Combinatorial CRISPR/Cas9 Approach to Elucidate a Far-Upstream Enhancer Complex for Tissue-Specific Sox9 Expression

SRY-box 9 (SOX9) is a master transcription factor that regulates cartilage development. SOX9 haploinsufficiency resulting from breakpoints in a ∼1-Mb region upstream of SOX9 was reported in acampomelic campomelic dysplasia (ACD) patients, suggesting that essential enhancer regions of SOX9 for cartilage development are located in this long non-coding sequence. However, the cis-acting enhancer region regulating cartilage-specific SOX9 expression remains to be identified. To identify distant cartilage Sox9 enhancers, we utilized the combination of multiple CRISPR/Cas9 technologies including enrichment of the promoter-enhancer complex followed by next-generation sequencing and mass spectrometry (MS), SIN3A-dCas9-mediated epigenetic silencing, and generation of enhancer deletion mice. As a result, we could identify a critical far-upstream cis-element and Stat3 as a trans-acting factor, regulating cartilage-specific Sox9 expression and subsequent skeletal development. Our strategy could facilitate definitive ACD diagnosis and should be useful to reveal the detailed chromatin conformation and regulation. •CRISPR/Cas9-ChIP-seq approach revealed Sox9 enhancer candidate•SIN3A-dCas9-mediated epigenetic silencing and 3C confirmed far-upstream Sox9 enhancer•CRISPR/Cas9-mediated enhancer deletion mice showed an ACD-like phenotype•CRISPR/dCas9-ChIP-MS identified STAT3 as a trans-acting mediator of Sox9 enhancer Mochizuki et al. develop combinatorial and systematic CRISPR/Cas9-based approaches to identify tissue-specific enhancers. They apply this to exploring Sox9 regulation in chondrocytes and identify a cartilage-specific enhancer important for SOX9 expression and skeletal development. CRISPR/dCas9-ChIP-mass spectrometry analysis further implicated STAT3 in acting at the enhancer to regulate SOX9 expression.