Abstract
SummaryConventional 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.
Highlights
CRISPR activation (CRISPRa) uses a catalytically inactivated form of Cas9 fused with a transactivator domain that enables the activation of transcription from endogenous promoters (Bikard et al, 2013)
We showed that simultaneous targeting of the EEA motif and the promoter of the miR-302/367 cluster enhanced the reprogramming efficiency of fibroblasts and lymphoblastoid cell lines (LCLs) cells and accelerated the kinetics of induced pluripotent stem cell formation
CRISPRa reprogramming of LCL We began by validating the CRISPRa system for the reprogramming of LCL
Summary
CRISPR activation (CRISPRa) uses a catalytically inactivated form of Cas (dCas9) fused with a transactivator domain that enables the activation of transcription from endogenous promoters (Bikard et al, 2013). Advantages of CRISPRa over conventional reprogramming (Fusaki et al, 2009; Okita et al, 2011; Warren et al, 2010) include the direct transcriptional activation from endogenous loci, high multiplexing capability, and the potential to target non-coding regulatory elements. Low reprogramming efficiency hampers the use of CRISPRa. Several small molecules and genes, including pluripotent stem cell micro RNAs (miRNA), can improve reprogramming efficiency (Subramanyam et al, 2011).
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