Takafumi Hiramoto, Yuji Kashiwakura, Morisada Hayakawa, Nemekhbayar Baatartsogt, Nobuhiko Kamoshita, Tomoyuki Abe, Hiroshi Inaba, Hiroshi Nishimasu, Hideki Uosaki, Yutaka Hanazono, Osamu Nureki, Tsukasa Ohmori
Communications Medicine 3(1) 2023年4月19日
Abstract
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.
