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Effective restoration of dystrophin expression in iPSC Mdx-derived muscle progenitor cells using the CRISPR/Cas9 system and homology-directed repair technology.
Comput Struct Biotechnol J. 2020; 18:765-773.CS

Abstract

Duchenne muscular dystrophy (DMD) is a progressive myopathic disease caused by mutations in the gene encoding dystrophin protein that eventually leads to the exhaustion of myogenic progenitor cells (MPC). Autologous induced pluripotent stem cells (iPSCs) provide an endless source of MPC, which can potentially replenish the progenitor cell pool, repair muscle damage, and prevent DMD progression. Deletion of mutant exon 23 (ΔEx23) with clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) gene-editing technology can correct dystrophin gene expression in iPSCs. However, successful exon23 deletion and clonal isolation are very inefficient (~3%), and manual selection of each iPSC clone and genotyping to identify ΔEx23 is labor-intensive. To overcome these obstacles, we added a homology-directed repair (HDR) donor vector, which carries floxed fluorescent protein and antibiotic selection genes, thus allowing us to identify ΔEx23 iPSC with donor selective gene integration. Our results indicate that the HDR-mediated targeted integration enables ΔEx23 iPSC identification; the HDR donor vector increased the recognition efficiency of clonal isolation (>90% as confirmed by Sanger sequencing). After removal of the inserted genes by Cre-mediated recombination followed by doxycycline (Dox)-induced MyoD induction, ΔEx23 iPSC differentiated into MPC with restored dystrophin expression in vitro. Importantly, transplanted ΔEx23 iPSC-MPC express dystrophin in the muscles of a mouse model of DMD (Mdx mice). In conclusion, the use of HDR donor vector increased the efficiency of ΔEx23 gene correction by CRISPR/Cas9, and facilitate the identification of successfully edited iPSC clones for cell therapy of DMD.

Authors+Show Affiliations

Medical College of Georgia, Augusta University, Augusta, GA, USA.Medical College of Georgia, Augusta University, Augusta, GA, USA.Medical College of Georgia, Augusta University, Augusta, GA, USA.Medical College of Georgia, Augusta University, Augusta, GA, USA.Medical College of Georgia, Augusta University, Augusta, GA, USA.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

32280431

Citation

Jin, Yue, et al. "Effective Restoration of Dystrophin Expression in iPSC Mdx-derived Muscle Progenitor Cells Using the CRISPR/Cas9 System and Homology-directed Repair Technology." Computational and Structural Biotechnology Journal, vol. 18, 2020, pp. 765-773.
Jin Y, Shen Y, Su X, et al. Effective restoration of dystrophin expression in iPSC Mdx-derived muscle progenitor cells using the CRISPR/Cas9 system and homology-directed repair technology. Computational and structural biotechnology journal. 2020;18:765-773.
Jin, Y., Shen, Y., Su, X., Weintraub, N. L., & Tang, Y. (2020). Effective restoration of dystrophin expression in iPSC Mdx-derived muscle progenitor cells using the CRISPR/Cas9 system and homology-directed repair technology. Computational and Structural Biotechnology Journal, 18, 765-773. https://doi.org/10.1016/j.csbj.2020.03.012
Jin Y, et al. Effective Restoration of Dystrophin Expression in iPSC Mdx-derived Muscle Progenitor Cells Using the CRISPR/Cas9 System and Homology-directed Repair Technology. Computational and structural biotechnology journal. 2020;18:765-773. PubMed PMID: 32280431.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Effective restoration of dystrophin expression in iPSC Mdx-derived muscle progenitor cells using the CRISPR/Cas9 system and homology-directed repair technology. AU - Jin,Yue, AU - Shen,Yan, AU - Su,Xuan, AU - Weintraub,Neal L, AU - Tang,Yaoliang, Y1 - 2020/03/25/ PY - 2019/10/01/received PY - 2020/02/14/revised PY - 2020/03/17/accepted PY - 2020/4/14/entrez PY - 2020/4/14/pubmed PY - 2020/4/14/medline KW - Duchenne muscular dystrophy (DMD) KW - Homology directed repair (HDR) KW - Induced pluripotent stem cells KW - interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) KW - myogenic progenitor cells (MPC) SP - 765 EP - 773 JF - Computational and structural biotechnology journal VL - 18 N2 - Duchenne muscular dystrophy (DMD) is a progressive myopathic disease caused by mutations in the gene encoding dystrophin protein that eventually leads to the exhaustion of myogenic progenitor cells (MPC). Autologous induced pluripotent stem cells (iPSCs) provide an endless source of MPC, which can potentially replenish the progenitor cell pool, repair muscle damage, and prevent DMD progression. Deletion of mutant exon 23 (ΔEx23) with clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) gene-editing technology can correct dystrophin gene expression in iPSCs. However, successful exon23 deletion and clonal isolation are very inefficient (~3%), and manual selection of each iPSC clone and genotyping to identify ΔEx23 is labor-intensive. To overcome these obstacles, we added a homology-directed repair (HDR) donor vector, which carries floxed fluorescent protein and antibiotic selection genes, thus allowing us to identify ΔEx23 iPSC with donor selective gene integration. Our results indicate that the HDR-mediated targeted integration enables ΔEx23 iPSC identification; the HDR donor vector increased the recognition efficiency of clonal isolation (>90% as confirmed by Sanger sequencing). After removal of the inserted genes by Cre-mediated recombination followed by doxycycline (Dox)-induced MyoD induction, ΔEx23 iPSC differentiated into MPC with restored dystrophin expression in vitro. Importantly, transplanted ΔEx23 iPSC-MPC express dystrophin in the muscles of a mouse model of DMD (Mdx mice). In conclusion, the use of HDR donor vector increased the efficiency of ΔEx23 gene correction by CRISPR/Cas9, and facilitate the identification of successfully edited iPSC clones for cell therapy of DMD. SN - 2001-0370 UR - https://www.unboundmedicine.com/medline/citation/32280431/Effective_restoration_of_dystrophin_expression_in_iPSC_Mdx_derived_muscle_progenitor_cells_using_the_CRISPR/Cas9_system_and_homology_directed_repair_technology_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S2001-0370(19)30414-3 DB - PRIME DP - Unbound Medicine ER -
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