CRISPR-based transcriptional activation (CRISPRa) offers a precise strategy for enhancing endogenous gene expression without altering the genomic sequence. This approach holds significant potential for targeted gene regulation, either to test therapeutic strategies of validate patho-mechanistic properties. Here were present a new model for testing CRISPRa in a large animal model that has been created by genetic modification (GM) and provide evidence that transcriptional regulation can be induced with specific gRNAs for selected heart-relevant genes.
The pig model has been generated by placing a constitutively silent transgene, comprising a CAG promoter and a removable floxed STOP cassette, followed by a fusion gene expressing a Cas9 variant with inactivated DNA cutting sites and a VP64-p65-Rta (VPR) domain, stimulating transcription. The transgene was placed into the defined ROSA26 / THUMPD3-AS safe harbor. GM founder pigs were created by Somatic Cell Nuclear Transfer. 3 founder animals were delivered, allowing the isolation of primary kidney cells.
To test transcriptional regulation in vitro, we designed gRNAs binding near the transcriptional start site of USH1C and the cardiac-relevant PKP2 and MEG3 genes, using a combination of multi-species alignments, regulatory databases and previously conducted high throughput screening studies in human cell lines. In the experimental design, we combined 4 plasmids expressing different gRNA for a defined genes with a plasmid expressing Cre-recombinase to remove the STOP cassette and thereby induce dCas-VPR expression.
For the 3 tested genes, one set of gRNAs stimulated transcription from baseline whereas other sets of gRNAs did not indicate stimulatory capacity.
Thus, we here give evidence that transcription of designated genes can be induced by a CRISPRa system in pig. Design of gRNAs can be improved by a combination of bio-informatics and literature-based data.
