CRISPR gene editing now possible in cockroaches
Japanese and Spanish researchers have developed a CRISPR-Cas9 approach to enable gene editing in cockroaches, according to a new study published in Cell Reports Methods. The simple and efficient technique, named ‘direct parental CRISPR’ (DIPA-CRISPR), involves the injection of materials into female adults where eggs are developing, rather than into the embryos themselves.
Current approaches for insect gene editing typically require microinjection of materials into early embryos, limiting their application in many species; for example, past studies have not achieved genetic manipulation of cockroaches due to their unique reproductive system. In addition, insect gene editing often requires expensive equipment, a specific experimental set-up for each species and highly skilled laboratory personnel.
To overcome these limitations, researchers injected Cas9 ribonucleoproteins (RNPs) into the main body cavity of adult female cockroaches to introduce heritable mutations in developing egg cells. The results demonstrated that gene editing efficiency — the proportion of edited individuals out of the total number hatched — could reach as high as 22%. Meanwhile, in the red flour beetle, DIPA-CRISPR achieved an efficiency of more than 50%. Moreover, the researchers generated gene knockin beetles by co-injecting single-stranded oligonucleotides and Cas9 RNPs, though efficiency here needs to be further improved.
“We can now edit insect genomes more freely and at will,” said senior study author Takaaki Daimon of Kyoto University. “In principle, this method should work for more than 90% of insect species.”
The successful application of DIPA-CRISPR in two evolutionarily distant species demonstrates its potential for broad use, though the approach is not directly applicable to all insect species. In addition, the experiments showed that the most critical parameter for success is the stage of the adult females injected. As a result, DIPA-CRISPR requires good knowledge of ovary development, which can be challenging in some species.
Nevertheless, the researchers say that DIPA-CRISPR is accessible, highly practical and could be readily implemented in laboratories, extending the application of gene editing to a range of model and non-model insect species. The technique requires minimal equipment for adult injection, and only two components — Cas9 protein and single-guide RNA — simplifying procedures for gene editing. Moreover, commercially available, standard Cas9 can be used for adult injection, eliminating the need for custom engineering of the protein.
Daimon said the method may eventually enable genome editing in almost all of the more than 1.5 million species of insects, with a similar approach potentially used in other arthropods.
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