DataSheet1_Assessment of distant-site rescue elements for CRISPR toxin-antidote gene drives.DOCX

Gene drive is a genetic engineering technology that can enable super-mendelian inheritance of specific alleles, allowing them to spread through a population. New gene drive types have increased flexibility, offering options for confined modification or suppression of target populations. Among the most promising are CRISPR toxin-antidote gene drives, which disrupt essential wild-type genes by targeting them with Cas9/gRNA. This results in their removal, increasing the frequency of the drive. All these drives rely on having an effective rescue element, which consists of a recoded version of the target gene. This rescue element can be at the same site as the target gene, maximizing the chance of efficient rescue, or at a distant site, which allows useful options such as easily disrupting another essential gene or increasing confinement. Previously, we developed a homing rescue drive targeting a haplolethal gene and a toxin-antidote drive targeting a haplosufficient gene. These successful drives had functional rescue elements but suboptimal drive efficiency. Here, we attempted to construct toxin-antidote drives targeting these genes with a distant-site configuration from three loci in Drosophila melanogaster. We found that additional gRNAs increased cut rates to nearly 100%. However, all distant-site rescue elements failed for both target genes. Furthermore, one rescue element with a minimally recoded sequence was used as a template for homology-directed repair for the target gene on a different chromosomal arm, resulting in the formation of functional resistance alleles. Together, these results can inform the design of future CRISPR-based toxin-antidote gene drives.

基因驱动（Gene drive）是一项基因工程技术，可使特定等位基因呈现超孟德尔式遗传，使其能够在种群中快速扩散。新型基因驱动的灵活性显著提升，为限制性种群修饰或种群抑制提供了可行方案。其中最具应用前景的当属CRISPR毒素-抗毒素基因驱动（CRISPR toxin-antidote gene drive），这类驱动通过Cas9/gRNA靶向并切割关键野生型基因，使其功能丧失，进而提升驱动元件在种群中的频率。所有此类驱动均依赖于高效的拯救元件（rescue element），该元件由靶基因的重编码版本构成。该拯救元件可与靶基因位于同一基因组位点，以最大化高效拯救的概率；也可整合至远端基因组位点，从而实现诸多实用功能，例如轻松破坏另一关键基因或进一步提升驱动的限制性。此前我们的团队已开发出针对单倍体致死基因（haplolethal gene）的归巢拯救驱动（homing rescue drive），以及针对单倍体充足基因（haplosufficient gene）的毒素-抗毒素驱动。上述成功构建的驱动均具备功能正常的拯救元件，但驱动效率未达最优水平。本研究尝试以黑腹果蝇（Drosophila melanogaster）的三个基因座为靶点，采用远端位点构型构建针对上述两类基因的毒素-抗毒素驱动。实验结果显示，额外添加向导RNA（gRNA）可将靶基因的切割效率提升至接近100%。然而，针对两个靶基因的所有远端位点拯救元件均未能发挥预期功能。此外，一个带有最小化重编码序列的拯救元件被用作另一染色体臂上靶基因的同源定向修复（homology-directed repair）模板，最终形成了具有功能的抗性等位基因（resistance alleles）。综上，本研究结果可为未来基于CRISPR的毒素-抗毒素基因驱动的设计与优化提供重要参考。