CRISPR Activation Reverses Haploinsufficiency and Functional Deficits Caused by TTN Truncation Variants

TTN CRISPR activation rescued TTNtv-related functional deficits despite increasing truncated TTN levels, which provides evidence to support haploinsufficiency as a relevant genetic mechanism underlying heterozygous TTNtvs (TTN truncation variants). CRISPR activation could be developed as a therapeutic to treat a large proportion of TTNtvs. We adapted CRISPR activation using dCas9-VPR to functionally interrogate TTNtv pathogenicity and develop a therapeutic in human cardiomyocytes and 3-dimensional cardiac microtissues engineered from induced pluripotent stem cell models harboring a dilated cardiomyopathy–associated TTNtv. We performed guide RNA screening with custom TTN reporter assays, agarose gel electrophoresis to quantify TTN protein levels and isoforms, and RNA sequencing to identify molecular consequences of TTN activation. Cardiomyocyte epigenetic assays were also used to nominate DNA regulatory elements to enable cardiomyocyte-specific TTN activation. By obtaining sequences from chromatin immunoprecipitated DNA, we generated genome-wide maps of inter-molecular ligated DNA representing long-range interactions in chromatin with in-situ ChiIA-PET RNAPII technology.