Figure 3.

Characteristics of gRNA-Guided ^VP64dCas9-BFP^VP64-Mediated Activation of the Endogenous Myod1 Gene Locus

(A) VdC9BV-mediated activation of the endogenous murine Myod1 gene is stable and sustained following doxycycline removal. The red arrowheads indicate the time points on which addition and removal of doxycycline are performed. dCas9 expression kinetics is significantly different from that of transactivated Myod1 (p ≤ 0.0001, two-way ANOVA). Fold change in expression is relative to levels at day 0.5 posttransduction (n = 4 biological replicates, error bars represent SEM).

(B) Illustration of dCas9-based activators.

(C) The C-terminal-only fusion of VP64 to dCas9, dCas9-BFP^VP64, fails to activate the endogenous murine Myod1 gene in C3H10T1/2 cells even in the presence of multiple gRNAs. VdC9BV significantly activated transcription of the endogenous Myod1 gene locus when compared with all other groups (p ≤ 0.001, one-way ANOVA, Tukey’s post hoc test, n = 3). Control group: M2rtTA + Empty FUW. Fold change in expression is relative to the (−) control group.

(D) The N-terminal-only VP64 fusion to dCas9 (^VP64dCas9-BFP) also failed to match the ability of VdC9BV to transactivate significant levels of endogenous Myod1 expression in the presence of a single gRNA (p ≤ 0.001, one-way ANOVA, Dunnett’s post hoc test, n = 3). Control: M2rtTA +Empty FUW. Fold change in expression is relative to the (−) control group.

(E) Only the N- and C-terminal VP64 fusion to dCas9 (VdC9BV) significantly activated the human MYOD1 locus of HEK293T cells in the presence of a gRNA (p ≤ 0.01, one-way ANOVA, Dunnett’s post hoc test, n = 3).

(F) A gRNA targeting the antisense strand (gRNA4) can significantly activate endogenous Myod1 expression to levels similar to that achieved by the gRNA targeting the sense strand (gRNA3) (p ≤ 0.001, one-way ANOVA, Dunnett’s post hoc comparing all the groups to the [−] gRNA group, n = 3). (−) gRNA group: M2rtTA + VdC9BV + Empty FUW virus. Fold change in expression is relative to the (−) control group.

(G) In the absence of the BFP domain, ^VP64dCas9^VP64 activates Myod1 expression to a lesser extent than the VdC9BV group, although dCas9 is expressed at a significantly higher level using the same MOI (C3H10T1/2 cells, unpaired two-tailed t test p = 0.0051 [dCas9], p = 0.0044 [Myod1], n = 3). Fold change in expression is relative to the VdC9BV group. Measurements for (C–G) occurred at posttransduction day 6, and the data from independent biological replicates are represented as mean ± SEM.