Genetically Modified Conditional Sexing An. stephensi Line for PfSPZ Manufacture

Objectives

1) Establish a Y-linked docking strain in An. stephensi. Using CRISPR/Cas9 gene insertion we will create a site in the Y (male) chromosome into which larger gene drive cassettes can be inserted. 

2) Establish An. stephensi carrying Y-linked dominant-negative form of relish2 (dnRel2) under the control of Tet-on transactivator. Expression of dnRel2 in An. stephensi causes embryonic lethality. The Y-linked docking strain will be used to integrate a gene drive cassette containing a Tet-response element (TRE), a minimal promoter and dnRel2. Here, dnRel2 will be expressed only when both doxycycline and the Tet-On transactivator are available. 

3) Establish a transgenic An. stephensi line carrying Tet-On transactivator. Expression of dnRel2 in the transgenic line requires the binding of Tet-On transactivator to TRE. Therefore, another transgenic line will be established by inserting Tet-On transactivator under the control of bZP1, an An. stephensi early embryonic gene promoter. In this line, the expression of the transactivator will be tightly controlled by the bZP1 promoter which is expressed until 36 h after egg laying. A homozygous line for this transgene will be established.

4) Generate and test a transgenic conditional male-lethal sexing strain of An. stephensi. dnRel2 and the transactivator will be brought together by crossing dnRel2 males with Tet-On transactivator females. In the progenitor male eggs, the Tet-On transactivator will bind to TRE in the presence of doxycycline, driving the expression of dnRel2, thus causing lethality to the transgenic males.