Synergetic targeting of RBM20 in iPSC derived cardiomyocytes by CRISPR/Cas

Mutations that result in haploinsufficiency and the production of toxic proteins are common and difficult to correct epigenetically. Using CRISPR/Cas systems, our research provided proof-of-concept that simultaneous activation as well as silencing of disease-modifying factors represents a novel technology for improving therapeutics of complex diseases. Specifically, downregulation of the RBM20 p.R634L mutated form, associated with aberrant alternative splicing and cardiomyopathy was tested employing the CRISPR/Cas13 system, an RNA editing approach that results in RNA silencing. The system is based on CRISPR (cr)RNA sequences that match the target transcript for degradation. Accordingly, we designed crRNA that matched perfectly the mutant transcript but had a mismatch in the wild-type sequence in position 18 or 19 (crRNA#18/crRNA#19) in order to edit mainly the mutated RBM20 transcript, which has negative effects. The crRNA#18 and crRNA#19 significantly decreased the expression of a RBMB20 mutated reporter by 68% ± 12% and 77% ± 9%, respectively, and the wild-type reporter by 47% ± 9% and 71% ± 2%, respectively, compared to non-target controls. Thus, the crRNA#18 offered the best compromise between mutated and wild-type allele targeting. Next, a second-generation CRISPR activation (CRISPRa) system was tested across various cell lines to upregulate RBM20. Despite high transfection efficiencies, only a modest upregulation of 1.4-fold was observed in HEK293T-cells with a sgRNA targeting the distal promoter region compared to non-target (p < 0.03). No significant activation was detected in hiPSCs or hiPSC-derived cardiomyocytes, although a chromatin accessibility analysis indicated that the targeted RBM20 promoter regions were transcriptionally active. These results indicate the standard rules cannot be applied for activation of RBM20 and further investigation is needed. In conclusion, CRISPRa showed limited upregulation potential, while CRISPR/Cas13 exhibited strong knockdown of the mutated form. This is currently investigated using patient derived iPSC cardiomyocytes for testing the specific effects.