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PAM-flexible Cas9-mediated base editing of a hemophilia B mutation in induced pluripotent stem cells. | LitMetric

AI Article Synopsis

  • Base editing using CRISPR-Cas9 has potential for correcting genetic mutations, such as in severe hemophilia B, without causing chromosome damage, but traditional methods have limitations due to protospacer adjacent motif (PAM) restrictions.
  • The research involved creating induced pluripotent stem cells and testing a modified Cas9 (SpCas9-NG) that allows for broader PAM flexibility, successfully converting the specific mutation in both cell lines and animal models.
  • The results indicate that SpCas9-NG can effectively restore normal function in hemophilia B by correcting mutations, suggesting a promising avenue for treating various genetic diseases.

Article 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.

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Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC10115777PMC
http://dx.doi.org/10.1038/s43856-023-00286-wDOI Listing

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