Here, we performed AAV-CRISPR mediated homology-independent knock-in at a new target site in mAlb 3’UTR and demonstrated that single dose of AAVs enabled long-term integration and expression of hF9 transgene in both adult and neonatal hemophilia B mice (mF9 −/−), yielding high levels of circulating human Factor IX (hFIX) and stable hemostasis restoration during entire 48-week observation period. This evidence concerns the gene F9 and hemophilia.