396. Multiplex CRISPR/Cas9 Genome Engineering for Directing Myogenic Cellular Differentiation

Engineered DNA-binding proteins facilitate manipulation of the human genome and transcriptome. A new class of transcription factors has recently been developed based on the RNA-guided Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system. Through complementary base pairing, guide RNAs (gRNAs) localize the Cas9 protein to target DNA sequences. Cas9-based transcriptional activators are created by fusing a catalytically deactivated Cas9 (dCas9) to an activation domain such as VP64. Using myogenic differentiation as a model, we utilized the CRISPR/Cas9 system to direct cellular behavior in primary human cells. MyoD is the master transcription factor defining the myogenic lineage. Overexpression of MyoD in certain cell types upregulates the myogenic network and induces phenotypic myogenic differentiation. In comparison to overexpressing MyoD, directly targeting silenced regulatory regions in the endogenous MyoD promoter with a CRISPR/Cas9 transcriptional activator may induce the myogenic gene network in a more controlled manner. To test this hypothesis, we made use of our previously published platform that expresses a Cas9 transcriptional activator and four gRNAs from a single lentiviral vector. This approach mitigates the technical challenges associated with sustained delivery of the CRISPR/Cas9 components in difficult to transfect primary cells. We designed 30 gRNAs that span the entire proximal regulatory region (PRR) of the MyoD gene. To screen the gRNAs, we treated dermal fibroblast with virus co-expressing a single gRNA with a Cas9 transcriptional activator and assayed for MyoD expression using qRT-PCR. The seven gRNAs that induced the largest level of MyoD expression were further tested in combinations of four using the multiplex lentiviral platform. From this process, an optimal set of four gRNAs were selected. In fibroblasts, the optimized gRNAs induce expression of MyoD and its downstream marker myogenin. However, no other markers were detected and a myogenic phenotype was not observed. Interestingly, the upregulation of the myogenic network was enhanced in the presence a small-molecule cocktail known to promote cardiac cellular differentiation. In contrast to the dermal fibroblasts, delivery of the optimized gRNAs into human induced pluripotent stem cells (iPSCs), which have a partially open chromatin structure in the PRR, effectively induced myogenic differentiation in three days. The activation of several myogenic markers was detected and the resulting myofibers stained positive for myosin heavy chain, a terminal marker of myogenic differentiation. Since CRISPR/Cas9 transcriptional activators induce expression from the endogenous locus, we hypothesize that our multiplex CRISPR/Cas9 system will require a shorter duration of expression compared to a MyoD transgene to induce sustained expression from the endogenous locus, and that the resulting epigenetic landscape will closely mimic that of myoblasts. Ongoing studies are investigating the kinetics of induced differentiation and the global transcriptional and epigenetic state of these cells. CRISPR/Cas9 transcriptional activators effectively coordinate myogenesis and as such may provide a new avenue for obtaining donor cells for muscle cell therapies.