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Naively Does It! – New iPSC Generation Strategy Allows Better CRISPR Gene Editing



Review of “Naïve Induced Pluripotent Stem Cells Generated From β-Thalassemia Fibroblasts Allow Efficient Gene Correction with CRISPR/Cas9” from Stem Cells Translational Medicine by Stuart P. Atkinson

The correction of disease-associated mutations in human induced pluripotent stem cells (hiPSCs) generated from patient samples holds great promise as a means to produce functional replacement cells to treat diseases/disorders. Multiple strategies to correct mutations exist, although the most promising of these is the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 nuclease (CRISPR/Cas9) system. Although this system is relatively safe, quick, and easy, there is always room for improvement!

Now researchers from the laboratories of Shaorong Gao and Yixuan Wang (Tongji University, Shanghai, People’s Republic of China) have attempted, for the first time, to directly generate and correct hiPSCs from β-thalassemia patient-derived fibroblasts in their naïve state to assess whether this represents a better state for gene editing [1]. The normal state of hiPSCs (similar to human embryonic stem cells (hESCs) and mouse epiblast stem cells (mEpiSCs)) is known as the “primed” state and actually exhibits lower self-renewal ability and multi-differentiation capacity as compared to “naïve” iPSCs (similar to mouse ESCs and mouse iPSCs) [2]. 

Their direct strategy took inspiration from a culture-based strategy which can convert human primed cells to naïve cells using a combination of inhibitors and growth factors (5i/L/FA medium) [3]. To this end, the study reprogrammed the mutation-laden fibroblasts with using episomal vectors carrying OCT4, sh-p53, SOX2, KLF4, MYC, LIN28, and NANOG and cultured the emerging colonies in the previously formulated naïve medium.

This produced dome-shaped colonies like mESCs (See Figure) which could be passaged as single cells at high efficiency and expressed elevated levels of genes associated with the naïve pluripotent state (e.g. NANOG, KLF4, REX1, STELLA, KLF2, and TFCP2L1) as compared to the primed state. Furthermore, these hiPSCs had the ability to differentiate into cells representative of the three germ layers, the formed teratomas, and, importantly, they had the ability to form interspecies chimeras with widespread integration of human naive iPSCs (which primed iPSCs could not). Overall, this suggests that the applied strategy efficiently produced hiPSCs in the naïve state.

A great finding you may think, but this study went a little bit further and assessed the correction of the β-thalassemia mutation using CRISPR/Cas9 with a specific single-guide RNA in both naïve and primed hiPSCs. At the single allele level, the naïve hiPSCs displayed a correction efficiency of 57%, almost double that of primed hiPSCs (23%), and one naïve hiPSC colony displayed correction of both alleles. Furthermore, the primed hiPSCs had no detectable off-target events, while the primed hiPSCs colonies had 2 clones with off-target events. Importantly, the hematopoietic differentiation capacity of the iPSCs remained similar in the corrected naive iPSCs, parental naive iPSCs, and the primed iPSC lines.

Naively does it! This exciting study not only describes an effective method for the production of the more useful naïve hiPSCs but also demonstrates that this pluripotent state allows for improved gene editing. While only applied to β-thalassemia here, the possibilities are endless, and this study will hopefully represent an important milestone on the path towards the widespread clinical application of gene corrected hiPSCs.


  1. Yang Y, Zhang X, Yi L, et al. Naive Induced Pluripotent Stem Cells Generated From beta-Thalassemia Fibroblasts Allow Efficient Gene Correction With CRISPR/Cas9. Stem Cells Transl Med 2015;
  2. Nichols J and Smith A Naive and primed pluripotent states. Cell Stem Cell 2009;4:487-492.
  3. Theunissen TW, Powell BE, Wang H, et al. Systematic identification of culture conditions for induction and maintenance of naive human pluripotency. Cell Stem Cell 2014;15:471-487.