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CRISPR Activation Strategy Takes Induced Pluripotent Stem Cell Generation to another Level

Review of “Human pluripotent reprogramming with CRISPR activators” from Nature Communications by Stuart P. Atkinson

CRISPR activation employs a catalytically inactive Cas9 protein fused to an effector module to stimulate the expression of genes targeted using a short guide RNA (gRNA). While many groups have sought to apply this system to the reprogramming of human somatic cells into induced pluripotent stem cells (iPSCs) by targeting reprogramming factor genes, no study has yet reported complete pluripotent reprogramming. 

Now, new research led by Jere Weltner, Juha Kere, and Timo Otonkoski (University of Helsinki, Finland) sought to rectify this situation through the application of a new targeting strategy based on their previous study that established the enrichment of an Alu-motif in the promoter of genes that control early embryonic transcriptional networks [1]. Weltner et al. demonstrate that additional CRISPR activation targeting to this motif, called the embryo genome-activation (EEA) motif, improves the activation of reprogramming-associated transcription factor genes and permits the first generation of iPSCs via CRISPR activation [2].  

Here is a low down on how the authors employed CRISPR activation to take iPSC-generation to another level:

  • The reprogramming process electroplated primary human skin fibroblasts with:
    • an episomally replicating simple dCas9 effector module plasmid containing a TP53 short hairpin RNA
    • an EEA-motif targeting gRNA plasmid
    • a plasmid carrying gRNAs for the OCT4, MYC, KLF4, SOX2, and LIN28A reprogramming factors
    • an additional plasmid carrying gRNAs targeting KLF4 and MYC (KM)
  • This addition of the EEA-motif targeting gRNA improved low basal reprogramming efficiency (not sufficient to generate iPSCs) by an order of magnitude and permitted the emergence of iPSC-like colonies
    • Transcriptional analysis suggested that EEA-motif targeting promoted initial stages of the reprogramming process prior to colony formation
  • EEA-motif targeting efficiently activated NANOG and REX1 to boost reprogramming

Overall, this exciting new study demonstrates that CRISPR activation can take iPSC reprogramming to another level and highlights the importance of the EEA-motif to the process. Excitingly, the authors note that their core method can be improved by targeting additional pluripotency genes and regulatory elements and may synergize with transgenic factors, RNA interference, and small molecules to promote more efficient and specific reprogramming.

For more on how CRISPR activation can take iPSC generation to even higher levels, stay tuned to the Stem Cells Portal.


  1. Töhönen V, Katayama S, Vesterlund L, et al., Novel PRD-like homeodomain transcription factors and retrotransposon elements in early human development. Nature Communications 2015;6:8207.
  2. Weltner J, Balboa D, Katayama S, et al., Human pluripotent reprogramming with CRISPR activators. Nature Communications 2018;9:2643.