As our protocol shows, CRISPR CasRx technologies can be used to achieve ubiquitous and tissue specific gene transcript reductions in fruit flies. Our technique offers a starter package for anyone who is interested in using CRISPR-based gene transcript reduction in fruit flies while being compatible with further customization and optimization. To set up a ubiquitous in vivo RNA targeting using a two component CasRx system, collect 10 virgin adult female flies from the homozygous gRNA line of interest and five adult male flies from the balanced heterozygous Ubiq CasRx CurlyO.
Place the parental flies into a vial supplemented with 100 micrograms of dry yeast powder and rear the vials for 48 hours at 26 degrees Celsius. Observe the vials every day to see if any new adult progeny have emerged from the pubic of the F1 generation and anesthetize any of adults with carbon dioxide for 10 seconds. Once the flies become immobile empty the vial onto a fly pad connected to the carbon dioxide tank with continuous carbon dioxide and use a color camera connected to a fluorescent stereo microscope to score and image the phenotypes of the flies.
Then count the numbers of progeny with different phenotypes, according to the fluorescent signal expression. Based on Mendelian genetics, 50%of the progeny are expected to express the targeted RNA. Note that toxicity of the Ubiq CasRx system may reduce the ratio of targeted RNA expressing progeny to less than 50%To set up a ubiquitous in vivo exogenous RNA targeting using a three-component CasRx system, collect eight to 10 Virgin adult female flies from a dual luciferase expressing line and four to five adult male flies from the balanced heterozygous Ubiq CasRx Curly plus TM6, STB line that exhibits white eyes, curly wings, and ds red fluorescence simultaneously.
Place the parental step one cross-by in a vial supplemented with 100 micrograms of dry yeast powder and rear this cross for 48 hours at 26 degree Celsius. At the end of the incubation, remove all of the parental flies from each vial and return the vials to the 26 degree Celsius incubator for at least 14 days. Over the course of 14 days, as mentioned earlier collect eight to 10 female virgins from the homozygous firefly luciferase targeting gRNA line three times.
Observe the step one cross vials every day to see if any new adult progenies have emerged from the pupae, anesthetizing with carbon dioxide to allow the collection of four to five male flies expressing both the Ubiq CasRx and the dual Luciferase reporter as available. Place the flies into a new vial with 10 Virgin females from the fruit fly luciferase targeting gRNA line, and place these step two crosses at 26 degrees Celsius for 48 hours. Collect another five one day old males expressing both the Ubiq CasRx and dual luciferase reporter from the step one cross vials and incubate the flies for two to four days alone at 26 degree Celsius before transferring them into a 1.5 milliliter centrifuge for minus 80 degree Celsius storage.
At the end of the incubation, remove all of the parental vials from each of the step two cross vials. And return the vials to the 26 degree Celsius incubator for at least 20 days. Observe the step two cross vials every day to see if any new adult progeny have emerged from the pubic, anesthetizing the flies with carbon dioxide as they become available, and scoring and imaging their phenotypes as demonstrated.
Mendelian genetics suggests that, if all of the flies are viable, 25%of the progeny should express the targeted RNA. To perform a luciferase assay, generate the triple trans heterozygous flies as well as the control flies as demonstrated. Collecting the male flies at birth, and aging them until three days old.
Transfer the three day old flies into 1.5 milliliter centrifuge tubes, and lyse them using a buffer in the luciferase lysis buffer from a commercially available luciferase assay kit. Then use five microliters of lysed tissue from each sample and luminometer to measure both the fruit flies and ran Luciferase activity, according to standard luciferase assay protocols. F1, trans heterozygous CasRx, have significantly lower survival rates compared to trans heterozygous dCasRx flies.
Confirming the toxicity of the Ubiq CasRx system of the three target genes, the U6 gRNA Y heterozygous flies are nonviable and do not grow beyond the second instar larval stage. The surviving Ubq CasRx and U6 gRNA W heterozygous flies, demonstrate a distinct fully penetrant wide eyed phenotype. In addition, a significant reduction of target gene transcripts for three target genes is also observed.
Evidence of off target activity induced by CasRx is also observed when differential expressed transcripts between samples from CasRx expressing flies and samples from dCasRx expressing flies are compared. In the two step cross, only the combination of all three trans genes results in a 100%lethality. When tissue specific RNA targeting using a three component CasRx system design is employed, the level of toxicity is reduced when the overall CasRx expression is lowered using the UAST promoter compared to that of the Ubiq promoter.
It is important to set up the genetic process correctly using flies of correct gender and correct phenotypes.
