The best way to study the function of a gene is by generating a mutation or knockdown/knockout so that the amount of protein coded by that gene is compromised. Several model systems are employed by scientists to study the function of human genes. Drosophila has been used as a genetic model for more than 100 years to decipher many biological phenomena. Drosophila has always been convenient for genetic analysis and this resulted in the generation of many genetic tools, such as the UAS-GAL4 system, Generation of Transgenic insects, Flp-FRT technique, UAS-RNAi, and generating gene knockouts using p-element .
The recent advent of CRISPR/Cas technology to create complete knockout of genes in many model organisms has been a great addition to the already available toolkit. This allows us to study the function of different proteins in living conditions. Loss of function studies has always been an efficient way to decipher the function of a gene or protein coded by that gene. It is not possible to know the function of a gene in a wild-type organism. Appearance of marked visible phenotypeupon generating amutation in gene interest , indicates the possible function of that gene in the organ where we observe the phenotype. eg: After gene knockout, if we see a defect in the eye of Drosophila, suggests us that the gene which we mutated might be involved in the eye development of a fly.
What is CRISPR ?
CRISPR/Cas9 is an exciting new tool employed for carrying out targeted mutagenesis in Drosophila along with many other model systems. CRISPRs (clustered regularly inter-spaced short palindromic repeats) and the CRISPR-associated Cas9 endonuclease function as part of an adaptive immune system against invading viruses and plasmids in bacteria and archaea.
The best studied bacterial model for CRISPR is Streptococcus pyogenes. In this bacteria, the Cas9 endonuclease targets the incoming pathogen by virtue of crRNA (CRISPR RNA),which provides specificity to the endonuclease by base pairing with a 20 nt complimentary sequence within the target DNA (of pathogen) . Additionally one more component inside the bacteria termed as tracrRNA (trans-acting crRNA) forms a complex with the crRNA and targets its incorporation into the Cas9 complex.
This unique mechanism employed for self defense by various bacteria is also known to work in many other organisms, including Insect, fungal, plant and mammalian cells. Researchers around the world started adapting this technique to manipulate genome of their favorite model systems.
The CRISPR/Cas9 system was simplified by combining crRNA and tracrRNA into a approx. 100 nt synthetic single guide or chimeric RNA , which can be incorporated into fly using several means such as injecting the plasmid (DNA) encoding chimeric RNA into embryos of transgenic flies with cas9 protein or by generation a separate stable transgenic line for chimeric RNA driven by RNA pol III promoter from U6 gene.
Flies with both cas9 and chimeric RNA are crossed to wild-type flies and progeny of the cross leads to the formation of an active Cas9-gRNA complex specifically in germ cells, hence creating a mutant in the desired gene in a certain population of progeny.
Recent updates in Drosophila CRISPR
Performing large-scale genetics using CRISPR technique in Drosophila has been gaining importance in the last decade. The key advantage of using CRISPR when compared to UAS-RNAi or other knockdown tools is that CRISPR yields complete gene knockout, which is not always possible in UAS-RNAi. To study gene function it’s always advantageous to have a complete gene knockout (no protein is coded from that gene of interest in study) or null mutations.
This advantage with CRISPR encourages the scientists in the field to keep coming up with advanced strategies to make large-scale genetic screens simple and quick. Usually, screens involving CRISPR are done in cell lines and not in a living organism. To perform large-scale genetic screen in vivo conditions involves a tedious approach ( involves creating many clones and subsequent transgenic organisms for each clone) which eventually hinders the scope of the planned screen and its efficacy.
Large scale CRISPR genetic screens in Drosophila:
To facilitate genetic screens involving many genes, scientists around the world are creating useful toolkit reagents such as transgenic flies expressing Cas9, tissue-specific / ubiquitous expressing GAl4, Guide RNAs targeting individual genes covering the majority of ~ 15500 genes in the Drosophila genome. This led to an ever-increasing collection of transgenes in stock centers at Bloomington, VDRC, and NIG Japan. Special mention to scientists in Germany ( Fillip Port and Michael Boutros ), Isreal ( Oren Schuldiner), Harvard, and most recently Ethan Bier’s lab for the efforts to simplify and facilitate large genetic screens using CRISPR in flies. Now it is possible to perform a large-scale genetic screen to identify the role of different genes in particular biological pathways or in a tissue or organ of interest. With the availability of tissue-specific GAL4’s and controllable cas9 lines, it is now possible to knock down a gene in a region of interest for a particular time and also at the desired stage of drosophila development ( embryo, larval, pupae, or adult).
Resources and reference:
1. A-Z of CRISPR : http://www.crisprflydesign.org/
2. CRISPR fly stocks and resources for gene editing at Harvard : TRIP lines
3. A large-scale resource for tissue-specific CRISPR mutagenesis in Drosophila : doi: 10.7554/eLife.53865
4. Meltzer, H., Marom, E., Alyagor, I. et al. Tissue-specific (ts) CRISPR as an efficient strategy for in vivo screening in Drosophila. Nat Commun 10, 2113 (2019). https://doi.org/10.1038/s41467-019-10140-0
5. Terradas, G., Buchman, A.B., Bennett, J.B. et al. Inherently confinable split-drive systems in Drosophila. Nat Commun 12, 1480 (2021). https://doi.org/10.1038/s41467-021-21771-7