NEW YORK — A new CRISPR-based gene editing approach may allow gene editing of a wide range of insects, including cockroaches, by targeting their developing eggs.
Current approaches for gene editing insects largely rely on microinjecting the gene-editing machinery into early embryos. This has limited gene editing to insects whose embryos are easily accessible. Cockroaches, for instance, encapsulate their fertilized eggs in a hard egg case, which makes them inaccessible to microinjection.
But researchers from Japan and Spain have found that Cas9 ribonucleoproteins (RNPs) can instead be injected into adult female insects when their oocytes are developing. This approach, dubbed direct parental CRISPR or DIPA-CRISPR, had an efficiency of more than 20 percent in cockroaches and more than 50 percent in a type of beetle, as the researchers reported in Cell Reports Methods on Monday. Further, DIPA-CRISPR works with commercially available Cas9 and needs minimal equipment, which could allow it to be adopted by a wide range of labs.
"In a sense, insect researchers have been freed from the annoyance of egg injections," senior study author Takaaki Daimon from Kyoto University said in a statement. "We can now edit insect genomes more freely and at will. In principle, this method should work for more than 90 percent of insect species."
The researchers injected commercial Cas9 ribonucleoproteins targeting an eye color gene into 16 mature female German cockroaches, Blattella germanica, that were not carrying any fertilized eggs. Following injection, five of the cockroaches then produced ootheca, or fertilized eggs encapsulated in a hard casing, which showed a gene editing efficiency of 2.3 percent.
Following this initial result, the researchers further tested their approach in mature female B. germanica at different stages of their reproductive cycle. They found that they could boost the gene editing efficiency to 21.8 percent by focusing on female cockroaches four days after ootheca drop.
Through crossing experiments, they further found that subsequent progeny of the edited cockroaches also had disrupted cinnabar eye color genes, indicating that DIPA-CRISPR could be used to develop knockout cockroaches.
The researchers additionally applied their approach to the red flour beetle, Tribolium castaneum, which is evolutionarily distant from cockroaches. When they injected RNPs — this time targeting the cardinal eye color gene on the X chromosome — into female beetles four or five days after they emerged as adults, the researchers saw gene editing efficiencies of 50.8 percent and 71.4 percent, respectively. This, they noted, is similar to the efficiencies observed in embryo injection approaches in this species and suggests that DIPA-CRISPR may be a generalizable insect editing approach.
Using T. castaneum, they also found that DIPA-CRISPR could be applied to generate gene knock-in insects, though they noted the efficiency of 1.2 percent was low and needs to be improved.
Still, as DIPA-CRISPR uses commercially available Cas9 and requires minimal equipment, the researchers said it could be applied to a range of insects.
"By improving the DIPA-CRISPR method and making it even more efficient and versatile, we may be able to enable genome editing in almost all of the more than 1.5 million species of insects, opening up a future in which we can fully utilize the amazing biological functions of insects," Daimon added. "In principle, it may be also possible that other arthropods could be genome edited using a similar approach. These include agricultural and medical pests such as mites and ticks, and important fishery resources such as shrimp and crabs."
However, the researchers also noted that the approach has some limitations, as it requires knowledge of ovary development in the target species and may not be directly applicable to some insects with other reproductive strategies.