scAAV8-Cre

Genetically engineered mouse models (GEMMs) have transformed the study of disease phenotypes in living organisms but are limited by their lengthy generation process in embryonic stem cells. Here, researchers describe methods for rapid and scalable genome engineering in liver and pancreatic somatic cells by delivering CRISPR components into living mice. They developed methods and protocols for liver and pancreatic CRISPR delivery using AAV, enabling somatic cell genome engineering, disease modeling, and a range of cancer-related applications. It would be ideal to achieve high transduction and editing efficiencies via a simple route of administration (without the need for complex surgeries such as retrograde catheterization or intraparenchymal injections). To this end, the researchers benchmarked a robust protocol based on intraperitoneal injection of scAAV, optimizing it to outperform other delivery routes (e.g., intravenous) in terms of efficiency, reproducibility, and ease of use.

Figure 1 shows functional outputs of scAAV8-Cre delivery to the pancreas and liver of Rosa26^mT/mG mice 28 d after infection. In Rosa26^mT/mG reporter mice, Cre expression converts membrane localized tdTomato to membranous/cytoplasmic EGFP fluorescence. Intraperitoneal injection of 1012 viral genomes (vg) resulted in complete (～100%) or near-complete (～90%) recombination in the liver and in the pancreas, respectively (Figure 1c). Furthermore, reducing viral titers allows dosage-dependent generation of mosaic phenotypes (Figure 1d). Target cells in the pancreas are mainly acinar cells, and to a lower degree islet cells (Figure 1d), whereas hepatocytes (but not cholangiocytes or endothelial cells) are infected in the liver (Figure 1e). Enrichment of transduced cells in specific areas of a tissue (e.g., around pericentral veins upon intravenous injection in the liver) is much less of an issue for intraperitoneal administration of AAV as compared to HTVI or electroporation. Nonetheless, infections with multiple viral copies per cell may be more likely to occur in central regions of the liver.

Figure 1. Efficacy of viral and nonviral nucleic acid delivery methods for the pancreas and liver of mice. (Kaltenbacher T, et al., 2022)