Introduction of the Human Sickle Cell Mutation in Sheep Beta-globin by CRISPR Cas9Models Human SCD in Cloned Sheep

Sickle cell disease (SCD) affects approximately 100,000 patients in the US and millions worldwide. Disease burden for SCD patients continues to be high, and treatment options limited. Thus, the development of models able to provide novel insights into the pathophysiology of the disease and/or in which to test new therapies is crucial. Recently, molecular dynamic simulations demonstrated that introducing the human SCD mutation into sheep beta-globin in silico, caused alterations consistent with Hb polymerization driving RBC sickling. Using CRISPR/Cas9 to introduce the human SCD mutation into b-globin locus of sheep fetal fibroblasts, followed by somatic cell nuclear transfer, produced cloned animals with clinical and laboratory parameters that recapitulate human SCD. Animals exhibit a positive SickleDex test, sickled erythrocytes and polychromasia, abnormal Hb with the same migration pattern as human HbS, and the presence of HbF at birth, which was replaced by HbS. Ektacytometry demonstrated a leftward shift of the elongation index-osmolality curves similar to those found in SCD patients. Moreover, the offspring of a SCD ram bred with wild-type ewes display milder phenotypic alterations similar to humans with the SC trait (SCT). These findings thus support the translational relevance of the SCD sheep model and highlight the potential for its future use in the development of novel SCD therapies.