First, more gene promoters were targeted by adding the corresponding sgRNAs. It was first developed by Stanley Qi and colleagues in the laboratories of Wendell Lim, Adam Arkin, Jonathan Weissman, and Jennifer Doudna. 15 Alternative CRISPRa technologies include the SAM (synergistic activator mediator) system, which combines optimized dCas9-transactivation domain fusion and recruitment of heteromeric transactivation domains comprising VP64, p65, and HSF1 (SPH [SunTag … Figure 5. We further explored the ability to titrate the system by modulating the expression of either dCas9VPR or gRNA and selected 2 gRNA for Mef2d (Fi+Ei) in Neuro2a cells. (D) Detection of Sox2 expression by immunofluorescent staining in presence of doxycycline (scale bar, 100 μm). Structure and functions of powerful transactivators: VP16, MyoD and FoxA. B, TRISPR construct induced MEF2D protein increase in C3H/10T/1/2 and C2C12 constitutively expressing dCas9VPR as well as in Neuro2a cells that were transiently transfected with both components. CRISPRa has largely been limited to in vitro settings due to the large size of Cas9 activators, which largely precludes delivery by adeno-associated vectors (AAV). Just recently, promising in vivo approaches in mouse models have been presented, with a demonstration of transcription regulation in the brain and liver using CRISPRa18,19 or epigenetic modulation for the treatment of diabetes mellitus, muscular dystrophy, and acute kidney disease.20. Nppa and Nppb (natriuretic peptides a and b), as well as the fibrosis marker, Ctgf (connective tissue growth factor), normally upregulated in pathological heart remodeling, were significantly elevated in TRISPR Mef2d AAV9-injected Myh6-dCas9VPR-2A-tdTomato transgenic mouse hearts compared with controls (Figure 5C through 5E and Online Figure VIIB). F, Total Mef2d transcripts as well as Mef2d splice variants mRNA levels were elevated compared with controls. Ejection fraction and fractional shortening were significantly reduced at both time points as compared with saline-injected Myh6-dCas9VPR-2A-tdTomato, AAV9-injected, as well as saline-injected wild-type littermates (Figure 5A and 5B and Online Figure VIIA). However, we also confirmed activation of Klf15 expression and its target metabolic genes in dCas9VPR expressing mice. We tested several gRNAs covering the 5′ upstream regions of the respective TSS, since no algorithm is defined for the design of gRNAs targeting promoter regions of genes. First of all, whether simultaneous remodeling of a large number of pluripotency-related loci is necessary or precise remodeling of a single locus is sufficient for iPSC induction is not clear. This mouse expresses a modified version of the “Suntag” Cas9 activator that recruits up to twenty copies of the p65-HSF1 transcriptional activator to each genome-bound dCas9 (see Figure 1a for the transgenic construct). Combining several crRNA sequences targeting the same gene can enhance transcriptional activation. Image, Download Hi-res We next designed and validated gRNAs binding to the 5′ upstream region of the transcriptional start site (TSS) of 2 transcription factors: one implicated in cardiomyocyte hypertrophy and another in metabolic homeostasis, that is, Mef2d (myocyte enhancer factor 2 D) and Klf15 (Krueppel like factor 15). D, Titration of CRISPRa components revealed a stronger dependency of Mef2d activation (ii) on dCas9VPR presence than (i) on gRNA expression, while remaining components were co-transfected at constant level. As we aimed for traceable transcriptional activation efficiency for this proof-of-concept study, the most potent combination of gRNAs, Ci, Fi, and Gi for Mef2d, and A, B, and C for Klf15 were co-transfected in isolated adult cardiomyocytes of Myh6-dCas9VPR-2A-tdTomato animals along with control gRNAs. A detailed description of the methods is available in the Online Data Supplement. The primary purpose of our study was to generate a tool to normalize expression of genes which are reduced during heart remodeling. TRISPR-Klf15-gRNAs showed an activation of up to 3-fold in C3H/10T1/2 fibroblasts and up to 14-fold in C2C12 myoblasts compared with cells transfected with a construct expressing nontargeted gRNA (TRISPR control; Figure 3A). In this study, we aimed to generate a mouse model for dCas9-enhanced endogenous gene activation in cardiomyocytes, with the ultimate goal of normalizing expression of genes, which are reduced on cardiac remodeling and are essential to maintain homeostasis. The linearized Myh6-dCas9VPR-2A-tdTomato construct was injected into the pronucleus of mouse zygotes from B6C3F1 or C57/BL6N mice (both Charles River), which were implanted into pseudo-pregnant ICR mice (Envigo). OG2 Mice (B6;CBA-Tg(Pou5f1-EGFP)2Mnn/J) and B6 Albino mice (B6(Cg)-. At stable dCas9VPR concentration and increasing amount of gRNA (0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, and 100 ng), transfected cells showed increasing MEF2D activation, reaching a maximum activation of ≈5-fold over control at 10 ng or higher. A, Mef2d-activated hearts exhibit decreased fractional shortening and ejection fraction at 7–8 wk post-injection compared with control groups. Addgene is a nonprofit plasmid repository. Document S1. This site uses cookies. Quality control criteria included primarily the percentage of reads at different stages of the analysis, including those with sample barcodes, uniquely mapped to genome, associated with exons/introns and mitochondrial/ribosomal-associated reads, as well as presence of cardiomyocyte markers within the analyzed cells.