To test whether the direction of the

To test whether the direction of the genomic strand targeted by the gRNA molecule was a factor in its capacity to recruit dCas9 and activate expression of the endogenous locus, we designed a gRNA molecule (gRNA4) targeting the minus strand of the same region for which gRNA3 was designed (PAM separated by 14 bases) (Figure S1D). Interestingly, gRNA4 was also able to activate Myod1 expression and initiate reprogramming, indicating the system is insensitive to the target strand (Figures 3F and S3B). We also evaluated the effect of BFP fusion on the activity of VP64dCas9-BFPVP64. Following transduction at equivalent multiplicity of infection (MOIs), the construct without BFP showed statistically higher levels of dCas9 expression, although it was less efficacious than the BFP-fused form in inducing expression of Myod1 (Figures 3B and 3G). However, both forms led to reprogramming (Figure S3C). The omission of BFP from the C-terminal-only VP64 fusion protein (dCas9VP64) failed to impart ability to activate the endogenous Myod1 locus (Figure S3D). We speculate that the positive effect of BFP on Myod1 expression may be due to increased spacing between the two VP64 domains or increased flexibility of the domains. We also compared the VP64dCas9-BFPVP64-mediated activation of endogenous Myod1 gene to transgenic MYOD1 overexpression for its ability to reprogram cells. C3H10T1/2 kisspeptin were transduced with VP64dCas9-BFPVP64 and M2rtTA. The resultant BFP-positive cells were sorted for BFP expression and the BFP+ cells were transduced with either gRNA or doxycycline-inducible transgenic human MYOD1 at equivalent MOIs (MOI = 10) (Figures 4A and 4B). During the induction phase of the reprogramming process, endogenous Myod1 expression over the entire duration was significantly higher in the VP64dCas9-BFPVP64/gRNA-mediated activation group than the transgenic MYOD1 overexpression group. Expression kinetics of Myog and Desmin were similar in both the groups (Figure 4C). The effects of limiting the activation of the VP64dCas9-BFPVP64/gRNA system (by varying the duration of doxycycline exposure) on myogenic gene expression were compared with the MYOD1 overexpression system. Results showed that even a single day of doxycycline exposure (from day 2 to day 3 posttransduction) was adequate to activate the downstream myogenic genes (Myog and Desmin) to similar levels in both the groups and similar to levels achieved after 8 days of induction (Figure 4D). Almost all the markers on day 18 posttransduction after a maximum 8 days of induction had similar expression levels in both the groups (Figures 4D and S4A). However, both Myod1 expression and the percentage of MYOD1 TF+ nuclei were approximately 3-fold higher in the Myod1 transactivation group than the MYOD1 overexpression group (Figures 4D, 4E, and S4B). It indicates that the higher expression of Myod1 on quantitative RT-PCR (qRT-PCR) was probably a result of more MYOD1 TF+ cells rather than higher expression levels in individual cells. Myog expression in C3H10T1/2 followed a pattern similar to Myod1 (Figures 4D and 4E). Similar differential Myod1 expression was also observed in the reprogrammed MEFs. However, expression of all the other myogenic markers and the percentage of MYOG+ nuclei were similar in both groups (Figures S4C and S4D). It may be speculated that VP64dCas9-BFPVP64/gRNA action renders the endogenous Myod1 locus more receptive to MYOD1 TF-positive feedback response (Zingg et al., 1994). However, other cellular factors needed in conjunction with MYOD1 TF to initiate skeletal reprogramming may not be present in adequate quantities, thereby explaining the lack of a significant difference for other downstream late myogenic genes. This study shows that the dual fusion of the VP64 transactivation domain to both the N and C terminus of dCas9 enables a high level of endogenous Myod1 activation for the direct conversion of primary murine fibroblasts into SkMs. This potentiation of the transactivation process can be explained by an increased probability of the TFs homing onto two VP64 domains compared with just one VP64 domain. As a result, transcription can be initiated more frequently in the presence of VP64dCas9-BFPVP64. Moreover, a synergistic transactivation effect resulting from favorable interactions of the TF complexes assembled on both the terminus of VP64dCas9-BFPVP64 can also help in explaining its potency. Improved nuclear localization of dCas9 afforded by increasing the number of NLS sequences, efficient lentiviral transgene delivery, inclusion of a BFP spacer sequence to decrease steric hindrance, and the identification of an efficient gRNA together complemented the effect of an additional VP64 domain in VP64dCas9-BFPVP64 to efficiently transactivate the endogenous Myod1 locus. The results also demonstrate that CRISPR/Cas9-based transactivation performs comparably to the traditional MYOD1 overexpression-based skeletal reprogramming in upregulating some major myogenic genes. It augurs well for the potential use of this tool in reprogramming protocols that require complex and multiple TF activation.