Department of Entomology and AgriLife Research, Texas A&M University, College Station, Texas, United States of America.
Department of Industrial and Systems Engineering, Texas A&M University, College Station, Texas, United States of America.
PLoS Genet. 2024 Nov 7;20(11):e1011450. doi: 10.1371/journal.pgen.1011450. eCollection 2024 Nov.
Here, we demonstrate that single strand annealing (SSA) can be co-opted for the precise autocatalytic excision of a drive element. We have termed this technology Repeat Mediated Excision of a Drive Element (ReMEDE). By engineering direct repeats flanking the drive allele and inducing a double-strand DNA break (DSB) at a second endonuclease target site within the allele, we increased the utilization of SSA repair. ReMEDE was incorporated into the mutagenic chain reaction (MCR) gene drive targeting the yellow gene of Drosophila melanogaster, successfully replacing drive alleles with wild-type alleles. Sequencing across the Cas9 target site confirmed transgene excision by SSA after pair-mated outcrosses with yReMEDE females, revealing 4% inheritance of an engineered silent TcG marker sequence. However, phenotypically wild-type flies with alleles of indeterminate biogenesis also were observed, retaining the TGG sequence (16%) or harboring a silent gGG mutation (~0.5%) at the PAM site. Additionally, ~14% of alleles in the F2 flies were intact or uncut paternally inherited alleles, indicating limited maternal deposition of Cas9 RNP. Although ReMEDE requires further research and development, the technology has some promising features as a gene drive mitigation strategy, notably its potential to restore wild-type populations without additional transgenic releases or large-scale environmental modifications.
在这里，我们证明单链退火（SSA）可以被用于精确的自催化切除驱动元件。我们将这项技术命名为重复介导的驱动元件切除（ReMEDE）。通过在驱动等位基因侧翼工程化直接重复序列，并在等位基因内的第二个内切酶靶位点诱导双链 DNA 断裂（DSB），我们增加了 SSA 修复的利用率。ReMEDE 被整合到针对黑腹果蝇黄色基因的诱变链反应（MCR）基因驱动中，成功地用野生型等位基因替换了驱动等位基因。在与 yReMEDE 雌性进行配对杂交后的 Cas9 靶位点进行测序，证实了 SSA 通过同源重组修复切除了转基因，揭示了大约 4%的工程化沉默 TcG 标记序列的遗传。然而，也观察到表型野生型的具有不定生源等位基因的果蝇，保留了 TGG 序列（约 16%）或在 PAM 位点上具有沉默的 gGG 突变（约 0.5%）。此外，在 F2 代果蝇中约有 14%的等位基因是完整的或未切割的父系遗传等位基因，表明 Cas9 RNP 的母源沉积有限。尽管 ReMEDE 需要进一步的研究和开发，但作为一种基因驱动缓解策略，该技术具有一些有前途的特点，特别是它有可能在不进行额外的转基因释放或大规模环境改造的情况下恢复野生型种群。
