SaCas9 and AsCas12a Ultra Enable Highly Efficient and Specific Gene Editing in Hematopoietic Stem and Progenitor...

Gene editing therapies for treating single-gene disorders have primarily utilized Streptococcus pyogenes Cas9 (SpCas9), while the performance of Cas orthologs Staphylococcus aureus Cas9 (SaCas9) and Acidaminococcus sp. Cas12a (AsCas12a) has not been systematically compared with SpCas9. Here, we compared the efficiency and specificity of SaCas9 and AsCas12a with SpCas9 in hematopoietic stem and progenitor cells from patients with sickle cell disease. SaCas9 achieved higher efficiencies in correcting the sickle mutation and targeting the BCL11A erythroid enhancer, whereas AsCas12a showed superior editing at the HBG promoter and induced the highest levels of fetal hemoglobin. Both SaCas9 and AsCas12a exhibited reduced off-target and unintended on-target editing relative to SpCas9, suggesting improved safety profiles. We also assessed the persistence of large deletions generated during BCL11A enhancer editing after 20 weeks of engraftment in NBSGW mice. Together, these findings establish SaCas9 and AsCas12a as promising alternatives to SpCas9 for therapeutic gene editing applications.