While we gained detailed insight into the single cell transcriptional profiles of heart cells in health and disease in recent years, endogenous control over single gene expression or gene networks remains challenging. The complementation of the descriptive work with functional testing of enhanced gene (re-)expression is highly attractive for designing novel therapeutic strategies in various heart diseases. We previously established endogenous gene activator models in murine and human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM) for in vivo and in vitro transcriptional control of gene expression. These systems recruit enzymatically inactive Cas9 fused to transcription activators (dCas9VPR) to any promoter region of choice by nucleotide programmable guide (g) RNAs. We hypothesize that iterative testing of gRNAs can sharpen our design rules for novel gRNAs and target genes to efficiently use these tools and models in cardiovascular research. While testing multiple gRNAs per target gene in HEK293T (human) and Neuro2a cells (murine), we were able to plot transcriptional activation efficiency (as determined by fold-change mRNA expression over controls including non-targeting gRNA) compared to nucleobase distance to the respective transcriptional start site (TSS). With more than 15 mouse and 10 human genes tested, we identified the -100 bp region upstream of the TSS to be the most effective region for dCas9VPR gene activation recruitment (n ≥ 3 experiments and 2-10 gRNAs tested per candidate gene). Notably, this distance commonly harbours the proximal promoter region of eukaryotic genes. We also identified candidate target genes which were strongly expressed at baseline, and which showed a reduced dCas9VPR gene activation efficiency in this region. This suggested that dCas9VPR cannot overcome endogenous transcription factor mediated gene transcription and therefore allows gene activity modulation within the physiological boundaries. Selecting the most effective predicted and validated gRNAs with our pipeline, we observed consistent gene activation in hiPSC-derived cardiomyocytes and dCas9VPR mice according to the transcript analyses in our validation experiments. This highlighted the usefulness of careful experimental pre-screening of gRNAs for these approaches to achieve robust gene activation in advanced in vitro and in vivo models. More importantly, we also observed gene activation titratability by using up to three gRNAs tiled in a specific 5’ TSS upstream region of the same gene. The degree of gene activation varied greatly and was target gene rather than target cell type or model dependant based on our analysis (i.e. transcription factor KLF15: tested in HEK293T - applied in hiPSC-CM, Ribosome Binding Protein 1 (Rrbp1): tested in Neuro2a - applied in murine hearts, mouse Insulin Like Growth Factor Binding Protein 5 (Igfbp5): tested in Neuro2a - applied in murine hearts, and human IGFBP5: tested in HEK293T - applied in hiPSC-CM). Importantly, for target gene products with available antibodies, we confirmed a translation of the enhanced mRNA expression levels to the protein level in both, test systems and applied in vivo or in vitro models. With this work, we gained further insight into basic transcriptional dynamics of enhanced gene expression and provide experimentally validated application rules for effective CRISPR-based endogenous gene activation approaches in basic and translational cardiovascular research.