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Meiotic Drive Impacts Expression and Evolution of X-Linked Genes In Targeting Drive Associated Genes using CRISPR-Cas9 David Akanonu & Dr. Josie Reinhardt Introduction Methods Results Teleopsis Dalmanni (also known as Stalk-eyed Flies) are creatures that have these long stalks growing out of their heads. Attached to these stalks are their eyes which is not Figure 6. Selected Genes noticeable at first glance. My focus is on selfish gene these Generate Hypothesis… What From Gene Pool Gene Do I need to Target? What flies carry known as a meiotic drive gene. According to Phenotype? Mendel’s Law of segregation in species where chromosomes determine sex, the chances of receiving a male or a female is half and half. However, in this case that half and half is now 10 and 90. Meiotic drive is a Figure 7. caused by a selfish gene on the X-chromosome that also DAVID causes specific tradeoffs in the sexes: males have reduced Find Genes that are associated with meiotic drive Functional fertility whereas in females have higher fertility. In Annotation addition, the way these flies are attracted to each other are Tool due to their stalks. Female stalk eyed flies are attracted to Results on long stalks on males. Males with the drive associated X- the Yuri chromosome will have shorter stalks and therefore reduce and their chances of reproducing with other females. However, Design and order gRNAs as Chiffon almost nothing is known about the genetic causes of any of well as PCR primers Gene these consequences of carrying a meiotic drive chromosome. In my project, we plan on modifying a standard stalk-eyed fly and hope to replicate meiotic drive. In addition, we look at impacts on the other traits described Figure 8. Nano drop above. What we want to do with these stalk-eyed flies is concentration graph Pre- modify candidate genes using CRISPR-Cas9. We’ll use Plasmid DNA Prep Digest CRISPR to induce a frameshift mutation, and that is an insertion or a deletion of a nucleotide in a DNA sequence. All things considered, modifying genomes to flies is just a A B small portion of the project. Figure 4. Plasmid Map Dad (sperm): Aa Digest Plasmid A a AX a* Figure 9. Nano drop ): A AA Aa A X Aa* concentrations Post Figure 10. Bacterial Transformations Unsuccessful Yuri Gene Transformation (A) and eggs Digest. Ligate gRNA insert Transform Aa into Bacteria Successful Chiffon Gene Transformation (B) Mom Mom ( a aA aa a X aa* Figure 5. gRNA Mock Design + Figure 1. Effect of meiotic drive on gene inheritance r Chiffon F colonies Chiffon B Chiffon e m13 rev/for colonies F v Strategy 2: No gel extraction, ligation, Ladder m13 colonies / Ladder Strategy 1: Gel extraction, ligation, transformation rev/spacer m13 f rev/for o transformation r yuri_52_Left&Right yuri_52_Left&Right chif_127_Left&Right chif_127_Left&Right 3 3 yuri_52_Left&Right 3 yuri_52_Left&Right 2 g h 2 chif_127_Left&Right 2 chif_127_Left&Right g 1 chif_1620_Left&Right 1 chif_1620_Left&Right h yuri_b_5220_Left&Right yuri_b_5220_Left&Right g1 chif_1620_Left&Right chif_1620_Left&Right g1 h 4 h4 yuri_b_5220_Left&Right yuri_b_5220_Left&Right h4 + g r Colony PCR Chiffon B colonies Chiffon B colonies e v Figure 12. PCR m13 rev/for m13 rev/spacer Ladder Chiffon F colonies / Primer Gel m13 rev/spacer s Figure ?. Colony PCR Results p “g” means DNA with a Unfortunately Colony PCR did not work 5 + 16S1F R 16S1 g5 + 16S1F g5 R 16S1 g master mix and “h” c for the Chiffon Gene. However, the “Mini-Prep” Plasmid DNA, then sequence the e means DNA w/o master r ladders set in place showed bands. DNA “Mini-Prep” Plasmid DNA, then sequence the mix DNA Figure 11. Colony PCR on m13 rev/for & m13 rev/ spacer primers for Chiffon colonies Figure 2. Effect of meiotic drive on sex ratio Inject plasmid and cas9 into fly Conclusion embryos The Yuri transformation was unsuccessful however, the Chiffon David Pst1 David BbS1 transformation was successful. Moving forward, we want to re-do References our Yuri ligation and transformation, • Reinhardt, J. A., Brand, C. L., Paczolt, K. A., Johns, P. M., Baker, R. H., & Wilkinson, G. S. (n.d.). Meiotic Drive Impacts Expression and Evolution of Figure 13. Digest of PCFD3 using BbS1 X-Linked Genes in Stalk-Eyed Flies sequence colonies from the chiffon • David, P., Bjorksten, T., Fowler, K., Pomiankowski, A. (2000). Condition-dependent signalling of genetic variation in stalk-eyed flies. Nature, 406, 186- and Pst1 188. transformation. We need to • Wilkinson, Gerald & S Presgraves, D & Crymes, Lili. (1998). Male eye span in stalk-eyed flies indicates genetic quality by meiotic drive suppresion. Nature. 391. 276-279. 10.1038/34640. troubleshoot our PCR approach and • Port, F., Chen, H., Lee, T., & Bullock, S. L. (2014). Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila. hopefully be ready to inject modified Figure 3. Effect of meiotic drive on fertility Proceedings of the National Academy of Sciences,111(29). doi:10.1073/pnas.1405500111 • Warren, I. A., Fowler, K., & Smith, H. (2010). Germline transformation of the stalk-eyed fly, Teleopsis dalmanni. BMC Molecular Biology,11(1), 86. plasmids into fly embryos! • Wilkinson, G. S., Johns, P. M., Kelleher, E. S., Muscedere, M. L., & Lorsong, A. (2006). Fitness effects of X chromosome drive in the stalk-eyed fly, Cyrtodiopsis dalmanni. Journal of Evolutionary Biology, 19(6), 1851-1860..
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