Skip to main content
. 2024 Jul 30;111(8):1673–1699. doi: 10.1016/j.ajhg.2024.06.018

Figure 2.

Figure 2

SMGs are conducive to gene transactivation

(A) The dCas9-ST-PH-gRNA gene transactivation system. Co-expression of three transgenes results in the assembled transactivation complex on a gene promoter consisting of (1) the enzymatically dCas9 fused to a SunTag array (10 copies of GCN4 epitopes; dCas9-ST), (2) the hybrid p65 and HSF1 (heat shock factor 1) transcriptional transactivation domains (TADs) fused to a single-chain variable fragment (scFv) that recognizes the GCN4 epitope, and (3) the short gRNA, which directs the dCas9-ST-PH complex to the target gene promoter. The dCas9-ST can recruit up to ten copies of the hybrid transactivator P65-HSF1.

(B) HEK293T and HDF clonal cell lines stably expressing dCas9-ST-PH. Representative images of HEK293TdCas9−ST-PH and HDFdCas9−ST-PH cell lines showing stable co-expression of transgenes encoding dCas9-ST (as reported by mCherry encoded in cis) and P65-HSF1 (as reported by EGFP encoded in cis).

(C–E) Transactivation screen using single-cell transcriptomics. A pooled gRNA expression plasmid library (160 gRNAs; 4 gRNAs per gene, targeting 40 SMGs) was delivered by lentivirus to the stable HEK293TdCas9−ST-PH and HDFdCas9−ST-PH using a low multiplicity of infection to deliver ∼1 gRNA vector per cell. >20,000 cells per cell line were subjected to single-cell Perturb-seq using the 10× Genomics platform. (C) Cells expressing >6 molecules of a given gRNAs species were analyzed for expression of their target gene. ∗p < 0.05 (adjusted for multiple comparisons). (D) In general, the number of gRNAs per cell is positively associated with target gene expression and negatively associated with cell number analyzed. Data are pooled from all four gRNAs per gene. Dark blue lines are the number of cells, light blue lines are transcripts per million (TPM), and x axis is gRNA expression. (E) Expression levels of the 40 targeted SMGs in single cells. Each dot represents the expression of the target gene in a single cell analyzed.

(F) Transactivation screen using bulk-cell transcriptomics. The pooled gRNA expression plasmid library was transfected to the stable HEK293TdCas9−ST-PH or transduced into HDFdCas9−ST-PH cells at high efficiency. Isolated RNA was subjected to srRNA-seq. Bar graph showing the transactivation of 40 SMGs (n = 4 biological replicates; ∗p < 0.05, Genewise statistical test).