Fig. 3: CRISPR-DREAM is portable to orthogonal dCas9 proteins and amenable to miniaturization.
a, The SadCas9-DREAM system is schematically depicted. Nuclease-inactivating mutations are indicated by yellow bars with dots above. b, HBG1 or TTN gene activation using the SadCas9-DREAM or SadCas9-SAM system, when recruited using pools of 4 promoter-targeting gRNAs. c, HBG1 or TTN gene activation using the SadCas9-DREAM or SadCas9-VPR system, when recruited using pools of 4 MS2-modifed (SadCas9-DREAM) or standard promoter-targeting gRNAs (SadCas9-VPR), respectively. d, The CjdCas9-DREAM system is schematically depicted. Nuclease-inactivating mutations are indicated by yellow bars with dots above. e, HBG1 or TTN gene activation using the CjdCas9-DREAM or CjdCas9-SAM system, when recruited using pools of 3 MS2-modified promoter-targeting gRNAs. f, HBG1 or TTN gene activation using the CjdCas9-DREAM or CjdCas9-VPR system, when recruited using pools of 3 MS2-modifed (SadCas9-DREAM) or standard promoter-targeting gRNAs (CjdCas9-VPR), respectively. g, A 3x 9aa TAD derived from MYOCD and MRTF-B TADs is schematically depicted; GS, glycine-serine linker. h, HBG1 or TTN gene activation when the 3x 9aa TAD was fused to MCP and recruited using dCas9 and a pool of 4 MS2-modified promoter-targeting gRNAs. i, The mini-DREAM system is schematically depicted. MCP-eN3x9 is a fusion protein consisting of MCP, eNRF2 and the 3x 9aa TAD from g. j, HBG1 or TTN gene activation when either the mini-DREAM or CRISPR-DREAM system was recruited using a pool of 4 MS2-modified promoter-targeting gRNAs. k, A simplified biosynthetic pathway for progesterone production is schematically depicted. l, The workflow to build progesterone-producing HEK293T cell factories using the mini-DREAM platform and corresponding gRNA array is shown. m, STAR, CYP11A1 and HSD3B2 gene activation after mini-DREAM-transduced HEK293T cells were transfected with the indicated gRNA array or a nontargeting gRNA control plasmid. n, Secreted progesterone levels after mini-DREAM-transduced HEK293T cells were transfected with the indicated gRNA array or a nontargeting gRNA control plasmid. All samples were processed for qPCR or ELISA at 72 h post-transfection. Data are the result of 4 biological replicates for b, c, e, f, j, m and n, and 3 or 4 biological replicates for h. See the source data for more information. Data are presented as mean ± s.e.m. P values were determined using unpaired two-sided t-test. BH, bridge helix; eN, engineered NRF2; M, MRTF-A; PI, PAM-interacting domain; REC, recognition lobe; S, STAT1.
