Background: Base editing via CRISPR-Cas9 has garnered attention as a method for correcting disease-specific mutations without causing double-strand breaks, thereby avoiding large deletions and translocations in the host chromosome. However, its reliance on the protospacer adjacent motif (PAM) can limit its use. We aimed to restore a disease mutation in a patient with severe hemophilia B using base editing with SpCas9-NG, a modified Cas9 with the board PAM flexibility.
Methods: We generated induced pluripotent stem cells (iPSCs) from a patient with hemophilia B (c.947T>C; I316T) and established HEK293 cells and knock-in mice expressing the patient's F9 cDNA. We transduced the cytidine base editor (C>T), including the nickase version of Cas9 (wild-type SpCas9 or SpCas9-NG), into the HEK293 cells and knock-in mice through plasmid transfection and an adeno-associated virus vector, respectively.
Results: Here we demonstrate the broad PAM flexibility of SpCas9-NG near the mutation site. The base-editing approach using SpCas9-NG but not wild-type SpCas9 successfully converts C to T at the mutation in the iPSCs. Gene-corrected iPSCs differentiate into hepatocyte-like cells in vitro and express substantial levels of F9 mRNA after subrenal capsule transplantation into immunodeficient mice. Additionally, SpCas9-NG-mediated base editing corrects the mutation in both HEK293 cells and knock-in mice, thereby restoring the production of the coagulation factor.
Conclusion: A base-editing approach utilizing the broad PAM flexibility of SpCas9-NG can provide a solution for the treatment of genetic diseases, including hemophilia B.
In patients with hemophilia B, the blood does not clot properly, leading to excessive bruising and bleeding. Hemophilia B is caused by an error in a gene called coagulation factor IX (F9). To treat patients with hemophilia B, we might be able to use a technology called CRISPR-Cas9 to edit and correct this genetic error, restoring factor IX function and improving clotting. Here, we test a specific CRISPR-Cas9 approach in cells and animals. We show that we are able to correct the genetic error in F9 in cells isolated from a patient with severe hemophilia B. We also show that we can fix the error in mice and that this increases levels of factor IX in the blood of the mice. With further testing, this gene-editing approach may be a viable therapy for patients with hemophilia B or similar genetic disorders.
© 2023. The Author(s).