Precision and efficacy of RNA-guided DNA integration in high-expressing muscle loci
We explored a novel gene-editing approach using endogenous muscle gene promoters to achieve high-level gene expression. By targeting the muscle creatine kinase (Ckm) and myoglobin (Mb) sites in mouse muscle both in vitro and in vivo with CRISPR-Cas9 technology, we integrated genes without relying on their own promoters. Using the Homology-Independent Targeted Integration (HITI) method, we utilized the Ckm and Mb promoters to express green fluorescent protein (GFP) and human Factor IX (hFIX) in mouse myoblasts and muscle tissue. To ensure the safety and efficacy of this method and the targeted sites, we performed RNA sequencing (RNAseq) and gDNA deep sequencing to evaluate changes in the global transcriptome and on-target and off-target integration.