CRISPR/Cas9 Genome Editing in LGMD2A/R1 Patient-Derived Induced Pluripotent Stem and Skeletal Muscle Progenitor Cells

Lampros Mavrommatis; Abdul Zaben; Urs Kindler; Marie-Cécile Kienitz; Julienne Dietz; Hyun-Woo Jeong; Pierre Böhme; Beate Brand-Saberi; Matthias Vorgerd; Holm Zaehres

doi:10.1155/2023/9246825

CRISPR/Cas9 Genome Editing in LGMD2A/R1 Patient-Derived Induced Pluripotent Stem and Skeletal Muscle Progenitor Cells

Stem Cells Int. 2023 Nov 9:2023:9246825. doi: 10.1155/2023/9246825. eCollection 2023.

Authors

Lampros Mavrommatis^{1

2

3}, Abdul Zaben^{1

2}, Urs Kindler¹, Marie-Cécile Kienitz⁴, Julienne Dietz^{2

5}, Hyun-Woo Jeong⁶, Pierre Böhme⁷, Beate Brand-Saberi¹, Matthias Vorgerd², Holm Zaehres^{1

3}

Affiliations

¹ Ruhr University Bochum, Medical Faculty, Institute of Anatomy, Department of Anatomy and Molecular Embryology, 44801 Bochum, Germany.
² Ruhr University Bochum, Medical Faculty, Department of Neurology with Heimer Institute for Muscle Research, University Hospital Bergmannsheil, 44789 Bochum, Germany.
³ Max Planck Institute for Molecular Biomedicine, Department of Cell and Developmental Biology, 48149 Münster, Germany.
⁴ Ruhr University Bochum, Medical Faculty, Department of Cellular Physiology, 44801 Bochum, Germany.
⁵ Witten/Herdecke University, Institute of Virology and Microbiology, Department of Human Medicine, Faculty of Health, 58453 Witten, Germany.
⁶ Max Planck Institute for Molecular Biomedicine, Sequencing Core Facility, 48149 Münster, Germany.
⁷ Ruhr University Bochum, Department of Psychiatry, Psychotherapy and Preventive Medicine, LWL University Hospital Bochum, 44791 Bochum, Germany.

Abstract

Large numbers of Calpain 3 (CAPN3) mutations cause recessive forms of limb-girdle muscular dystrophy (LGMD2A/LGMDR1) with selective atrophy of the proximal limb muscles. We have generated induced pluripotent stem cells (iPSC) from a patient with two mutations in exon 3 and exon 4 at the calpain 3 locus (W130C, 550delA). Two different strategies to rescue these mutations are devised: (i) on the level of LGMD2A-iPSC, we combined CRISPR/Cas9 genome targeting with a FACS and Tet transactivator-based biallelic selection strategy, which resulted in a new functional chimeric exon 3-4 without the two CAPN3 mutations. (ii) On the level of LGMD2A-iPSC-derived CD82+/Pax7+ myogenic progenitor cells, we demonstrate CRISPR/Cas9 mediated rescue of the highly prevalent exon 4 CAPN3 mutation. The first strategy specifically provides isogenic LGMD2A corrected iPSC for disease modelling, and the second strategy can be further elaborated for potential translational approaches.