Cartoon CRISPR in cockroaches. Credit: Shirai et al./Cell Reporting Methods
According to a research paper published in the journal Cell Reporting Methods By Cell Press on May 16yIn 2022, researchers created CRISPR-Cas9 technology to enable gene editing in cockroaches. The direct and effective CRISPR (DIPA-CRISPR) procedure involves injecting substances into adult females where the eggs grow rather than the embryos themselves.
“In a sense, the insect researchers were freed from the discomfort of injecting the eggs,” says senior study author Takaaki Daimon of Kyoto University. We can now modify insect genomes more freely and of our own volition. In principle, this method should work for more than 90% of insect species.”
“By improving the DIPA-CRISPR method and making it 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 take full advantage of the amazing biological functions of insects.” – Takaaki Damon
Current insect gene editing methods typically require microinjection of material into early embryos, severely limiting their application to many species. For example, previous studies have not investigated genetic manipulation of cockroaches due to their unique reproductive system. In addition, insect gene editing often requires expensive equipment, specific experimental setup for each species, and highly skilled laboratory personnel. “These problems with conventional methods have plagued researchers who want to perform genome editing on a variety of insect species,” says Damon.
To overcome these limitations, Damon and his collaborators injected Cas9 ribonucleoproteins (RNPs) into the main body cavity of adult female crickets to introduce genetic mutations into developing egg cells. The results showed that the gene-editing efficiency – the proportion of individuals modified out of the total number of individuals hatched – could be as high as 22%. In the red flour beetle, DIPA-CRISPR achieved an efficiency of over 50%. Furthermore, the researchers generated gene beetles by co-injecting single-stranded oligonucleotides and Cas9 RNPs, but the efficiency is low and should be further improved.
The successful application of DIPA-CRISPR in two evolutionarily distant species demonstrates its widespread use. But this approach is not directly applicable to all types of insects, including fruit flies. In addition, experiments have shown that the most important factor for success is the stage of injection of adult females. As a consequence, DIPA-CRISPR requires a good knowledge of ovarian development. This can be challenging in some species, due to the varied life history and reproductive strategies of insects.
Despite these limitations, DIPA-CRISPR is accessible, highly practical, and easily implemented in laboratories, expanding the application of gene editing to a variety of model and non-model insect species. This technology requires minimal equipment to inject adults, and only two components – Cas9 protein and a single guide[{” attribute=””>RNA—greatly simplifying procedures for gene editing. Moreover, commercially available, standard Cas9 can be used for adult injection, eliminating the need for time-consuming custom engineering of the protein.
“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 says. “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.”
Reference: “DIPA-CRISPR is a simple and accessible method for insect gene editing” by Yu Shirai, Maria-Dolors Piulachs, Xavier Belles and Takaaki Daimon, 16 May 2022, Cell Reports Methods.
DOI: 10.1016/j.crmeth.2022.100215
This work was supported by funding from JSPS KAKENHI, JSPS Open Partnership Joint Research Projects, Spanish Ministry of Innovation and Competitiveness, and CSIC-Spain, and in part by Cabinet Office, Government of Japan, Cross-ministerial Moonshot Agriculture, Forestry and Fisheries Research and Development Program.