Non-viral ex vivo genome-editing in mouse bona fide hematopoietic stem cells with CRISPR/Cas9

• Published in: Molecular therapy. Methods & clinical development 2021-03, Vol.20, p.451-462
• Main Authors: Byambaa, Suvd, Uosaki, Hideki, Ohmori, Tsukasa, Hara, Hiromasa, Endo, Hitoshi, Nureki, Osamu, Hanazono, Yutaka
• Format: Article
• Language: English
• Subjects: CRISPR/Cas9; hematopoietic stem cells; homology-independent targeted integration; non-viral genome editing; Original; X-linked severe combined immunodeficiency
• ISSN: 2329-0501; 2329-0501
• DOI: 10.1016/j.omtm.2021.01.001

We conducted two lines of genome-editing experiments of mouse hematopoietic stem cells (HSCs) with the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9). First, to evaluate the genome-editing efficiency in mouse bona fide HSCs, we knocked out integrin alpha 2b (Itga2b) with Cas9 ribonucleoprotein (Cas9/RNP) and performed serial transplantation in mice. The knockout efficiency was estimated at approximately 15%. Second, giving an example of X-linked severe combined immunodeficiency (X-SCID) as a target genetic disease, we showed a proof-of-concept of universal gene correction, allowing rescue of most of X-SCID mutations, in a completely non-viral setting. We inserted partial cDNA of interleukin-2 receptor gamma chain (Il2rg) into intron 1 of Il2rg via non-homologous end-joining (NHEJ) with Cas9/RNP and a homology-independent targeted integration (HITI)-based construct. Repaired HSCs reconstituted T lymphocytes and thymuses in SCID mice. Our results show that a non-viral genome-editing of HSCs with CRISPR/Cas9 will help cure genetic diseases. [Display omitted] The gene-knockout efficiency with CRISPR-Cas9 in mouse true HSCs was about 15%, as assessed by serial transplantation experiments. X-SCID mice were cured phenotypically by the knockin of the cDNA under the natural promoter in the HSCs with CRISPR-Cas9, which was a non-viral, universal method applicable to basically any mutations.