Virally Induced CRISPR/Cas9-Based Knock-In of Fluorescent Albumin Allows Long-Term Visualization of Cerebral Circulation in Infant and Adult Mice

Albumin, a protein primarily produced by liver hepatocytes, is the most abundant protein in blood plasma. We have previously engineered a liver-targeting adeno-associated viral vector (AAV) that expresses fluorescent protein-tagged albumin to visualize blood plasma in mice longitudinally. While this approach is versatile for imaging in adult mice, transgene expression vanishes when AAV is administered in neonates due to dilution of the episomal AAV genome in the rapidly growing liver. Here, we use CRISPR/Cas9 genome editing to insert the fluorescent protein mNeonGreen (mNG) gene into the albumin (Alb) locus of hepatocytes to produce fluorescently labeled albumin (Alb-mNG). We constructed a CRISPR AAV that includes ~1 kb homologous arms around Alb exon 14 to express Alb-mNG. Subcutaneous injection of this AAV with AAV-CMV-Cas9 in postnatal day 3 mice resulted in two-photon visualization of the cerebral cortex vasculature within 10 days. The expression levels of Alb-mNG were persistent for at least 3 months after infection and were robust enough to assess vasomotion and capillary blood flow transcranially in early postnatal mice. This knock-in approach provides a powerful means for micro- and macroscopic imaging of cerebral vascular dynamics in postnatal and adult mice.