Engineered CRISPR/Cas9-based transcriptional activators can potently and specifically activate endogenous fate-determining genes to direct differentiation of pluripotent stem cells. Here, we demonstrate that endogenous activation of the PAX7 transcription factor results in stable epigenetic remodeling and differentiates human pluripotent stem cells into skeletal myogenic progenitor cells. Compared with exogenous overexpression of PAX7 cDNA, we find that endogenous activation results in the generation of more proliferative myogenic progenitors that can maintain PAX7 expression over multiple passages in serum-free conditions while preserving the capacity for terminal myogenic differentiation. Transplantation of human myogenic progenitors derived from endogenous activation of PAX7 into immunodeficient mice resulted in a greater number of human dystrophin⁺ myofibers compared with exogenous PAX7 overexpression. RNA-sequencing analysis also revealed transcriptome-wide differences between myogenic progenitors generated via CRISPR-based endogenous activation of PAX7 and exogenous PAX7 cDNA overexpression. These studies demonstrate the utility of CRISPR/Cas9-based transcriptional activators for controlling cell-fate decisions.

基于工程化 CRISPR/Cas9 的转录激活剂可以有效且特异性地激活内源性命运决定基因，以指导多能干细胞的分化。在这里，我们证明PAX7转录因子的内源性激活导致稳定的表观遗传重塑，并将人类多能干细胞分化为骨骼肌祖细胞。与PAX7 cDNA 的外源性过表达相比，我们发现内源性激活导致产生更多增殖性肌源性祖细胞，其可以在无血清条件下多次传代维持 PAX7 表达，同时保留终末肌源性分化的能力。源自内源性激活的人肌源性祖细胞的移植与外源性PAX7过表达相比，将PAX7注入免疫缺陷小鼠导致更多的人肌营养不良蛋白⁺肌纤维。RNA 测序分析还揭示了通过基于 CRISPR 的PAX7内源性激活和外源性PAX7 cDNA 过表达产生的肌源性祖细胞之间在转录组范围内的差异。这些研究证明了基于 CRISPR/Cas9 的转录激活剂在控制细胞命运决定方面的效用。