Active Genetic Neutralizing Elements for Halting or Deleting Gene Drives

In this study, genetic elements with which gene drives in the genome of fruit flies can either be switched off or completely removed are presented. These mechanisms are based on the transmission of specific guide RNAs (gRNAs, signposts for the CRISPR/Cas9 gene scissors), which determine the target region(s) in the genome and are intended here to end the chain reaction of a CRISPR/Cas-based gene drive.
A CRISPR/Cas based gene drive that has been released, even only once, in the environment is no longer controllable, and it is not entirely predictable how the gene drive will spread in wild populations. Such gene drive organisms carry the genetic information of the gene scissors CRISPR/Cas9 as well as gRNA(s), which lead to the insertion of the gene drive construct in specific regions of the DNA and replicates itself in each generation. The Mendelian inheritance rules are circumvented. Often a target gene is destroyed by the insertion of the gene drive, which can cause the generation of male offspring in the progeny (see for example Kyrou, et al (2018). A CRISPR-Cas9 gene drive targeting doublesex causes complete population suppression in caged Anopheles gambiae mosquitoes. Nature Biotechnology).
The control mechanisms presented in the study use the activity of CRISPR/Cas9. As a result, the gene scissors effectively knock-out themselves. With the control mechanisms, no additional genetic information for CRISPR/Cas9 is introduced; the mechanisms are only active as long as CRISPR/Cas9 is active. If the gene drive has been switched off or replaced by one of the two neutralizing elements, the remaining genetic elements in the genome are passed on to subsequent generations according to Mendelian rules. So they are passed on with normal frequency and may be lost after a few generations.
Two different approaches are presented in the study. In both cases, initially gene drive organisms are present in a population and further GM organisms equipped with either of the two different control mechanisms are to be exposed. These should then spread through the affected population and deactivate the original gene drive:
e-CHACR: Inactivation of CRISPR/Cas
e-CHACRs (erasing construct hitch-hiking on the autocatalytic chain reaction) consist of two different gRNAs and use the activity of CRISPR/Cas9: One gRNA causes the e-CHACR to be inserted in the genome of the fruit flies, the second gRNA leads the gene scissors to its own gene and causes its alteration. In other words, the gene scissors destroys its own DNA. Thus, the gene scissors can no longer be formed. Both the e-CHACR construct and the deactivated gene drive remain in the genome of the flies. It is hard to say how these synthetic genetic elements will behave in the wild in the long term and whether they will be influenced by external influences. They may be lost after a few generations.
ERACR: Replacement of the gene drive construct
ERACR (element reversing the autocatalytic chain reaction) also consists of two gRNAs. In this case, the already formed CRISPR/Cas9 is led by them to the left and right border of the gene drive construct and cuts both sites. Afterwards the ERACR element is integrated into the genome by homology directed repair at that part of the genome. Thus, the gene drive construct is deleted from the genome and replaced by another genetic element. It turned out that the chromosome on which the gene drive construct was integrated can be damaged during the genome editing process and various unintended changes can be triggered: When the gene drive construct is cut from the genome, mistakes can happen when the DNA is being reassembled. Despite these errors, the succesfully introduced synthetic ERACR construct remains in the genome and is inherited according to Mendelian rules.
The study showed that both systems work in principle and that they can prevail in caged experiments with fruit flies within ten generations. How well the mechanisms presented here can neutralize an invasive gene drive in a real scenario remains unanwsered. The results are extensive and interesting. However, they also show that unintended changes can occur through the cell’s own mechanisms, the effects of which are difficult to assess, according to the authors.

Xu X-RS, Bulger EA, Gantz VM, Klanseck C, Heimler SR, Auradkar A, Bennett JB, Miller LA, Leahy S, Juste SS, Buchman A, Akbari OS, Marshall JM, Bier E (2020) Active Genetic Neutralizing Elements for Halting or Deleting Gene Drives. Molecular Cell. doi: