Rationale Loss-of-function studies in cardiac myocytes (CMs) are currently limited by the need for appropriate conditional knockout alleles. The factors that regulate CM maturation are poorly understood. Prior studies on CM maturation have been confounded by heart dysfunction caused by whole organ gene inactivation. Objective To develop a new technical platform to rapidly characterize cell-autonomous gene function in postnatal murine CMs and apply it to identify genes that regulate T-tubules, a hallmark of mature cardiac myocytes. Methods and Results We developed CASAAV (CRISPR/Cas9-AAV9-based somatic mutagenesis), a platform in which AAV9 delivers tandem guide RNAs targeting a gene of interest and cardiac troponin T promoter (cTNT)-driven Cre to RosaCas9GFP/Cas9GFP neonatal mice. When directed against junctophilin-2 (Jph2), a gene previously implicated in T-tubule maturation, we achieved efficient, rapid, and CM-specific JPH2 depletion. High-dose AAV9 ablated JPH2 in 64% CMs and caused lethal heart failure, whereas low-dose AAV9 ablated JPH2 in 22% CMs and preserved normal heart function. In the context of preserved heart function, CMs lacking JPH2 developed T-tubules that were nearly morphologically normal, indicating that JPH2 does not have a major, cell-autonomous role in T-tubule maturation. However, in hearts with severe dysfunction, both AAV-transduced and non-transduced CMs exhibited T-tubule disruption, which was more severe in the transduced subset. These data indicate that cardiac dysfunction disrupts T-tubule structure, and that JPH2 protects T-tubules in this context. We then used CASAAV to screen 8 additional genes for required, cell-autonomous roles in T-tubule formation. We identified ryanodine receptor 2 (RYR2) as a novel, cell-autonomously required T-tubule maturation factor. Conclusions CASAAV is a powerful tool to study cell-autonomous gene functions. Genetic mosaics are invaluable to accurately define cell-autonomous gene function. JPH2 has a minor role in normal T-tubule maturation but is required to stabilize T-tubules in the failing heart. RYR2 is a novel T-tubule maturation factor.

The forward genetic screen is a powerful, unbiased method to gain insights into biological processes, yet this approach has infrequently been used in vivo in mammals because of high resource demands. Here, we use in vivo somatic Cas9 mutagenesis to perform an in vivo forward genetic screen in mice to identify regulators of cardiomyocyte (CM) maturation, the coordinated changes in phenotype and gene expression that occur in neonatal CMs. We discover and validate a number of transcriptional regulators of this process. Among these are RNF20 and RNF40, which form a complex that monoubiquitinates H2B on lysine 120. Mechanistic studies indicate that this epigenetic mark controls dynamic changes in gene expression required for CM maturation. These insights into CM maturation will inform efforts in cardiac regenerative medicine. More broadly, our approach will enable unbiased forward genetics across mammalian organ systems.

In vivo loss-of-function studies are currently limited by the need for appropriate conditional knockout alleles. CRISPR/Cas9 is a powerful tool commonly used to induce loss-of-function mutations in vitro. However, the CRISPR components have been difficult to deploy in vivo. To address this problem, we developed CASAAV (CRISPR/Cas9-AAV-based somatic mutagenesis), a platform in which recombinant adeno-associated virus (AAV) is used to deliver tandem guide RNAs targeting a gene of interest and Cre, driven by a cell type selective promoter, to RosafsCas9GFP postnatal mice. Using this system, within one month it is possible to induce temporally controlled cell type-selective knockout of virtually any gene using only one mouse line. Here, we focus on knockout of genes in cardiomyocytes, although with minimal modifications the system could be adapted in inactivate genes in other AAV-transduced cell types.