CAS9-MEDIATED GENOME EDITING OF THE MOSQUITO GERMLINE VIA RECEPTOR-MEDIATED ENDOCYTOSIS
Open Access
- Author:
- Chaverra Rodriguez, Duverney De
- Graduate Program:
- Entomology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- May 15, 2018
- Committee Members:
- Jason Laurence Rasgon, Dissertation Advisor/Co-Advisor
Jason Laurence Rasgon, Committee Chair/Co-Chair
Christina M Grozinger, Committee Member
Andrew Fraser Read, Committee Member
Yinong Yang, Outside Member - Keywords:
- CRISPR/Cas9
germline gene editing
Endosomal escape
Oocyte development
genetic engineering - Abstract:
- CRISPR/Cas9 is a revolutionary system used for gene editing and gene regulation in prokaryotic and eukaryotic organisms. In insects, germline gene editing is strongly limited by delivery of Cas9 ribonucleoprotein (RNP) complex by embryonic microinjection, a technique which is technically challenging, not appropriate for non-viviparous species, and which requires expensive equipment and training to implement. As alternative to embryo injection, I explored Receptor-Mediated Endocytosis (RME) as a pathway to specifically deliver RNP into the developing oocytes of female mosquitoes for heritable editing of the offspring’s maternal and paternal chromosomes. RME is exceptionally important during oocyte maturation. The size of the developing oocyte increases up to 300-fold due to the receptor-mediated uptake from the hemolymph of several yolk protein precursors (YPP) which act as ligands that bind to receptors present on the oocyte membrane. I hypothesized that if an YPP ligand is fused to Cas9 RNP, and injected into vitellogenic females, the enzyme complex would be transduced into the oocyte at the levels necessary to achieve genome editing in the embryo. I studied three YPP ligands reportedly recognized by receptors in the mosquitoes’ oocytes: Aedes aegypti vitellogenin A1 (VgA1), Aedes aegypti vitellogenic carboxypeptidase 1 (VC1), and Drosophila melanogaster Yolk Protein 1 (DmYP1). Compared to VgA1 and VC1, I found that “P2C” (a small peptide derivative of DmYP1) was a very efficient ligand to transduce EGFP into the ovaries of seven species of mosquitoes. When fused to Cas9, P2C-Cas9 was localized in granules in the oocyte, an outcome expected as an effect of endosomal retention of the protein. To induce endosomal release of the RNP complex, I tested fusogenic peptides and several membrane destabilizing reagents. After optimization, ovary transduction of Cas9 RNP reliably generated maternal chromosome mutations, producing on average 0.3 mutants per injected female. Also, the RNP injected in females showed activity after egg fertilization. Overall, this work demonstrates that targeted delivery of Cas9 RNP by RME is a reliable alternative to embryo injection to generate heritable germline mutations in the mosquito Ae. aegypti. Further optimization of this technology will make CRISPR/Cas9 gene editing techniques accessible to a wider research community.