CRISPR/Cas9 creation of a mouse lacking a unique motif on alpha globin demonstrates its critical role in regulating nitric oxide in vivo

Alpha globin and eNOS form molecular complexes in microvascular endothelial cells that can regulate nitric oxide (NO) availability. On the alpha globin protein we have identified the amino acids 35–44 (FASFPTTKTY) as being essential for eNOS interaction. Using in silico prediction, we identified the 37–40 amino acid motif (SFPT) as being especially crucial for eNOS interaction, which we confirmed with purified protein binding assays. To confirm the importance of this motif in vivo, we used CRISPR/Cas9 to create a mouse with alpha globin missing amino acids 37–40 in the Hba1 gene. Whole genome sequencing confirmed the mutation and demonstrated a lack of any off-target effects. We found that homozygous Hba1Δ37–40 mice were not viable (0 mice born from 8 different heterozygous pairs; 16 litters) and were re-absorbed in utero by approximately day E12. The lethal homozygous Hba1Δ37–40 embryos had significantly dilated arteries and cardiac ventricles. For this reason we utilized Hba1WT/Δ37–40 heterozygous anima...