Fig. 5: Enhancing transcription activation through crRNA engineering and protein engineering. | Nature Communications

Fig. 5: Enhancing transcription activation through crRNA engineering and protein engineering.

From: Engineered minimal type I CRISPR-Cas system for transcriptional activation and base editing in human cells

Fig. 5

a Schematic illustration of the speculated structures of Cascade-VPR with various spacer lengths. Orange: Cas7; Yellow: Cas6; Cyan: Cas5; Blue: VPR. b Fold activations with various spacer lengths. c Left: The crRNA-DNA hybridization double strand was stabilized by aromatic residues (Blue: Y149, F160, and F161) in the Cas7 subunit of Spu Cascade (pale green) through base stacking30. Right: The interactions between the protein and the hybridized double strand after replacing Spu Cas7 with Mos350 Cas7 (pink) using Pymol. In both diagrams, the left orange strand depicts the DNA strand, and the right orange strand depicts the crRNA strand. d Fold activations using Cascade-VPR carrying Cas7 mutations, compared to wild-type [P values from left to right: 0.0056, 0.2076; 0.0003, 0.3364]. e EMSA to compare the binding ability of Mos350 Cascade (gray) and Mos350 Cascade L175F (yellow) to FAM-labeled dsDNA. The concentrations of the two complexes were, from left to right, 0, 5, 50, 100, 150, 200, 250, 500, 1000, 2000 nM. f Fold activations using three different tools. First: dCas9-VPR; Second: Mos350 Cascade-VPR with L175F and 32 bp spacer (L-F S32); Third: Mos350 Cascade-VPR with L175F and 50 bp spacer (L-F S50) [P values from left to right: 0.0010; 0.1005]. g Fold activations of the ASCL1 gene (data from f) and potential off-target genes [P values from left to right: 0.0051, <0.0001, 0.0382]. Ctrl or NT: Only the plasmid expressing the Cascade-VPR or dCas9-VPR was transformed. All values are represented as mean ± s.d. (n = 3 biological replicates), except for results of S44 targeting IL1B gene of two biological replicates (b). Statistical significance was assessed by unpaired two-tailed Student’s t-test (n.s., not significant; *P < 0.05; **P < 0.01; ***P < 0.001). In (b), (d), and (f–g), each bar chart shows the results of samples treated in the same batch. In (b) and (d–g), each experiment was conducted at least in two batches with similar results. Source data are provided as a Source data file.

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