Genetically Modified Conditional Sexing A. stephensi Line for PfSPZ Manufacture

迫切需要新的工具来预防、控制和消除疟疾：“..在 尽管全球疟疾控制取得了前所未有的成功，但进展却停滞不前。2016年，有一个 估计有2.16亿疟疾病例，比2015年增加约500万例。死亡人数达到445人 000“，比2015年增加15,000人。恶性疟原虫(PF)子孢子(SPZ)是唯一 已经在90%的人类受者中预防了肺炎支原体感染。Sanaria®PfSPZ疫苗，由辐射组成 减毒、无菌、纯化、低温保存的PfSPZ反复提供高水平的保护 控制人类疟疾感染(CHMI)和自然传播异质性肺吸虫。PfSPZ生长在 无菌饲养斯氏按蚊。在这个项目中，我们计划让无菌蚊子 通过将雄性蚊子从系统中移除，在不增加成本的情况下将饲养过程的效率提高100% 在胚胎阶段使用致命的基因插入，可以通过应用药物多西环素来控制。 在我们的四个具体目标中，我们将：1)在斯氏拟青霉中建立Y连锁对接菌株。vbl.使用 CRISPR/Cas9基因插入我们将在Y(男性)染色体上创建一个较大的基因驱动的位置 可以插入盒式磁带；2)建立携带Y连锁显性-负性形式的嗜好的斯氏A.stephensi (DnRel2)在Tet-on反式激活子的控制下。DnRel2在斯氏假丝酵母中的表达 胚胎致死。Y连锁对接菌株将被用来整合含有 Tet-Response元件(Tre)，最小启动子和dnRel2。在这里，dnRel2将仅在同时 可利用强力霉素和Tet-on反式激活剂；3)建立转基因斯氏针茅品系 携带Tet-On反激活剂。DnRel2在转基因株系中的表达需要Tet-on的结合 超激活器转特雷。因此，通过插入Tet-on反式激活子将建立另一转基因系。 在bZP1的控制下，斯氏沼虾早期胚胎基因启动子。在这一行中， 反式激活子受bZP1启动子的严格控制，直到产卵后36h才能表达。一个 将建立该转基因的纯合子系；以及4)产生并检测转基因条件 雄性致死斯氏吸虫性别鉴定品系。DnRel2和反式激活子将通过交叉结合在一起 DnRel2带有Tet-on反式激活子的雄性雌性。在先祖雄性卵子中，Tet-on反式激活因子将 在强力霉素存在的情况下，与tre结合，驱动dnRel2的表达，从而导致对 转基因雄性。在没有多西环素的情况下，两性都会存活下来，因为Tet-on反式激活因子是 不能绑定到tre。来自这种品系的雌性蚊子将被评估它们藏匿高空的能力。 肺炎支气管炎的强度感染。来自该项目中产生的所有品系和品系的卵子将被冷冻保存。

There is an the urgent need for new tools for prevention, control, and elimination of malaria: “..after an unprecedented period of success in global malaria control, progress has stalled. In 2016, there were an estimated 216 million cases of malaria, an increase of about 5 million cases over 2015. Deaths reached 445 000”, 15,000 more than in 2015. Plasmodium falciparum (Pf) sporozoites (SPZ) are the only immunogens that have prevented Pf infection in >90% of human recipients. Sanaria® PfSPZ Vaccine, composed of radiation attenuated, aseptic, purified, cryopreserved PfSPZ has repeatedly conferred high level protection against controlled human malaria infection (CHMI) and naturally transmitted heterogeneous Pf. PfSPZ are grown in aseptically reared Anopheles stephensi mosquitoes. In this project we plan to make the aseptic mosquito rearing process up to 100% more efficient at no additional cost by removing male mosquitoes from the system at the embryonic stage using a lethal gene insert that can be controlled by application of the drug, doxycycline. In our four specific aims we will: 1) Establish a Y-linked docking strain in A. 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 A. stephensi carrying Y-linked dominant-negative form of relish2 (dnRel2) under the control of Tet-on transactivator. Expression of dnRel2 in A. 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 A. 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 A. 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; and 4) Generate and test a transgenic conditional male-lethal sexing strain of A. 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. In the absence of doxycycline both sexes will survive because the Tet-On transactivator is not able to bind to TRE. Female mosquitoes from this strain will be assessed for their capacity to harbor high intensity infections of Pf. Eggs from all lines and strains generated in this project will be cryopreserved.