Hiramoto Takafumi, Kashiwakura Yuji, Hayakawa Morisada, Baatartsogt Nemekhbayar, Kamoshita Nobuhiko, Abe Tomoyuki, Inaba Hiroshi, Nishimasu Hiroshi, Uosaki Hideki, Hanazono Yutaka, Nureki Osamu, Ohmori Tsukasa
Department of Biochemistry, Jichi Medical University School of Medicine, 3311-1 Yakushiji, Shimotsuke, Tochigi, 329-0498, Japan.
Center for Gene Therapy Research, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke, Tochigi, 329-0498, Japan.
Commun Med (Lond). 2023 Apr 19;3(1):56. doi: 10.1038/s43856-023-00286-w.
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.
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.
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.
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.
通过CRISPR-Cas9进行碱基编辑作为一种校正疾病特异性突变而不引起双链断裂的方法受到关注，从而避免宿主染色体中的大缺失和易位。然而，其对原间隔相邻基序（PAM）的依赖可能会限制其应用。我们旨在使用具有广泛PAM灵活性的改良型Cas9即SpCas9-NG通过碱基编辑来恢复一名重症B型血友病患者的疾病突变。
我们从一名B型血友病患者（c.947T>C；I316T）中生成了诱导多能干细胞（iPSC），并建立了表达患者F9 cDNA的HEK293细胞和基因敲入小鼠。我们分别通过质粒转染和腺相关病毒载体将胞嘧啶碱基编辑器（C>T），包括Cas9的切口酶版本（野生型SpCas9或SpCas9-NG）转导到HEK293细胞和基因敲入小鼠中。
在此我们展示了SpCas9-NG在突变位点附近具有广泛的PAM灵活性。使用SpCas9-NG而非野生型SpCas9的碱基编辑方法成功地将iPSC中的突变C转换为T。基因校正的iPSC在体外分化为肝细胞样细胞，并在肾包膜下移植到免疫缺陷小鼠后表达大量F9 mRNA。此外，SpCas9-NG介导的碱基编辑校正了HEK293细胞和基因敲入小鼠中的突变，从而恢复了凝血因子的产生。
利用SpCas9-NG广泛的PAM灵活性的碱基编辑方法可为包括B型血友病在内的遗传性疾病的治疗提供一种解决方案。
