Researchers Make Knockout Stem Cell Lines in One Step

Combining gene editing and stem-cell induction improves efficiency of functional genetic analyses.

By | December 1, 2017

Plasmids encoding reprogramming factors and plasmids encoding gene-editing machinery are transfected together into fibroblast cells. Approximately three weeks later, induced pluripotent stem cell (iPSC) colonies grown from single cells are apparent. These clones can be individually picked from the dish for further isolated growth and study.
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© GEORGE RETSECK

In theory, mutating a gene of interest inside stem cells enables researchers to analyze the effects of that mutation on the development of particular cell types. In the laboratory of Jack Parent at the University of Michigan Medical School, for example, postdoctoral researcher Andrew Tidball is using such an approach to investigate how gene mutations associated with epileptic encephalopathy affect brain cell development. But while trying to introduce the specific mutations into human induced pluripotent stem cells (iPSCs), he ran into difficulties.

A major problem, Tidball says, is that after transfecting iPSCs with gene-editing plasmids, individual cells need to be isolated, but “stem cells don’t like to be [alone]. They die unless you add some components to help them along.” Even then, he adds, “a very low percentage survive.” Furthermore, says Parent, “not all genes are amenable to gene editing [in] iPSCs.”

Tidball realized that instead of first transfecting cells with plasmids containing reprogramming genes and then later adding plasmids with CRISPR-Cas9 components, he could combine the two steps into one. Because iPSC induction already involves the production of single cell–derived colonies, he could, in one fell swoop, create gene-edited stem cell lines.

Tidball, Parent, and colleagues have now used the technique to generate multiple cell lines containing epilepsy-associated mutations, and have found that not only is the combination strategy more time-efficient and reproducible than the sequential approach, it is also more successful: more clones carry the intended mutations. The team’s investigations suggest that this improvement may be due to increased chromatin accessibility at the time of reprogramming, allowing the gene-editing machinery to reach its target DNA more easily.

The technique also preserves genome integrity, says the University of Wisconsin’s Anita Bhattacharyya, who was not involved in the work. “We know that these pluripotent stem cells, over time, tend to acquire chromosomal abnormalities,” she says, so doing both processes at once reduces the likelihood of aberrations. “For those people who work in disease modeling of single-gene mutations, this is a really important move forward.” (Stem Cell Rep, 9:725-31, 2017) 

GENE EDITING OF iPSCs


HOW IT WORKS

SUCCESS AND REPRODUCIBILITY

CONTROLS
 
Subsequent to iPSC creation An established iPSC line is transfected with a gene-editing plasmid and isolated into single-cell clones for sequence analysis. Between less than 1 percent and 20 percent of the clones that survive single-cell isolation (very few) carry desired mutations. The nontransfected iPSC line is often used as an isogenic control, but it will not have been subjected to the exact same culturing conditions as the mutant line.
Simultaneous with iPSC creation Differentiated cells are transfected with both
reprogramming and gene-editing plasmids.
Plating of the transfected cells results in single iPSC clones which are picked for sequence analysis.
Approximately 45 percent of clones carry at least one mutated allele. Homozygous mutants, heterozygous mutants, and wild-type isogenic controls are all made during the same experiment.

 

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