Gene Drive Control Worry Eased by Genetic Neutralizing Elements

Reducing the burden of mosquito-borne infectious diseases is the goal of many researchers. Strategies such as the distribution of mosquito nets and repellents have had some success, while many are innovating new ways to suppress the mosquito population in an effort to quell the spread of disease. A relatively new method has developed CRISPR-Cas9-based tools that control the balance of genetic inheritance. These gene drives possess the inherent capacity to spread progressively throughout target populations, a trait that has some worried about their ability to be controlled once distributed in the wild. Now, the same team that designs these gene drives has designed two self-copying (or active) guide RNA-only genetic elements that act as a mechanism of control.

The work, from the University of California, San Diego (UCSD), is published in the paper titled, “Active Genetic Neutralizing Elements for Halting or Deleting Gene Drives” in Molecular Cell.

Gene drive technology, when moved from the laboratory into the wild, could suppress devastating, mosquito-borne diseases. And, a lthough the newest gene drives have been proven to spread efficiently as designed in laboratory settings, concerns have been raised regarding the safety of releasing such systems into wild populations. Questions have emerged about the predictability and controllability of gene drives and whether, once let loose, they can be recalled in the field if they spread beyond their intended application region.

“One way to mitigate the perceived risks of gene drives is to develop approaches to halt their spread or to delete them if necessary,” said Ethan Bier, PhD, science director for the Tata Institute for Genetics and Society. “There’s been a lot of concern that there are so many unknowns associated with gene drives. Now we have saturated the possibilities, both at the genetic and molecular levels, and developed mitigating elements.”

Bier and his colleagues have developed two new active genetic systems that address such risks by halting or eliminating gene drives in the wild, offering two new solutions based on elements developed in the common fruit fly. The two self-copying (or active) guide RNA-only genetic elements, are called e-CHACRs and ERACRs. These elements, the authors write, “use Cas9 produced in trans by a gene drive either to inactivate the cas9 transgene (e-CHACRs) or to delete and replace the gene drive (ERACRs).”

The first neutralizing system, called e-CHACR (erasing Constructs Hitchhiking on the Autocatalytic Chain Reaction) is designed to halt the spread of a gene drive-by “shooting it with its own gun.” e-CHACRs, which can be inserted at various genomic locations and carry two or more gRNAs, use the CRISPR enzyme Cas9 carried on a gene drive to copy itself, while simultaneously mutating and inactivating the Cas9 gene.

“Without a source of Cas9, it is inherited like any other normal gene,” said Shannon (Xiang-Ru) Xu, research associate in the Bier lab. “However, once an e-CHACR confronts a gene drive, it inactivates the gene drive in its tracks and continues to spread across several generations ‘chasing down’ the drive element until its function is lost from the population.”

The second neutralizing system, ERACR (Element Reversing the Autocatalytic Chain Reaction), is designed to eliminate the gene drive altogether. ERACRs are designed to be inserted at the site of the gene drive, where they use the Cas9 from the gene drive to attack either side of the Cas9, cutting it out. Once the gene drive is deleted, the ERACR copies itself and replaces the gene-drive.

“If the ERACR is also given an edge by carrying a functional copy of a gene that is disrupted by the gene drive, then it races across the finish line, completely eliminating the gene drive with unflinching resolve,” said Bier.

The researchers rigorously tested and analyzed e-CHACRs and ERACRs, as well as the resulting DNA sequences, in meticulous detail at the molecular level. Still, Bier cautions there are unforeseen scenarios that could emerge, and the neutralizing systems should not be used with a false sense of security for field-implemented gene drives.

“Such braking elements should just be developed and kept in reserve in case they are needed since it is not known whether some of the rare exceptional interactions between these elements and the gene drives they are designed to corral might have unintended activities,” Bier said.

According to Emily Bulger, a graduate student at the University of California, San Francisco, gene drives have enormous potential to alleviate suffering, but responsibly deploying them depends on having control mechanisms in place should unforeseen consequences arise. ERACRs and eCHACRs offer ways to stop the gene drive from spreading and, in the case of the ERACR, can potentially revert an engineered DNA sequence to a state much closer to the naturally-occurring sequence.

“Because ERACRs and e-CHACRs do not possess their own source of Cas9, they will only spread as far as the gene drive itself and will not edit the wild type population,” said Bulger. “These technologies are not perfect, but we now have a much more comprehensive understanding of why and how unintended outcomes influence their function and we believe they have the potential to be powerful gene drive control mechanisms should the need arise.”