SUPER to Probe the Site-Specific DNA-Binding Protein Complex [scRNA-seq]
Site-specific DNA binding by proteins is critical for regulating genetic activity and cell fate decision. However, identifying proteins bound to specific genomic regions (e.g., promoter or enhancer) remains challenging. To address this, we developed the Site-specific non-canonical amino acid resolved Protein EnRichment (SUPER) system, incorporating a photo-crosslinking amino acid into nuclease-deficient dCas9 mutant. Human pluripotent stem cells (hPSCs) carrying SUPER enables the capture of proteins bound to, in theory, any genomic location, facilitating the study of the cell context-dependent DNA-protein interactions. Using SUPER, we identified OCT4/SOX2/CARHSP1 complex binding to the NANOG promoter to maintain pluripotency in hPSCs. During ectoderm differentiation, ZIC2 acts as a competitive inhibitor, binding the same promoter to downregulate NANOG expression and promote differentiation. Additionally, SUPER identified ZNF8 binding to the distal regulatory region of OCT4 and maintain naïve pluripotency. In summary, SUPER provides a robust system for uncovering the cell context-dependent, site-specific genome regulators, offering valuable insights into gene regulation networks driving cell fate transitions. Overall design: We first incorporated a random 22 nucleotide barcodes (GBC) into the backbone plasmid pPS. The pooled sgRNA mixture was PCR amplified and ligated into this plasmid. The sgRNA-GBC correlation reference table was generated by sequencing. Next, we cloned an EGFP coding sequence along with the EF1a promoter and inserted it between the sgRNA and GBC elements. The final construct was packaged into lentivirus in 293T cells. A separated plasmid containing the CRIPSR/Cas9 coding gene and co-expressed mCherry was packaged into lentivirus. The hPSC cells were infected with lentivirus and sorted based on fluorescence protein expression. The sorted cells were recovered for one week, followed by induced differentiation towards ectoderm lineage. Ectoderm cells were collected and prepared for single cell RNA-seq .