Editors' ChoiceGENETIC ENGINEERING

Shining a light on stem cell biology

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Science Translational Medicine  05 Aug 2015:
Vol. 7, Issue 299, pp. 299ec133
DOI: 10.1126/scitranslmed.aac9320

Human pluripotent stem cells (hPSCs) and gene modification have lit up the world of human disease and development. However, immediately flipping the gene knockout switch has kept later-onset diseases in the dark. Chen et al. have effectively put a timer on that light switch, presenting a process for developing clonal hPSC lines with an inducible knockout system at any differentiation stage. Genes and mutations affecting natural development and a host of pathologies can now take their turn under the lights.

Chen et al. developed multiple strategies using the CRISPR/Cas9 gene targeting technique to bypass the typical structural and temporal difficulties associated with modeling late stage diseases and development. The authors found a dual-sg RNA targeting strategy that combines CRISPR/Cas9–mediated genome editing with the Flp/FRT and Cre/LoxP system essential to overcoming individual sgRNA's low targeting efficiency, lack of homozygous clones, or loss of a recombination recognition insertion sites. The two sgRNAs generated the proper clonal lines with a precise biallelic knockin of FRT and worked for a variety of gene structures. The authors then turned typical sequential targeting into a single-step process to generate homozygous hPSC lines that allow flanking of the targeted gene's exon. They took full structural and temporal control of targeted cells through the addition of an activity-controllable enhanced-flippase recombinase cassette and the simultaneous deletion of the drug-resistance expression cassette. These methods established predictable and clonal inducible knockout hPSC lines for the study of exons large and small, expressed and not, individually and in combination, throughout every stage of differentiation.

In just 10 to 12 weeks, researchers can now engineer a cell line in which gene knockout requires nothing more than a simple inducing chemical. Continuing efforts to maintain high-quality cell lines is imperative for these materials to be shared broadly to the biomedical community as qualified reagents. This proof-of-concept study in several hPSC systems is a great start to using these cells as model systems and can accelerate our understanding of human tissue development and somatic mutational changes that give rise to pathology. Chen et al. have shown that a single bulb on a timer can provide more visibility than a spotlight.

Y. Chen et al., Engineering human stem cell lines with inducible gene knockout using CRISPR/Cas9. Cell Stem Cell 10.1016/j.stem.2015.06.001 (2015). [Abstract]

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