School of Medicine, University of Adelaide, North Terrace, Adelaide, SA, Australia.
South Australian Genome Editing Facility, North Terrace, Adelaide, SA, Australia.
Methods Mol Biol. 2022;2495:203-230. doi: 10.1007/978-1-0716-2301-5_11.
Gene drives are genetic elements that are transmitted to greater than 50% of offspring and have potential for population modification or suppression. While gene drives are known to occur naturally, the recent emergence of CRISPR-Cas9 genome-editing technology has enabled generation of synthetic gene drives in a range of organisms including mosquitos, flies, and yeast. For example, studies in Anopheles mosquitos have demonstrated >95% transmission of CRISPR-engineered gene drive constructs, providing a possible strategy for malaria control. Recently published studies have also indicated that it may be possible to develop gene drive technology in invasive rodents such as mice. Here, we discuss the prospects for gene drive development in mice, including synthetic "homing drive" and X-shredder strategies as well as modifications of the naturally occurring t haplotype. We also provide detailed protocols for generation of gene drive mice through incorporation of plasmid-based transgenes in a targeted and non-targeted manner. Importantly, these protocols can be used for generating transgenic mice for any project that requires insertion of kilobase-scale transgenes such as knock-in of fluorescent reporters, gene swaps, overexpression/ectopic expression studies, and conditional "floxed" alleles.
基因驱动是指能够传递给超过 50%后代的遗传元件，具有改变或抑制种群的潜力。虽然基因驱动是自然发生的，但最近出现的 CRISPR-Cas9 基因组编辑技术使包括蚊子、苍蝇和酵母在内的一系列生物能够产生合成基因驱动。例如，在疟蚊中的研究表明，CRISPR 工程基因驱动构建体的传递率超过 95%，为疟疾控制提供了一种可能的策略。最近发表的研究还表明，有可能开发入侵性啮齿动物（如老鼠）的基因驱动技术。在这里，我们讨论了在老鼠中开发基因驱动的前景，包括合成的“归巢驱动”和 X 碎纸机策略，以及自然发生的 t 单倍型的修饰。我们还提供了通过靶向和非靶向方式将质粒转基因为老鼠生成基因驱动的详细方案。重要的是，这些方案可用于生成任何需要插入千碱基规模转基因的转基因老鼠，如荧光报告基因的敲入、基因交换、过表达/异位表达研究以及条件性“floxed”等位基因。
