biostudies-literatureUnknown21Uchida NNational Institute of Diabetes and Digestive and Kidney DiseasesNational Heart, Lung, and Blood InstituteNational Institutes of HealthGene editing with the CRISPR-Cas9 system could revolutionize hematopoietic stem cell (HSC)-targeted gene therapy for hereditary diseases, including sickle cell disease (SCD). Conventional delivery of editing tools by electroporation limits HSC fitness due to its toxicity; therefore, efficient and non-toxic delivery remains crucial. Integrating lentiviral vectors are established for therapeutic gene delivery to engraftable HSCs in gene therapy trials; however, their sustained expression and size limitation preclude their use for CRISPR-Cas9 delivery. Here, we developed a Cas9 protein delivery non-integrating lentiviral system encoding guide RNA and donor DNA, allowing for transient endonuclease function and inclusion of all editing tools in a single vector (all-in-one). We demonstrated efficient one-time correction of the SCD mutation in the endogenous βs-globin gene up to 42% at the protein level (p < 0.01) with the Cas9 protein delivery non-integrating lentiviral all-in-one system without electroporation. Our findings improve prospects for efficient and safe genome editing.Molecular therapy. Methods & clinical development121-132https://www.ebi.ac.uk/biostudies/studies/S-EPMC8005818biostudies-literatureCas9 protein delivery non-integrating lentiviral vectors for gene correction in sickle cell disease.PMC8005818Yapundich MUchida NNassehi THinds MDemirci SDrysdale CMDiNicola JHaro-Mora JJShibata YGamer JGudmundsdottir BTisdale JFfalseCas9 protein delivery non-integrating lentiviral vectors for gene correction in sickle cell disease.Gene editing with the CRISPR-Cas9 system could revolutionize hematopoietic stem cell (HSC)-targeted gene therapy for hereditary diseases, including sickle cell disease (SCD). Conventional delivery of editing tools by electroporation limits HSC fitness due to its toxicity; therefore, efficient and non-toxic delivery remains crucial. Integrating lentiviral vectors are established for therapeutic gene delivery to engraftable HSCs in gene therapy trials; however, their sustained expression and size limitation preclude their use for CRISPR-Cas9 delivery. Here, we developed a Cas9 protein delivery non-integrating lentiviral system encoding guide RNA and donor DNA, allowing for transient endonuclease function and inclusion of all editing tools in a single vector (all-in-one). We demonstrated efficient one-time correction of the SCD mutation in the endogenous βs-globin gene up to 42% at the protein level (p < 0.01) with the Cas9 protein delivery non-integrating lentiviral all-in-one system without electroporation. Our findings improve prospects for efficient and safe genome editing.2021-01-01T00:00:00Z2021 Jun2024-11-09T06:57:44.421Z2022-02-09T11:07:35.641ZS-EPMC80058183381664510.1016/j.omtm.2021.02.022