Controlling CRISPR and gene therapy with an amino acid switch

brown mouse
The switch, an amino acid called BOC, is cheap and nontoxic and "should work in any protein in any species." (Alexandra/München)

Gene editing and gene therapy have a range of potential applications, from editing out disease-causing mutations to treating spinal cord injuries. But the ability to control these treatments is a key concern, as they may stay switched on after they have had their intended effect, causing unwanted side effects. Scientists from the University of Bath and Cardiff University have created a switch that controls protein expression in cultured cells and mouse embryos and could one day be used in human gene therapies, the researchers believe. 

The team tested the switch, an amino acid called BOC, in mice that had been genetically engineered to glow green under ultraviolet light. They used a method called genetic code expansion to create mouse embryos whose fluorescence gene had been edited out—but only in the presence of BOC. Edited embryos that were exposed to BOC were able to develop into mice that did not glow, but those not exposed to BOC remained green. 

BOC is cheap and nontoxic and "should work in any protein in any species," the researchers said in a statement. Many of the switch's most immediate applications are environment-related—it could be used to keep the mosquito population down by propagating a gene that makes them infertile, they said. But it could also be used to control Cas9 proteins used in CRISPR-Cas9 gene editing. The study is published in Scientific Reports. 

Adding switches to gene therapies is not a new idea, but current approaches have kinks that need to be addressed before they can be used in humans. Researchers from King's College, for example, are working to treat spinal cord injuries using a gene therapy with an antibiotic-based "off" switch. The treatment triggers production of an enzyme that breaks down scar tissue, allowing neurons to regenerate. The antibiotic doxycyline is delivered after treatment to trigger the "off" switch. 

While the treatment restored some hand function to rats that had suffered a spinal cord injury, a small portion of the targeted gene remained active even after the therapy had been switched off. The King's team is working to shut off the gene completely and move into trials in larger animals. 

As for gene editing, several approaches are in development to address the possibility of runaway editing. A team from the Salk Institute devised an "epigenetic CRISPR-Cas9" tool that edits DNA without cutting it as traditional CRISPR does, breaking the DNA and making it vulnerable to off-target mutations. The system is based on dead, or inactivated, Cas9. And the Broad Institute is using a CRISPR-based system dubbed REPAIR to edit RNA and bring about reversible changes in DNA. CRISPR pioneer Feng Zhang and colleague David Liu launched their startup, Beam Therapeutics, in May, which will use REPAIR and other technologies to edit nucleotide bases and correct point mutations.

Others are also focusing on enzymes to make CRISPR safer. Scientists at the Institute of Bioorganic Chemistry in Poland used a variant of Cas9 that cuts one strand of DNA rather than both strands.

The U.K. team sees the potential for BOC in several scientific and clinical settings. In addition to controlling gene therapy, the switch could be used to switch on and off proteins in cells to study aging, or to enhance regenerative medicine. The researchers now plan to "iron out wrinkles" in the system before testing it in broader applications, they said in a statement.