Non-viral Genome-editing in Mouse Bona Fide Hematopoietic Stem Cells with CRISPR/Cas9

Overview

Journal Mol Ther Methods Clin Dev

Publisher Cell Press

Date 2021 Feb 22

PMID 33614821

Citations 4

Authors

Suvd Byambaa

Hideki Uosaki

Tsukasa Ohmori

Hiromasa Hara

Hitoshi Endo

Osamu Nureki

Yutaka Hanazono

Affiliations

Soon will be listed here.

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 () 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 () into intron 1 of 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.

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