Cardiac-Specific Gene Editing via an AAV9-Tnnt2-SaCas9-miR122TS Vector

The adeno-associated virus serotype 9 (AAV9)-delivered gene expression driven by the cardiac troponin T (Tnnt2) promoter is broadly considered to be cardiac-specific. However, in cases where low AAV expression is sufficient to trigger a profound biological effect in CRISPR/Cas9 gene editing, the ectopic AAV9-Tnnt2 expression and gene editing in the liver becomes non-negligible. MicroRNA122 is a microRNA that is specifically expressed in the liver. The incorporation of the microRNA122 target sequence (miR122TS) into the 3' untranslated region (UTR) of the AAV transgene could reduce ectopic gene expression in the liver. Here, we provide a protocol for sgRNA design, plasmid construction, AAV packaging, and in vivo validation of a new AAV9-Tnnt2-SaCas9-miR122TS vector using publicly available materials and tools. The application of this new vector enables cardiac-specific gene editing while circumventing leakages in the liver. Key features • This protocol describes a detailed procedure to construct and validate AAV-based cardiac-specific gene editing in mice. • MicroRNA-122 target sequences (miR122TS) in combination with a Tnnt2 promoter are used to enhance the cardiac specificity in genome editing. • Amplicon sequencing analysis is applied to precisely and sensitively quantify the genome editing efficiency and tissue specificity in mice.