北京大学Jackson Champer小组发现，带有复用gRNA的一个归巢拯救基因驱动在笼养种群中达到高频率，但会产生功能抗性。该研究于2024年4月8日发表于国际学术期刊《遗传学报》。

据研究人员介绍，CRISPR同源基因驱动是一种强大的技术，在管理具有重要医学和农业意义的昆虫种群方面具有相当大的潜力。它能诱导后代中驱动等位基因的遗传偏向，在整个种群中迅速传播所需的基因构建体。归巢驱动通过Cas9的切割，然后进行同源定向修复，将驱动等位基因复制到野生型染色体上。然而，末端连接修复形成的抗性等位基因对驱动的传播构成了巨大障碍。

为了应对这一挑战，研究人员在黑腹果蝇中创建了一种以必需但缺乏单倍体的hairy基因为目标的同源驱动。这个策略包括消除无功能的抗性等位基因（隐性致死），同时用重新编码的hairy基因拯救携带驱动力的个体。驱动力遗传率适中，多代笼养研究显示驱动力扩散到了96%-97%的种群中。然而，尽管使用了四种gRNA，但由于形成了功能性抗性等位基因，驱动未能覆盖整个群体。

这些等位基因有较大的缺失，但很可能利用了另一个起始密码子，它们与驱动基因相比具有较小的适应性优势。因此，为了避免这种功能性抗性，即使使用复用gRNA，也可能需要针对目标基因的更重要区域修改设计策略。用其他物种的同源物替代原生3' UTR的拯救元件，可使驱动遗传性提高13%-24%。这可能是因为拯救元件与附近的原生DNA之间的同源性降低了，这也可能是利用拯救元件的基因驱动在设计时需要考虑的一个重要因素。

附：英文原文

Title: A homing rescue gene drive with multiplexed gRNAs reaches high frequency in cage populations but generates functional resistance

Author: Jackson Champer

Issue&Volume: 2024/04/08

Abstract: CRISPR homing gene drive is a potent technology with considerable potential for managing populations of medically and agriculturally significant insects. It induces a bias in the inheritance of the drive allele in progeny, rapidly spreading the desired gene construct throughout the population. Homing drives operate by Cas9 cleavage followed by homology-directed repair, copying the drive allele to the wild-type chromosome. However, resistance alleles formed by end-joining repair pose a significant obstacle to the spread of the drive. To address this challenge, we create a homing drive targeting the essential but haplosufficient hairy gene in Drosophila melanogaster. Our strategy involves eliminating nonfunctional resistance alleles, which are recessive lethal, while rescuing drive-carrying individuals with a recoded version of hairy. The drive inheritance rate is moderate, and multigenerational cage studies show drive spread to 96%–97% of the population. However, the drive fails to reach the whole population due to the formation of functional resistance alleles, despite use of four gRNAs. These alleles have a large deletion but likely utilize an alternate start codon, and they have a small fitness advantage over the drive. Thus, revised design strategies targeting more essential regions of a target gene may often be necessary to avoid such functional resistance, even when using multiplexed gRNAs. Replacement of the native 3' UTR of the rescue element with a homolog from another species increases drive inheritance by 13%–24%. This was possibly because of reduced homology between the rescue element and nearby native DNA, which could also be an important design consideration for gene drives that utilize rescue elements.

DOI: 10.1016/j.jgg.2024.04.001

Source: https://www.sciencedirect.com/science/article/abs/pii/S1673852724000705