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.