Department of Life Sciences, Imperial College London, Sir Alexander Fleming Building, South Kensington Campus, London, UK.
Department of Entomology, Robert H. Smith Faculty of Agriculture, Food and Environment, Hebrew University of Jerusalem, Rehovot, Israel.
BMC Biol. 2021 Apr 16;19(1):78. doi: 10.1186/s12915-021-01010-7.
Genetic sex ratio distorters are systems aimed at effecting a bias in the reproductive sex ratio of a population and could be applied for the area-wide control of sexually reproducing insects that vector disease or disrupt agricultural production. One example of such a system leading to male bias is X-shredding, an approach that interferes with the transmission of the X-chromosome by inducing multiple DNA double-strand breaks during male meiosis. Endonucleases targeting the X-chromosome and whose activity is restricted to male gametogenesis have recently been pioneered as a means to engineer such traits.
Here, we enabled endogenous CRISPR/Cas9 and CRISPR/Cas12a activity during spermatogenesis of the Mediterranean fruit fly Ceratitis capitata, a worldwide agricultural pest of extensive economic significance. In the absence of a chromosome-level assembly, we analysed long- and short-read genome sequencing data from males and females to identify two clusters of abundant and X-chromosome-specific sequence repeats. When targeted by gRNAs in conjunction with Cas9, cleavage of these repeats yielded a significant and consistent distortion of the sex ratio towards males in independent transgenic strains, while the combination of distinct distorters induced a strong bias (~ 80%).
We provide a first demonstration of CRISPR-based sex distortion towards male bias in a non-model organism, the global pest insect Ceratitis capitata. Although the sex ratio bias reached in our study would require improvement, possibly through the generation and combination of additional transgenic lines, to result in a system with realistic applicability in the field, our results suggest that strains with characteristics suitable for field application can now be developed for a range of medically or agriculturally relevant insect species.
遗传性别比例干扰物是旨在影响种群生殖性别比例的系统，可用于对传播疾病或破坏农业生产的有性生殖昆虫进行区域控制。导致雄性偏向的此类系统的一个例子是 X 粉碎，这是一种通过在雄性减数分裂过程中诱导多个 DNA 双链断裂来干扰 X 染色体传递的方法。最近，针对 X 染色体的内切核酸酶及其活性仅限于雄性配子发生，已被开创性地用作工程此类特征的手段。
在这里，我们在地中海实蝇 Ceratitis capitata 的精子发生过程中启用了内源性 CRISPR/Cas9 和 CRISPR/Cas12a 活性，地中海实蝇 Ceratitis capitata 是一种具有广泛经济意义的世界性农业害虫。在没有染色体水平组装的情况下，我们分析了来自雄性和雌性的长读和短读基因组测序数据，以鉴定两个丰富且 X 染色体特异性序列重复的簇。当与 Cas9 一起针对 gRNA 时，这些重复的切割导致独立转基因品系中的性别比例朝着雄性方向发生显著且一致的扭曲，而不同干扰物的组合诱导出强烈的偏向（~80%）。
我们首次在非模式生物地中海实蝇 Ceratitis capitata 中展示了基于 CRISPR 的性别向雄性偏向的扭曲。尽管我们研究中的性别比例偏向需要改进，可能通过产生和组合额外的转基因系来实现，但在田间具有实际应用的系统，我们的结果表明，现在可以为一系列具有医学或农业相关意义的昆虫物种开发具有适合田间应用特征的品系。
