Development of CRISRP/Cas9-based TP53-knockout pig muscle stem cells for use in the cultured meat industry.

Abstract

Muscle satellite cells (MSCs) are essential for cultured meat production although their restricted lifespan and diminished stemness during prolonged culture pose significant limitations. This study established immortalized porcine MSCs using TP53 gene deletion with the CRISPR/Cas9 technology. Several TP53-knockout (KO) clones were generated, exhibiting indel alterations and monoallelic and biallelic deletions. These clones exhibited markedly prolonged cellular lifespans compared to wild-type cells, overcoming the constraints of senescence. The growth rates and the proliferation marker (ki67) gene expression (passage 20) in the TP53-KO clones were dramatically elevated compared to the WT cells. All TP53-KO clones demonstrated a loss of stemness, proliferation, and muscle differentiation marker gene expression during long-term cell culture except for Desmin expression in the TP53-KO 42 clone. Immortalized TP53-KO clones maintained the ability to express muscle-specific protein markers compared to wild-type cells. Moreover, all clones had non-tumorigenic behavior, except TP53-KO clones 41 and 42, which displayed tumorigenic potential. TP53-KO clones demonstrated enhanced myogenic differentiation efficiency after multiple passages in comparison to wild-type cells. The results highlight the potential of TP53-KO MSCs as a cellular resource for future cultured meat production.

Supplementary information: The online version contains supplementary material available at 10.1007/s13205-025-04225-5.