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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., Throughput of in vivo genetic screens is a barrier to efficient application. Here the authors use a high-throughput CRISPR-based in vivo forward genetic screen in mice to identify transcriptional regulators of cardiomyocyte maturation, including the epigenetic modifiers RNF20 and RNF40.

CRISPR/Cas9-based genome editing technologies provide powerful tools for genetic manipulation. Delivery of Cas9 and a homology directed repair (HDR) template using adeno-associated virus (AAV; CASAAV-HDR), was recently shown to enable creation of precise genomic edits, even within postmitotic cells. Here we studied CASAAV-HDR in cardiomyocytes. We constructed an AAV9 vector containing a gRNA targeting the ventricle specific Myl2 gene, and a promoterless HDR template that replaces the native Myl2 stop codon with a self-cleaving 2A peptide followed by mScarlet, a red fluorescent protein. When this vector was injected into Cas9 expressing newborn mice, we observed mScarlet expression within a remarkably high fraction of cardiomyocytes, approximately 45%. Expression was ventricle specific, consistent with the Myl2 expression profile. Similarly, when we targeted the atrial specific Myl7 gene, we observed mScarlet expression in ~20% of atrial cardiomyocytes. Amplicon sequencing of Myl2 and Myl7 transcripts showed that the vast majority of transcripts with an insertion were mutation-free, indicating that CASAAV-HDR is precise. Furthermore, CASAAV-HDR efficiency was comparable when AAV was delivered to fetal, neonatal, or mature mice. Next we targeted seven additional loci: Yap1, Tmem43, Nfatc3, Bdh1, Mkl1, Ttn, and Pln, fusing either an HA tag or mScarlet to each. Insertion efficiency varied dramatically between loci, with HDR efficiency generally correlating with target gene expression. TTN-mScarlet and mScarlet-PLN fusion proteins localized to the sarcomere and sarcoplasmic reticulum, respectively, consistent with the localization of the endogenous proteins. Collectively these data indicate that systemic delivery of CASAAV-HDR vectors can achieve efficient, precise, in vivo somatic genome modification that does not require cardiomyocyte proliferation. We successfully used this technology to monitor protein localization and anticipate it will be useful for many other applications, such as precise introduction of mutations to model disease or probe gene function. CASAAV-HDR may also enable efficient, permanent, and precisely targeted delivery of therapeutic transgenes to validated loci.

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.