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

Abstract

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.

Design of Liposome Formulations for CRISPR/Cas9 Enzyme Immobilization: Evaluation of 5-Alpha-Reductase Enzyme Knockout for Androgenic Disorders

“…For Cas9-mediated gene knockout with RNP transfection, it has been shown that the knockout efficiency ranges from 15 to 90% in various cell types . Similarly, in mouse bona fide hematopoietic stem cells (HSCs), the knockout efficiency of integrin alpha 2b (Itga2b) using Cas9/RNP was estimated to be approximately 15% . In human HEK293 cells, the knockout efficiencies achieved with Cas9/gRNA were 10–25%. , Higher gene knockout levels were also obtained depending on the cell type and delivery method.…”

Unveiling Clonal Cell Fate and Differentiation Dynamics: A Hybrid NeuralODE-Gillespie Approach

“…Compared with meta-clone 0 (which is multi-potent but more biased towards the Monocyte lineage), meta-clone 4 is also a multi-potent clone but rather biased towards erythrocytes, mast cells and megakaryocytes. Significant genes include Itga2b, which is specifically up-regulated during megakaryopoiesis 56,57 , and Gzmb, which is highly expressed in mast cells and used to degrade hemidesmosomal proteins 58,59 . Interestingly, Akr1c13 and Hbb-bt were also found to be significantly expressed (Fig.…”