Researchers at the University of Bath and Cardiff University say they have invented a biological switch that can turn protein expression on at will. The switch enables control of genome-editing tools that might one day regulate desired genetic changes through entire populations, they add.
The team points out that this new switching method should work for any protein in any species and uses a cheap, nontoxic amino acid as the control switch—the “on” mode requires the presence of a lysine derivative.
Their study (“Switchable Genome Editing via Genetic Code Expansion”) is published in Scientific Reports.
“Multiple applications of genome editing by CRISPR-Cas9 necessitate stringent regulation and Cas9 variants have accordingly been generated whose activity responds to small ligands, temperature or light. However, these approaches are often impracticable, for example in clinical therapeutic genome editing in situ or gene drives in which environmentally-compatible control is paramount. With this in mind, we have developed heritable Cas9-mediated mammalian genome editing that is acutely controlled by the cheap lysine derivative, Lys(Boc) (BOC),” write the investigators.
“Genetic code expansion permitted non-physiological BOC incorporation such that Cas9 (Cas9BOC) was expressed in a full-length, active form in cultured somatic cells only after BOC exposure. Stringently BOC-dependent, heritable editing of transgenic and native genomic loci occurred when Cas9BOC was expressed at the onset of mouse embryonic development from cRNA or Cas9BOC transgenic females. The tightly controlled Cas9 editing system reported here promises to have broad applications and is a first step towards purposed, spatiotemporal gene drive regulation over large geographical ranges.”
In contrast to other reported switches, this method does not use antibiotics, removing risks of selecting for bacterial antibiotic resistance. Neither is it “leaky,” a situation in which proteins are expressed even when in “off” mode, a problem faced by current methods that depend on temperature or light, according to Tony Perry, Ph.D., who led the Bath team from the department of biology and biochemistry.
The research teams in Bath and Cardiff successfully demonstrated the switch in both cultured cells and in early-stage mouse embryos without any detectable target protein expression activity in the absence of BOC.
The method extends a principle called genetic code expansion. To demonstrate the principle, the team used transgenic mice carrying a gene that makes their skin glow green under ultraviolet (UV) light. When the genetic code expansion toolkit adapted for genome editing was present in embryos from the mice, their genomic DNA was efficiently edited to remove the fluorescence gene, but only when BOC was present. In the absence of BOC, no editing occurred. Embryos edited in this way could develop into mice that did not fluoresce, but without BOC no editing had occurred, so these mice remained green.
The switch offers the potential to control a host of biological processes with the addition of BOC, report the scientists. These may include research and practical applications in the laboratory test tube, whole animals, or both. For example, it might be used to address how certain proteins effect aging of cells in culture or animals. Clinically, it may provide a means to switch on proteins to enhance regenerative processes and could offer a new tier of control in gene therapy.
One potential application would be in using the switch in gene-drive technology. Gene drives can use the CRISPR-Cas9 system in a way that ensures all offspring in sexually reproducing species inherit a particular genetic segment, overcoming the 50% chance of inheritance that sexual reproduction might otherwise confer upon it.
Characteristics endowed by a gene drive can spread rapidly through a population regardless of whether or not they are advantageous. For example, trials have been done using gene drives to spread genes in mosquitoes that make females infertile, intending to crash the population of the malaria-spreading insect.
However, multiple challenges must be met before gene-drive usage is likely to be authorized. Once initiated, gene drives are difficult or impossible to control and may work over wider areas than desired, for example, straying across international borders. They may have unintended environmental consequences or give rise to resistance. Regulating gene drives by making Cas9 BOC-switchable promises to ameliorate these problems, says Dr. Perry.
"Our switch is a way of controlling the expression of any protein via genetic code expansion,” he explains. "What sets our work apart is the potential for this as an environmentally friendly switch across large distances, which no previous method really enables. For example, you can imagine controlling gene drive activity in livestock herds by adding or removing BOC from feedstuffs as required.
"Gene editing has enormous potential across biological science, from biomedicine to food security, in insects, plants, and animals."