Holland Kendreze L, Blancher Ines, McKesey Marisa, Silas Michael, Gandhi Siddhant, Nickerson Axum, Jackson Kennedy, Blazeck John
Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332, United States.
Bioengineering Program, Georgia Institute of Technology, Atlanta, Georgia 30332, United States.
ACS Synth Biol. 2025 Sep 19;14(9):3387-3400. doi: 10.1021/acssynbio.5c00122. Epub 2025 Aug 18.
is a model organism commonly used to study gene regulation and function recently via CRISPR-()Cas9 technologies. Modulating the expression of multiple gene targets simultaneously is often important for synthetic biology and metabolic engineering applications and is crucial for genetic interaction studies. CRISPR-based systems can be used to target multiple genetic loci via expression of multiple single-guide RNAs (sgRNAs) in a single cell. However, there are currently a limited number of well-characterized RNA polymerase III (Pol III) promoters (e.g., pSNR52) for sgRNA expression in . Herein, we characterize 20 RNA Pol III promoters from different yeast species, from itself or from mammals, for their utility toward effectively mediating CRISPR activation and repression in . We show that the Pol III promoter cross-species functionality is impacted by promoter architecture and inclusion of core sequence motifs and that scaffold-mediated recruitment of multiple effectors can rescue poor promoter function in some contexts. Also, we highlight two Pol III promoters that mediate CRISPR function as well as the gold standard pSNR52 and previously described tRNA promoters. Finally, we show that these non-native promoters enable effective simultaneous CRISPR-mediated activation and repression of endogenous genes to enhance resistance to hydrogen peroxide. The Pol III promoters described here highlight the cross-species compatibility of genetic units in simple eukaryotes and will be useful for synthetic biology and phenotype engineering applications in yeast.
是一种常用于通过CRISPR-()Cas9技术研究基因调控和功能的模式生物。同时调节多个基因靶点的表达对于合成生物学和代谢工程应用通常很重要，并且对于遗传相互作用研究至关重要。基于CRISPR的系统可用于通过在单个细胞中表达多个单向导RNA（sgRNA）来靶向多个基因位点。然而，目前在中用于sgRNA表达的特征明确的RNA聚合酶III（Pol III）启动子数量有限（例如，pSNR52）。在此，我们表征了来自不同酵母物种、自身或哺乳动物的20个RNA Pol III启动子，以评估它们在中有效介导CRISPR激活和抑制的效用。我们表明，Pol III启动子的跨物种功能受到启动子结构和核心序列基序包含情况的影响，并且在某些情况下，支架介导的多种效应物募集可以挽救不良的启动子功能。此外，我们重点介绍了两个介导CRISPR功能的Pol III启动子以及金标准pSNR52和先前描述的tRNA启动子。最后，我们表明这些非天然启动子能够有效地同时进行CRISPR介导的内源性基因激活和抑制，以增强对过氧化氢的抗性。这里描述的Pol III启动子突出了简单真核生物中遗传单位的跨物种兼容性，将有助于酵母中的合成生物学和表型工程应用。
