Expanding the CRISPR Toolbox in Culicine Mosquitoes: In Vitro Validation of Pol III Promoters.,ACS Synthetic Biology

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Expanding the CRISPR Toolbox in Culicine Mosquitoes: In Vitro Validation of Pol III Promoters.
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2020-03-04 , DOI: 10.1021/acssynbio.9b00436
Michelle A E Anderson ₁ , Jessica Purcell ₁ , Sebald A N Verkuijl _{1,

2} , Victoria C Norman ₁ , Philip T Leftwich _{1,

3} , Tim Harvey-Samuel ₁ , Luke S Alphey ₁

Affiliation

CRISPR-Cas9-based "gene drive" technologies have been proposed as a novel and effective means of controlling human diseases vectored by mosquitoes. However, more complex designs than those demonstrated to date-and an expanded molecular toolbox with which to build them-will be required to overcome the issues of resistance formation/evolution and drive spatial/temporal limitation. Foreseeing this need, we assessed the sgRNA transcriptional activities of 33 phylogenetically diverse insect Polymerase III promoters using three disease-relevant Culicine mosquito cell lines (Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus). We show that U6 promoters work across species with a range of transcriptional activity levels and find 7SK promoters to be especially promising because of their broad phylogenetic activity. We further show that U6 promoters can be substantially truncated without affecting transcriptional levels. These results will be of great utility to researchers involved in developing the next generation of gene drives.

中文翻译：

在Culicine蚊子中扩展CRISPR工具箱：Pol III启动子的体外验证。

基于CRISPR-Cas9的“基因驱动”技术已经被提出作为控制蚊子传播的人类疾病的一种新颖而有效的手段。然而，将需要比迄今为止证明的设计更复杂的设计，以及用于构建它们的扩展分子工具箱，以克服抗性形成/发展和驱动空间/时间限制的问题。预见到这种需求，我们使用三种与疾病相关的Culicine蚊子细胞系（埃及伊蚊，白纹伊蚊和库蚊）评估了33个系统发育不同的昆虫聚合酶III启动子的sgRNA转录活性。我们显示，U6启动子可跨具有一定转录活性水平的物种工作，并且发现7SK启动子因其广泛的系统发育活性而特别有前途。我们进一步表明，U6启动子可以被基本上截断而不影响转录水平。这些结果对于参与开发下一代基因驱动器的研究人员将具有巨大的实用性。

更新日期：2020-03-05

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