A chemical approach facilitates CRISPRa-only human iPSC generation and minimizes the number of targeted loci required

<b>Aim:</b> We explored the generation of human induced pluripotent stem cells (iPSCs) solely through the transcriptional activation of endogenous genes by CRISPR activation (CRISPRa). <b>Methods:</b> Minimal number of human-specific guide RNAs targeting a limited set of loci were used with a unique cocktail of small molecules (CRISPRa-SM). <b>Results:</b> iPSC clones were efficiently generated by CRISPRa-SM, expressed general and naive iPSC markers and clustered with high-quality iPSCs generated using conventional reprogramming methods. iPSCs showed genomic stability and robust pluripotent potential as assessed by <i>in vitro</i> and <i>in vivo</i>. <b>Conclusion:</b> CRISPRa-SM-generated human iPSCs by direct and multiplexed loci activation facilitating a unique and potentially safer cellular reprogramming process to aid potential applications in cellular therapy and regenerative medicine. Combined chemical and CRISPRa-mediated approach leads to efficient generation of human iPSCs. Human iPSCs are conventionally derived through transient expression of exogenous reprogramming factors. We previously generated iPSCs from murine fibroblasts using SunTag-based CRISPRa method. With a minimal number of human-specific guide RNAs targeting a limited set of loci, combining CRISPRa with a unique cocktail of small molecules (CRISPRa-SM), bona fide iPSC colonies were successfully derived from human fibroblasts. iPSC clones expressed general and naive iPSC markers and clustered with high-quality iPSCs generated using conventional reprogramming methods. The CRISPRa-SM iPSCs displayed genomic stability and robust pluripotent potential as assessed by <i>in vitro</i> tri-lineage differentiation and <i>in vivo</i> teratoma assays. The CRISPRa-SM system generated human iPSCs by direct and multiplexed loci activation facilitating a unique and potentially safer cellular reprogramming process to aid potential applications in cellular therapy and regenerative medicine.

<b>目的：</b>本研究探索了仅通过CRISPR激活技术（CRISPRa）对内生基因进行转录激活以生成人类诱导多能干细胞（iPSCs）的可行性。<b>方法：</b>使用针对有限位点的最少数量的人类特异性向导RNA，并结合一种独特的小分子混合物（CRISPRa-SM）。<b>结果：</b>CRISPRa-SM可高效生成iPSC克隆，这些克隆表达通用及原始iPSC标志物，并与传统重编程方法生成的高质量iPSC聚为一类。经<i>体外</i>和<i>体内</i>评估，该iPSC表现出基因组稳定性及强大的多能性潜力。<b>结论：</b>CRISPRa-SM系统通过直接且多靶点的位点激活生成人类iPSC，促成了一种独特且潜在更安全的细胞重编程过程，有助于其在细胞治疗和再生医学中的潜在应用。化学与CRISPRa介导的联合方法可高效生成人类iPSC。人类iPSC传统上通过外源性重编程因子的瞬时表达获得。我们此前已利用基于SunTag的CRISPRa方法从小鼠成纤维细胞生成iPSC。使用针对有限位点的最少数量的人类特异性向导RNA，结合CRISPRa与独特的小分子混合物（CRISPRa-SM），成功从人类成纤维细胞获得了真实的iPSC集落。iPSC克隆表达通用及原始iPSC标志物，并与传统重编程方法生成的高质量iPSC聚为一类。经<i>体外</i>三系分化和<i>体内</i>畸胎瘤实验评估，CRISPRa-SM iPSC表现出基因组稳定性及强大的多能性潜力。CRISPRa-SM系统通过直接且多靶点的位点激活生成人类iPSC，促成了一种独特且潜在更安全的细胞重编程过程，有助于其在细胞治疗和再生医学中的潜在应用。