CRISPR/Cas9‐Mediated Genome Editing of the Murine Pulmonary Circulation

CRISPR/Cas9 is increasingly being tested for in vivo genome editing and we describe here the optimization, pharmacokinetic properties, and efficiency of this system to target the pulmonary vasculature in mice. We used cationic liposomes to deliver a PX458 vector expressing Cas9, eGFP, and gRNA to the pulmonary circulation and studied both its bio‐distribution and time needed to knock down proteins. Different amounts of liposomes were injected containing 25, 50, 75, and 100μg of plasmid DNA. After 4 days, mice were sacrificed and several organs were studied for eGFP expression. We found a dose‐dependent increase in pulmonary expression. The only other organ with detectable expression was the liver, but expression was at least 7‐fold higher in the lungs compared to the liver even at the highest dose tested. We found no eGFP expression in kidney, heart, brain, or skeletal muscle (n=4 animals/dose). Within the lung, we used immunohistochemistry to show that endothelial cells and to a lesser degree pulmonary artery smooth muscle cells were selectively targeted. We did not detect any expression in airway epithelial cells or airway macrophages. Lung expression was very temporary, with expression peaking at 4 days and returning to baseline after 28 days (n=4 mice/group), minimizing the chances of side effects, more specifically non‐specific alterations in the DNA that could result from continued expression of the endonuclease Cas9. Using mT mice that ubiquitously express tandem dimer Tomato fluorescent protein, were able to show a downregulation of fluorescence specifically in the majority of the pulmonary blood vessels after just 7 days (n=3 mice/group). Genomic alterations were confirmed by the T7 endonuclease assay and Next‐Generation Sequencing. Importantly, we did not observe any thrombosis or other adverse side‐effects that might be associated with expression of Cas9 in the pulmonary endothelium. In conclusion, we used a non‐viral delivery method to specifically deliver CRISPR/Cas9 to the pulmonary vasculature. The temporary expression of CRISPR/Cas9 led to a pronounced downregulation of the targeted fluorescent protein within 7 days, but could easily be adapted to target almost any gene. Currently available animal models do not allow to knock out a gene specifically in the pulmonary endothelial cells and effects on the pulmonary circulation might be compounded by effects in the systemic circulation. Therefore, our work provides a novel tool to specifically target and study genes in the pulmonary circulation. Support or Funding Information This work was supported in part by a Parker B. Francis fellowship and American Heart Scientist Development grant 15SDG23250002 (GM), NIH‐R01‐GM094220 (JR) and NIH‐R01‐HL045638, R01‐HL090152, R01‐HL118068, P01‐HL060678, P01‐HL077806 (ABM).