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The hard, protective egg cases of some insects, such as the cockroach Blatella germanica, makes it impossible to perform conventional CRISPR gene editing, as the components must be injected into the embryo. However, an international team of researchers has developed a method to edit insect genes by injecting Cas9 ribonucleoproteins and guide RNA directly into the hemolymph—circulating fluid akin to blood—of the mother instead of the embryo. With this technique, named “direct parental” CRISPR (or DIPA-CRISPR), the researchers were, for the first time, able to edit genes in cockroaches, as reported in a study first published online May 16 in Cell Reports Methods.
Injection of CRISPR components into adult insects to edit their offspring has been achieved before in Aedes aegypti mosquitoes, but the previously developed method, called Receptor-Mediated Ovary Transduction of Cargo (ReMOT), fuses the Cas9 protein to a species-specific ligand, which improves the efficiency of the edits by increasing uptake of Cas9 and single-guide RNAs into the developing egg. DIPA-CRISPR does away with the species-specific ligand, making the procedure more easily generalizable to other species, researchers tell The Scientist.
This exciting result opens up the possibility to use this technology in many other insect species which are difficult to inject at the embryo stage.
—Omar Akbari, University of California, San Diego
“What they are showing here is, in this system, you don’t really need the targeting ligand. They just inject a lot of [Cas9] into the hemolymph during the process of egg development. When the ovaries are taking in lots of material from the insect circulatory system, the Cas9 ribonucleoprotein complex seems to be being passively taken up,” says Jason Rasgon, a disease epidemiologist at The Pennsylvania State University who developed ReMOT but was not involved in the current study.
Rasgon also points out a previous study that demonstrated successful gene editing in spider mites (Tetranychus urticae) via adult injection. However, he adds that the current study is “the first time, to my knowledge, that it’s been shown to be efficient.” In spider mites, researchers only achieved an efficiency of about one half of a percent with CRISPR-Cas9, meaning that the desired gene edit successfully integrated into the target site one out of every 200 times. The new technique achieved an efficiency of up to 21.8 percent in cockroaches and over 70 percent in the red flour beetle Tribolium castaneum.
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Serendipity played a role in developing DIPA-CRISPR, says Xavier Belles, an expert in insect metamorphosis at the Institute of Evolutionary Biology in Barcelona, who is a coauthor of the new study. When the researchers were conducting systematic tests of various peptide fragments to serve as targeting ligands for cockroaches, they included a negative control group that didn’t receive a ligand at all. Unexpectedly, “the control one was more active than the others,” Belles tells The Scientist. “So we decided to work only with Cas9 and the RNA guide, and these work wonderfully.”
With DIPA-CRISPR, the researchers inject a commercially available Cas9 enzyme and guide RNA into the haemocoel—the main body cavity—of the cockroach mother. This injection must be timed just right such that it coincides with egg production, when large amounts of yolk proteins are incorporated into the eggs, the researchers found. “[T]he components of the CRISPR that we inject into the body are accompanying the proteins and enter also into the oocytes, and then affect the embryo that will develop later after copulation and fertilization,” says Belles.
The researchers also performed knock-in experiments by adding small fragments of DNA to the cockroach genome. “To my knowledge, this is the first time that’s been shown with an adult injection procedure,” says Rasgon. While only short stretches of DNA were added, the insertions were long enough to insert small functional sequences.
“This exciting result opens up the possibility to use this technology in many other insect species which are difficult to inject at the embryo stage,” Omar Akbari, a cell and developmental biologist at the University of California, San Diego, who was not involved in the study, tells The Scientist in an email.
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Takaaki Daimon, a geneticist at Kyoto University in Japan who coauthored the new study, tells The Scientist in an email that the researchers wanted to develop a genome editing method “that anyone can use and that is very easy and inexpensive.” Indeed, as Rasgon points out, researchers seeking to edit genomes with DIPA-CRISPR can use commercially available Cas9 and no longer have to set up protein expression systems to produce a species-specific ligand, as with ReMOT. Adult injection also works without microinjectors that would otherwise be needed to get CRISPR components into the tiny eggs of various species. Rasgon says that he would tell researchers who want to adapt the ReMOT method to their species “to try it just this way [with DIPA-CRISPR] first, and if that works, that’s great. And if not, come talk to us.” He adds that DIPA-CRISPR won’t work for every species, as he has tried direct injection without ligands in ticks and white flies without success.
Daimon expects that direct adult injection “can be done in most insects,” but lists several limitations. “The injections must be given to adult females at a specific time,” when yolks are forming, and “this period must be identified in advance for each target species,” he adds. “Some species do not have this stage; DIPA-CRISPR is not applicable to them.” As a result, DIPA-CRISPR is not suitable for the model Drosophila melanogaster or some higher Diptera, such as the stable fly Stomoxy calcitrans, the authors write in the paper. Nevertheless, Belles looks forward to using the new method in the non-model cockroach, for which RNA interference, a method that uses sequence-specific double-stranded RNA to silence gene expression but doesn’t edit the genome, has so far been the only way to reduce and probe gene function, telling The Scientist that “This opens up the possibility of studying the function of genes about which we still have no information.”