Controlling CRISPR and Gene Therapy with an Amino Acid Switch

Gene editing and gene therapy have a range of potential applications, ranging from the editing of pathogenic mutations to the treatment of spinal cord lesions. But the ability to control these treatments is a major concern because they can stay on after they've had the desired effect, causing unwanted side effects. Researchers from the University of Bath and Cardiff University have created a switch that controls the expression of proteins in cultured cells and mouse embryos and could one day be used in therapies human genes, according to the researchers.

The team tested the switch, an amino acid called BOC, in mice that had been genetically engineered to become green under ultraviolet light. They used a method called expansion of the genetic code to create mouse embryos whose fluorescence gene had been published – but only in the presence of BOC. The published embryos that were exposed to BOC could grow into mice that did not shine, but those that were 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 report. Most of the switch's most immediate applications are related to the environment – they could be used to reduce the mosquito population by propagating a gene that makes them infertile, they said. But it could also be used to control the Cas9 proteins used in editing the CRISPR-Cas9 gene. The study is published in Scientific Reports.

The addition of surrogates to gene therapies is not a new idea, but current approaches have problems that need to be solved before they can be used in humans. Researchers at King's College, for example, are working on the treatment of spinal cord injury using gene therapy with an "off" switch based on antibiotics. The treatment triggers the production of an enzyme that breaks down scar tissue, allowing neurons to regenerate. The antibiotic doxycyline is administered after the treatment to trigger the switch "off".

While treatment restores the function of the hand to rats that had suffered spinal cord injury, a small portion of the targeted gene remained active even after stopping the therapy. The King's team is working to completely shut down the gene and proceed to trials on larger animals.

With regard to gene editing, several approaches are being developed to address the possibility of an uncontrolled release. A Salk Institute team designed an epigenetic "CRISPR-Cas9" tool that modifies DNA without cutting it as does traditional CRISPR, breaking down DNA and making it vulnerable to out-of-target mutations . The system is based on dead, or inactivated, Cas9. And the Broad Institute uses a CRISPR-based system dubbed REPAIR to edit RNA and cause reversible changes in the DNA. CRISPR pioneer Feng Zhang and his colleague David Liu launched their start-up, 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. Scientists at the Institute of Bioorganic Chemistry in Poland used a variant of Cas9 that cuts a strand of DNA rather than both strands.

The UK team sees the potential of BOC in several scientific and clinical contexts. In addition to controlling gene therapy, the switch could be used to turn on and turn off proteins in cells to study aging, or to improve regenerative medicine. Researchers now plan to "smooth out wrinkles" in the system before testing it in larger applications, they said in a statement.