A Quick On/Off Switch for Genes

A rapid genome editing technique that can be switched on and off by a simple chemical cue holds great promise for stem cell studies

stem cell
Meng How Tan and colleagues have built on the CRISPR-Cas9 system (above) by fusing the enzyme Cas9 with an estrogen receptor which enables it to be switched on and off using a simple chemical. © MOLEKUUL/Science Photo Library/Getty

The ability to insert, replace or delete DNA in the genomes of living organisms provides incredible scope for developing synthetic organisms and new medical therapies. To maximize its usefulness, any genome editing technique should be both easy to implement and fast to act, as well as reversible and repeatable, enabling it to be switched on and off as required.

Now, Meng How Tan and co-workers at the A*STAR Genome Institute of Singapore, Nanyang Technological University and Ngee Ann Polytechnic have developed a new genome editing system, whose activity can be rapidly switched on and off within human cells by applying a simple chemical trigger1.

The team’s work builds on the CRISPR-Cas9 system — arguably the fastest, cheapest and most accurate gene editing system in use today. In this system, the enzyme Cas9 acts as molecular scissors to cut DNA at a specific location. The cell then recognizes that its DNA is damaged and tries to repair it. By interfering with this repair mechanism, scientists can introduce new genetic sequences.

“My lab is interested in identifying and studying genes that are essential at different stages of stem cell differentiation,” says Tan. “In these developmental studies, the precise timing of gene perturbation is important because many events happen really quickly. Hence, we need a genome editing system that we can trigger at precise times.”

When Tan and co-workers began their project in 2014, there were only two options available to them: an inducible promoter, which was too slow, or an optogenetic control system, which triggers genome editing by light but requires a complicated and expensive light setup. Instead, they speculated that by fusing an estrogen receptor called ERT2 to Cas9, they could change the enzyme’s activity so that it required the presence of a chemical called 4-hydroxytamoxifen (4-HT).

The resulting system, which the researchers named iCas, works extremely quickly and efficiently, and can be switched on and off repeatedly. The combined ERT2-Cas9 complex is normally unable to access the nucleus and edit DNA, but when 4-HT is present, it binds to the complex and allows rapid movement of Cas9 into the nucleus (see image). Then, when 4-HT is removed, the ERT2-Cas9 complex automatically moves back outside the nucleus so the Cas9 can’t cut DNA any more.

“As well as using our system to study stem cell differentiation more precisely, we can use it to study the reprogramming, or transdifferentiation, of one cell type into another,” says Tan.