic development.