ReMOT Control Gene Editing is Expanded to Anopheles stephensi

Mara Heilig is a graduate student in the laboratory of Dr. Peter Armbruster at Georgetown University and is studying the regulation of photoperiodic diapause in the Asian tiger mosquito, Aedes albopictus. MORE ABOUT THE AUTHOR

Traditional genome editing in insects involves directly injecting embryos.  However, most insect embryos are small and sensitive to manipulation.  This makes germ-line modifications difficult to achieve.  Therefore, the recent development of a new gene editing approach based on injecting adult mosquitoes to target oocytes is a major advance (Chaverra-Rodriguez et al. 2018). The researchers termed the method “ReMOT Control” (Receptor-mediated Ovary Transduction of Cargo, see Figure 1), and they originally optimized the technique in Aedes aegypti.

Macias et al (2019) recently adapted ReMOT Control for the malaria mosquito, Anopheles stephensiThis extension is significant for several reasons.  First, An. stephensi is especially difficult to micromanipulate, and genome editing is generally limited to labs with specialized equipment.  Further, the species is a major urban vector of malaria.  Thus, ReMOT Control can help expand medically important research using genome manipulation in the species.  Finally, this demonstrates that the method may be broadly applied to various other insects that are typically resistant to embryo injections.

To genetically modify the germline of adult insects, injection components must cross a barrier between the hemocoel and the ovaries.  ReMOT control facilitates this by fusing the Cas9 enzyme to a yolk protein fragment, P2C (1).  Since P2C is destined to enter the ovaries via receptor-mediated endocytosis, the fused Cas9 cargo can essentially “hitch a ride” (2).   The cargo then resides in an endosome (3), eventually escaping into the oocyte (4). Image Credit: Mara Heilig, created with


Macias et al (2019) optimized the ReMOT Control protocol for An. stephensi by adjusting three primary parameters: the timing of injections, the choice/concentration of the endosome escape reagent, and the final buffer concentration of the injection mix.  This allowed the team to minimize adult mortality and maximize the number of viable offspring.  Optimal conditions involve injecting females within 48 hours of a blood meal, reducing the concentration of the endosome escape reagent (saponin), and column exchanging the P2C-Cas9 elution buffer with water. Adjusting similar parameters will be critical for adapting the technique to other species.

To test the efficiency of ReMOT Control, Macias et al (2019) targeted enhanced cyan fluorescent protein (ECFP) in a transgenic line of An. stephensi.  The team used 3 sgRNAs and the optimized injection parameters to target ECFP.  About 1.4% of outcrossed progeny lost the function of ECFP, which was comparable to the efficiency of standard embryo injections (the ECFP marker was linked to a DsRed marker, so outcrossed individuals that lost ECFP function and retained DsRed could be distinguished from WT individuals).  In these individuals, Cas9 editing likely occurred prior to embryogenesis, since half their outcrossed progeny were WT and half were ECFP-/DsRed+.

Interestingly, there was also evidence that ReMOT Control targeted the germline of embryos after embryogenesis. Male and female progeny that displayed no somatic mutations (screened ECFP+/DsRed+) were outcrossed to WT mosquitoes.  A small percentage produced offspring lacked ECFP, suggesting that editing occurred after the embryos developed.  This indicates that the Cas9 cargo may remain active for a prolonged period of time in the progeny of injected adults.

Macias et al (2019) demonstrate that ReMOT Control can facilitate targeted and heritable gene editing in An. stephensi with a few alterations to the injection protocol, and without the need for difficult and cumbersome embryo injections.  The expansion of ReMOT Control in An. stephensi is a significant advance for making manipulative genome studies more accessible to diverse mosquito species.

  1. D. Chaverra-Rodriguez, V. M. Macias, G. L. Hughes, S. Pujhari, Y. Suzuki, D. R. Peterson, D. Kim, S. McKeand, J. L. Rasgon, Targeted delivery of CRISPR-Cas9 ribonucleoprotein into arthropod ovaries for heritable germline gene editing. Nat. Commun. 9, 1–11 (2018).
  2. V. M. Macias, S. Mckeand, D. Chaverra-rodriguez, G. L. Hughes, S. Pujhari, N. Jasinskiene, A. A. James, J. L. Rasgon, Cas9-mediated gene-editing in the malaria mosquito Anopheles stephensi by ReMOT Control. BioRxiv, 1–24 (2019).







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