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A CRISPR/Cas9-based system for reprogramming cell lineage specification.

Chakraborty S, Ji H, Kabadi AM, Gersbach CA, Christoforou N, Leong KW - Stem Cell Reports (2014)

Bottom Line: Gene activation by the CRISPR/Cas9 system has the potential to enable new approaches to science and medicine, but the technology must be enhanced to robustly control cell behavior.Targeted activation of the endogenous Myod1 gene locus with this system led to stable and sustained reprogramming of mouse embryonic fibroblasts into skeletal myocytes.The levels of myogenic marker expression obtained by the activation of endogenous Myod1 gene were comparable to that achieved by overexpression of lentivirally delivered MYOD1 transcription factor.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Duke University, Durham, NC 27708, USA; Department of Biomedical Engineering, Columbia University, New York, NY 10027, USA.

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Characteristics of gRNA-Guided VP64dCas9-BFPVP64-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-BFPVP64, 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, VP64dCas9VP64 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.
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fig3: Characteristics of gRNA-Guided VP64dCas9-BFPVP64-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-BFPVP64, 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, VP64dCas9VP64 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.

Mentions: To test whether transient expression of VP64dCas9-BFPVP64 is sufficient to induce sustained expression of Myod1, we induced transgene expression between days 2 and 10 posttransduction. We detected an initial robust upregulation of VP64dCas9-BFPVP64 expression followed by a gradual downregulation suggesting transgene silencing in the reprogramming cells (Figure 3A). Doxycycline removal coincided with rapid VP64dCas9-BFPVP64 decline to levels measured prior to initial induction. VP64dCas9-BFPVP64 expression also coincided with an increase in Myod1 expression. Importantly, Myod1 mRNA levels remained elevated for the duration of the 18-day experiment even after doxycycline removal (day 10), suggesting that maintenance of endogenous activation of Myod1 is stable and independent of VP64dCas9-BFPVP64 activity.


A CRISPR/Cas9-based system for reprogramming cell lineage specification.

Chakraborty S, Ji H, Kabadi AM, Gersbach CA, Christoforou N, Leong KW - Stem Cell Reports (2014)

Characteristics of gRNA-Guided VP64dCas9-BFPVP64-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-BFPVP64, 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, VP64dCas9VP64 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.
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fig3: Characteristics of gRNA-Guided VP64dCas9-BFPVP64-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-BFPVP64, 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, VP64dCas9VP64 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.
Mentions: To test whether transient expression of VP64dCas9-BFPVP64 is sufficient to induce sustained expression of Myod1, we induced transgene expression between days 2 and 10 posttransduction. We detected an initial robust upregulation of VP64dCas9-BFPVP64 expression followed by a gradual downregulation suggesting transgene silencing in the reprogramming cells (Figure 3A). Doxycycline removal coincided with rapid VP64dCas9-BFPVP64 decline to levels measured prior to initial induction. VP64dCas9-BFPVP64 expression also coincided with an increase in Myod1 expression. Importantly, Myod1 mRNA levels remained elevated for the duration of the 18-day experiment even after doxycycline removal (day 10), suggesting that maintenance of endogenous activation of Myod1 is stable and independent of VP64dCas9-BFPVP64 activity.

Bottom Line: Gene activation by the CRISPR/Cas9 system has the potential to enable new approaches to science and medicine, but the technology must be enhanced to robustly control cell behavior.Targeted activation of the endogenous Myod1 gene locus with this system led to stable and sustained reprogramming of mouse embryonic fibroblasts into skeletal myocytes.The levels of myogenic marker expression obtained by the activation of endogenous Myod1 gene were comparable to that achieved by overexpression of lentivirally delivered MYOD1 transcription factor.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Duke University, Durham, NC 27708, USA; Department of Biomedical Engineering, Columbia University, New York, NY 10027, USA.

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Related in: MedlinePlus