Reducing resistance allele formation in CRISPR gene drive
Reducing resistance allele formation in CRISPR gene drive
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DOI：
10.1073/pnas.1720354115
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发表时间：
2018-05-22
影响因子：
11.1
通讯作者：
Messer, Philipp W.
中科院分区：
文献类型：
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作者：
Champer, Jackson;Liu, Jingxian;Messer, Philipp W.
CRISPR归巢基因驱动可以将带有一个驱动等位基因副本的杂合细胞转化为纯合子，从而实现超孟德尔遗传。例如，这种机制可以用来在人群中迅速传播基因有效载荷，从而有望有效地控制媒介传播的疾病。然而，到目前为止，在昆虫中研究的所有CRISPR归巢基因驱动都产生了大量的抗性等位基因，这将限制它们的传播。在这项研究中，我们提供了一个实验证明，在果蝇CRISPR归巢基因驱动中，引导RNA的多路复用既可以显著提高驱动转换效率，又可以降低种系抵抗率。我们进一步表明，常染色体驱动可以在雄性生殖系中实现驱动转换，而不会通过父亲携带的Cas9在胚胎中形成抗性等位基因。最后，我们发现，与VAS启动子相比，Nanos启动子显著降低了体细胞Cas9的表达，这表明Nanos在驱动策略中提供了更好的选择，其中体细胞中的基因破坏可能会有适应成本。本研究和之前的一项研究中开发的不同结构之间的驱动参数比较表明，虽然不同基因组目标之间的驱动转化率和生殖系抵抗率相似，但胚胎抵抗率可能存在显著差异。综上所述，我们的结果标志着朝着开发能够在自然种群中发挥作用的有效基因驱动迈出了重要的一步，并为进一步控制耐药率提供了几种可能的途径。
CRISPR homing gene drives can convert heterozygous cells with one copy of the drive allele into homozygotes, thereby enabling super-Mendelian inheritance. Such a mechanism could be used, for example, to rapidly disseminate a genetic payload in a population, promising effective strategies for the control of vector-borne diseases. However, all CRISPR homing gene drives studied in insects thus far have produced significant quantities of resistance alleles that would limit their spread. In this study, we provide an experimental demonstration that multiplexing of guide RNAs can both significantly increase the drive conversion efficiency and reduce germline resistance rates of a CRISPR homing gene drive in Drosophila melanogaster. We further show that an autosomal drive can achieve drive conversion in the male germline, with no subsequent formation of resistance alleles in embryos through paternal carryover of Cas9. Finally, we find that the nanos promoter significantly lowers somatic Cas9 expression compared with the vasa promoter, suggesting that nanos provides a superior choice in drive strategies where gene disruption in somatic cells could have fitness costs. Comparison of drive parameters among the different constructs developed in this study and a previous study suggests that, while drive conversion and germline resistance rates are similar between different genomic targets, embryo resistance rates can vary significantly. Taken together, our results mark an important step toward developing effective gene drives capable of functioning in natural populations and provide several possible avenues for further control of resistance rates.