Speth Zachary J, Rehard David G, Norton Patricia J, Franz Alexander W E
Department of Veterinary Pathobiology, University of Missouri, Columbia, Missouri, United States of America.
PLoS Genet. 2025 Jun 26;21(6):e1011757. doi: 10.1371/journal.pgen.1011757. eCollection 2025 Jun.
Aedes aegypti is the predominant vector for arboviruses including dengue, Zika, and chikungunya viruses, which infect over 100 million people annually. Mosquito population replacement in which arbovirus-susceptible mosquitoes in the field are replaced by laboratory-engineered refractory mosquitoes represents a novel genetic control measure to interrupt arboviral disease cycles. For this approach, the engineered mosquitoes need to harbor two genetic components: an antiviral effector construct which is linked to a gene drive (GD). We tested the performance of two single-locus CRISPR/Cas9 based GD for Ae. aegypti population replacement in small cage populations for up to 16 generations. Starting from a low release threshold of 1:9 GD bearing males to wild-type males, we observed two GD constructs in which Cas9 was expressed from two different germline promoters, nanos and zpg, to increase in frequency in all cage populations. By G16, an average of 72% and 82% of individuals from the zpg-GD and nanos-GD populations, respectively, harbored at least one GD copy with corresponding increases in allele frequencies. This indicated that the two single-locus, CRISPR/Cas9-based homing GD exhibited continuous super-Mendelian inheritance in populations of Ae. aegypti. Gene drive blocking indel (GDBI, a.k.a. "resistant alleles") frequency was measured for each discrete generation in pooled samples from the six populations harboring GD. We found that populations with Cas9 expression under control of the nanos-promoter accumulated GDBI at more than twice the rate of those populations harboring the zpg-promoter driven GD. Based on preexisting data sets for homing and GDBI frequencies in addition to the cage trial observations, the relative contributions of sex-specific homing rates, maternal Cas9 deposition and potential fitness effects were modeled in MGDrivE for both GD, further explaining their divergent performance. Our study demonstrates the feasibility of low-threshold, single-locus CRISPR/Cas9 based GD for Ae. aegypti population replacement.
埃及伊蚊是包括登革热、寨卡和基孔肯雅病毒在内的虫媒病毒的主要传播媒介，这些病毒每年感染超过1亿人。用实验室改造的抗性蚊子取代野外易感染虫媒病毒的蚊子的蚊子种群替换法，是一种中断虫媒病毒疾病传播周期的新型遗传控制措施。对于这种方法，改造后的蚊子需要携带两个遗传成分：一个与基因驱动（GD）相连的抗病毒效应构建体。我们测试了两种基于单基因座CRISPR/Cas9的基因驱动在埃及伊蚊种群替换中的性能，在小笼种群中进行了长达16代的测试。从携带基因驱动的雄性与野生型雄性1:9的低释放阈值开始，我们观察到两种基因驱动构建体，其中Cas9由两个不同的种系启动子nanos和zpg表达，在所有笼子种群中的频率都有所增加。到第16代时，来自zpg-GD和nanos-GD种群的个体分别平均有72%和82%至少携带一个基因驱动拷贝，等位基因频率相应增加。这表明这两种基于单基因座、CRISPR/Cas9的归巢基因驱动在埃及伊蚊种群中表现出持续的超孟德尔遗传。在携带基因驱动的六个种群的混合样本中，对每个离散世代测量了基因驱动阻断插入缺失（GDBI，又称“抗性等位基因”）频率。我们发现，在nanos启动子控制下表达Cas9的种群积累GDBI的速度是那些由zpg启动子驱动基因驱动的种群的两倍多。除了笼子试验观察结果外，基于现有的归巢和GDBI频率数据集，在MGDrivE中对两种基因驱动的性别特异性归巢率、母体Cas9沉积和潜在适应性效应的相对贡献进行了建模，进一步解释了它们不同的性能。我们的研究证明了基于低阈值、单基因座CRISPR/Cas9的基因驱动用于埃及伊蚊种群替换的可行性。
