Current approaches for insect gene editing require microinjection of materials into early embryos. This severely limits the application of gene editing to a great number of insect species, especially to those whose reproduction systems preclude access to early embryos for injection. To overcome these limitations, researchers from Kyoto University and the Institute of Evolutionary Biology at the CSIC-Universitat Pompeu Fabra have developed a simple and accessible method for insect gene editing. Termed ‘direct parental’ CRISPR (DIPA-CRISPR), it involves the injection of materials into female adults where eggs are developing rather than into the embryos themselves.
Recent advances of genome editing tools have enabled sophisticated engineering of insect genomes.
However, current approaches rely on embryo injection, which requires expensive equipment, a specific experimental setup for each species, and highly skilled laboratory personnel.
Furthermore, embryo injection must be completed in a small time window, from oviposition to preblastoderm stage, which is not applicable to species that give live birth rather than lay eggs or species in which an access to very early embryos is highly challenging.
To overcome the limitations, Kyoto University’s Dr. Takaaki Daimon and colleagues injected Cas9 ribonucleoproteins (RNPs) into the main body cavity of adult female cockroaches to introduce heritable mutations in developing egg cells.
“In a sense, insect researchers have been freed from the annoyance of egg injections,” said Dr. Daimon, senior author of the study.
“We can now edit insect genomes more freely and at will. In principle, our method should work for more than 90% of insect species.”
The team’s results demonstrated that gene editing efficiency — the proportion of edited individuals out of the total number of individuals hatched — could reach as high as 22%.
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, but the efficiency is low and should be further improved.
The successful application of DIPA-CRISPR in two evolutionarily distant species demonstrates its potential for broad use. But the approach is not directly applicable to all insect species, including fruit flies.
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. This can be challenging in some species, given the diverse life histories and reproductive strategies in insects.
Despite these limitations, the new method is accessible, highly practical, and could be readily implemented in laboratories, extending the application of gene editing to a wide diversity of model and non-model insect species.
It requires minimal equipment for adult injection, and only two components — Cas9 protein and single-guide 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,” Dr. Daimon said.
“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.”
The team’s work was published in the journal Cell Reports Methods.
Yu Shirai et al. DIPA-CRISPR is a simple and accessible method for insect gene editing. Cell Reports Methods, published online May 16, 2022; doi: 10.1016/j.crmeth.2022.100215
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