CRISPR activation enables high-fidelity reprogramming into human pluripotent stem cells

Conventional reprogramming methods rely on the ectopic expression of transcription factors to reprogram somatic cells into induced pluripotent stem cells (iPSCs). The forced expression of transcription factors may lead to off-target gene activation and heterogeneous reprogramming, resulting in the emergence of alternative cell types and aberrant iPSCs. Activation of endogenous pluripotency factors by CRISPR activation (CRISPRa) can reduce this heterogeneity. Here, we describe a high-efficiency reprogramming of human somatic cells into iPSCs using optimized CRISPRa. Efficient reprogramming was dependent on the additional targeting of the embryo genome activation-enriched Alu-motif and the miR-302/367 locus. Single-cell transcriptome analysis revealed that the optimized CRISPRa reprogrammed cells more directly and specifically into the pluripotent state when compared to the conventional reprogramming method. These findings support the use of CRISPRa for high-quality pluripotent reprogramming of human cells. [Display omitted] •Efficient CRISPRa reprogramming with additional EEA and miR-302/367 targeting•Day 15 scRNA-seq reveals reprogramming progression•CRISPRa cells are reprogrammed with higher fidelity than transgenic cells Forced expression of transcription factors can lead to off-target gene activation and heterogeneous reprogramming, resulting in aberrant iPSCs. Activation of endogenous pluripotency factors by CRISPRa reduces this heterogeneity. Efficient reprogramming was dependent on the additional targeting of the EEA motif and the miR-302/367 locus, with single-cell analysis revealing that these cells reprogrammed more specifically compared to the conventional reprogramming method.