Mapping lncRNA interactions in the Sox2 promoter by CRIST-seq. (A) Schematic diagram of the CRIST-seq assay. (dCas9) The catalytically inactive CRISPR/Cas9; (gRNA) Cas9 guiding RNAs that target the target gene promoter; (pU6) RNA polymerase III U6 promoter; (pH1) human H1 RNA polymerase III promoter. Cells were transfected with the Cas9 gRNA cassette that targets the promoter of a given gene. In this study, we targeted the Sox2 promoter, a well-established core stem cell factor that is required for the maintenance of pluripotency. The Cas9 gRNA-expressing cells were crosslinked by formaldehyde to fix the RNA–DNA structure. After cell membrane lysis, the nuclei were isolated and the promoter-interacting RNAs were in situ reverse transcribed into cDNAs with biotin-dCTP. The promoter biotin-cDNA chromatin complex was immunoprecipitated by an antibody against FLAG, which binds to its target genes through a mechanism of base-pairing between the gRNA and target DNA. After Cas9-FLAG immunoprecipitation, the promoter-interacting biotin-cDNAs were separated from genomic DNAs by streptavidin beads. The CRIST-captured chromatin cDNAs were collected for library construction and sequenced to identify the lncRNAs that interact with the promoter of a target gene. (B) CRIST targeting vectors. (gRNA) Cas9 guiding RNAs that target the target gene promoter; (gCT) scrambled control gRNA. The Cas9-gCT vector was used as the CRIST control. The Cas9 vector that lacks the targeting gRNAs was used as the vector control. In the targeting vector, two Cas9 gRNAs are transcribed by human U6 and H1 promoters, respectively, and they guide the Cas9 to the promoters of target genes. (C) Specific CRIST targeting of the Sox2 promoter. (pSox2) The targeting site in the Sox2 promoter where the Cas9 gRNAs are designed; (5′-Ct) a fragment that is 14.6 kb away from the pSox2 target site and is used as the control site. (Cas9 vector) Cells that were treated with the Cas9 control vector that lacks the gRNAs; (Cas9-gRNA) cells that were targeted by both Cas9 and Sox2 gRNAs; (Cas9-gCT) cells that were treated with the random control gRNA vector. (Off-target) A CRIST control site that is 33.8 kb upstream of the housekeeping gene GAPDH. The Cas9 Sox2-gRNA iPSCs were fixed with formaldehyde and the chromatin complex was immunoprecipitated with a FLAG antibody and an IgG control antibody (without in situ reverse transcription). Cas9 enrichment signals were quantitated by real-time PCR using specific primers derived from the pSox2 targeting site, 5′-Ct control site, and off-target site. All data shown are mean ± SEM from three independent experiments by normalization over the IgG control. (**) P < 0.01 as compared with the Cas9 Vector and Cas9-gCT controls. Note the specific enrichment of Cas9 binding at the pSox2 site in Cas9-Sox2 gRNA targeting group. After confirming the specificity of the Cas9 gRNA, the Cas9 Sox2-gRNA iPSCs were then used for CRIST-seq assay.
