Efficient In Vivo Homology-Directed Repair Within Cardiomyocytes

RISPR/Cas9-based genome editing technologies provide powerful tools for genetic manipulation.Cas9 in vivo genome editing of cardiomyocytes through nonhomologous end joining efficiently creates insertion-deletion mutations at guide RNA-targeted sites. 1 However, precise addition of new genetic information, or mutation correction, requires alternate strategies.Delivery of Cas9 and a homology directed repair (HDR) template using adeno-associated virus (AAV) was recently shown to enable creation of precise genomic edits, even within postmitotic cells. 2 We studied CRISPR/Cas9 and AAV-based homology directed repair (CASAAV-HDR) in cardiomyocytes.Animal experiments were approved by the Institutional Animal Care and Use Committee and adhered to institutional guidelines.The authors will provide data, results, and reagents on reasonable request.We studied CASAAV-HDR at Myl2 (MLC2v), a highly expressed, ventricle-specific sarcomere gene.We constructed AAV9 with an Myl2-specific guide RNA and a promoterless HDR template that replaces the native stop codon with self-cleaving 2A peptide followed by mScarlet, a red fluorescent protein (Figure [A]).Subcutaneous delivery of the AAV to postnatal day 0 mice with cardiacrestricted Cas9 expression (Tnnt2Cre;Rosa26 fsCas9-P2A-GFP ) yielded strong mScarlet expression in postnatal day 7 ventricular cardiomyocytes (Figure [Bi and C]).mScarlet expression required Cas9 and Myl2 homology arms (Figure [Bii and Biii]).AAV-delivered Cas9 successfully directed mScarlet expression, albeit with reduced efficiency compared with Rosa26 fsCas9-P2A-GFP (Figure [Biv]).