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Inducible Gene Knockouts in Human Stem Cells: CRISPR Hits the Headlines Again

Review of “Engineering Human Stem Cell Lines with Inducible Gene Knockout using CRISPR/Cas9” from Cell Stem Cell by Stuart P. Atkinson.

Knocking out genes in pluripotent stem cells (PSCs) is a difficult and complex process, and depending on the gene in question, can leave cells unable to propagate or survive. Furthermore, many genes play different roles at different stages of development/differentiation, and therefore, precise temporal control of gene knockout is desirable. In a new study, published in Cell Stem Cell, Yuejun Chen and Su-Chun Zhang (University of Wisconsin, USA) demonstrate a fast and effective strategy to generate clonal inducible gene knockout (iKO) hPSC lines using CRISPR/Cas9-mediated genome editing to incorporating two different site-directed recombination systems [1, 2]. This exciting new technology will allow the study of the roles of single genes or groups of genes through inducible knockouts at any stage of differentiation [3]. 

The authors targeted a number of different genes to encompass various different genetic situations; multiple exons (PAX6, OTX2, and AGO2), large exons (SOX2), expressed in hPSCs ((SOX2, OTX2, and AGO2), and not expressed in hPSCs (PAX6). They found that CRISPR/Cas9-mediated genome editing using a dual-sgRNA targeting strategy was applicable to genes with differing structures. 

This allowed the integration of a donor plasmid containing the targeted  exon surrounded by two short flippase recognition target (FRT) sites to allow recombination directed by the flippase (Flp) recombinase (Flp-FRT recombination). This plasmid also introduces a PGK-puromycin drug resistance cassette which is itself flanked by LoxP sites allowing removal after Cre-recombinase expression. This strategy was further enhanced by the integration of D10A mutant nickase version of Cas9 (Cas9 nickase) which reduced off-target effects of CRISPR/Cas9.

The next stage involved the insertion of an “inducible” enhanced-flippase (Flpe) recombinase expression cassette (Flpe-ERT2) into the adeno-associated virus integration site 1 (AAVS1) viral integration site, a transcriptionally competent validated “safe harbor” for hosting DNA transgenes. Subsequent treatment with 4-hydroxytamoxifen (4-OHT) induces the expression of the flippase to knockout the targeted exon and inhibit gene function. For the introduction of the Flpe-ERT2 cassette, the researchers co-expressed a Cas9-2A-Cre plasmid that enabled co-expression of Cas9, and the integration of the Flpe-ERT2 element, and Cre recombinase, which removed the drug resistance cassette from the initial exon donor plasmid.

This strategy enabled the production of multiple inducible knockout human ESC or iPSC lines, and thorough analysis of off-target effects mediated through sgRNA-guided CRISPR/Cas9 editing found no deleterious occurrences. Encouragingly, 4-OHT treatment mediated the effective depletion of target genes at the PSC stage or in differentiated cells. To study the consequences of gene KO during differentiation, the authors assessed the role of OTX2, a gene required for the proper development of the brain, during early neural development. The implementation of this system at differing stages of development allowed the authors to dissect a role for OTX2 in dorsal-ventral patterning of forebrain neuroepithelia.

This efficient and effective strategy, which the authors describe in fine detail in the hopes that other laboratories can generate their own iKO PSCs, should open the floodgates for a milieu of new studies, new findings, and interesting applications. This includes the dissection of gene function during normal development, as shown for OTX2 in this study, and in the analysis of disease models, taking advantage of disease and patient-specific iPSCs. Furthermore, the study also demonstrated that multiple gene can be effectively targeted using this technique, leaving the door open for some inventive and highly exciting new studies to begin.


  1. Branda CS and Dymecki SM Talking about a revolution: The impact of site-specific recombinases on genetic analyses in mice. Dev Cell 2004;6:7-28.
  2. Capecchi MR Gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century. Nat Rev Genet 2005;6:507-512.
  3. Chen Y, Cao J, Xiong M, et al. Engineering Human Stem Cell Lines with Inducible Gene Knockout using CRISPR/Cas9. Cell Stem Cell 2015;17:233-244.