Abstract 106: Efficient In Vivo Homology-Directed Repair Within Cardiomyocytes

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.