In another advance for CRISPR technology, researchers have devised a way to edit genes in insects, including cockroaches. The technique involves editing the genomes of female adults that are carrying developing eggs (instead of editing the eggs), and is called "direct parental" CRISPR (DIPA-CRISPR). The method has been outlined in Cell Reports Methods.
"In a sense, insect researchers have been freed from the annoyance of egg injections," said senior study author Takaaki Daimon of Kyoto University. "We can now edit insect genomes more freely and at will." The study authors suggested that DIPA-CRISPR could be applied to over 90 percent of all insect species.
To use CRISPR, researchers have to inject a tiny cocktail of liquids that carry the CRISPR molecules into very early embryos, like zebrafish eggs that have just been spawned and collected. Once inside the cells, the CRISPR molecules can edit the genomes inside. But not all organisms produce eggs that can be edited during development in this way. Cockroaches, for example, have a unique reproductive system. Genome in editing in most insects can be tedious and expensive, which has stymied researchers that want to perform such experiments, said Daimon.
CRISPR takes advantage of an enzyme that cuts DNA, like Cas9, and a guide RNA, that moves the cutting enzyme to the right place in the genome. In this work, the researchers used a complex that combined the guide RNA and Cas9 into one molecule called a ribonucleoprotein (RNP). However, the technique also works with Cas9 enzymes and guide RNAs, a simpler and less expensive approach. In this study, the Cas9 RNPs were injected into adult female cockroaches' main body cavity. Then heritable mutations were introduced in the egg cells developing there.
The scientists determined that this approach successfully edited as many as 22 percent of the hatched insects. The work also showed that adult females have to be injected at a specific stage, so researchers using the tool must know about ovary development in the organism; that may present a different challenge. For a different insect called the red flour beetle, the efficiency of this method increased to over 50 percent. The researchers were also able to add genes to the beetle, creating 'knock-ins' with oligonucleotides and Cas9 RNPs, though the efficiency was very low.
While this demonstrates the method has potential and could be applied to many species, it does not work for fruit flies. Still, DIPA-CRISPR has now made it possible to edit the genomes of more species with very few reagents and tools.
Daimon noted that DIPA-CRISPR has opened up over "1.5 million species of insects" to genome editing, and "a future in which we can fully utilize the amazing biological functions of insects."