Simulations predict stronger CRISPRi transcriptional repression in plants for identical than heterogeneous gRNA target sites.

IF 2.6 Q2 BIOCHEMICAL RESEARCH METHODS
Synthetic biology (Oxford, England) Pub Date : 2025-04-18 eCollection Date: 2025-01-01 DOI:10.1093/synbio/ysae020
Helen Scott, Alessandro Occhialini, Scott C Lenaghan, Jacob Beal
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Abstract

Plant synthetic biologists have been working to adapt the CRISPRa and CRISPRi promoter regulation methods for applications such as improving crops or installing other valuable pathways. With other organisms, strong transcriptional control has typically required multiple gRNA target sites, which poses a critical engineering choice between heterogeneous sites, which allow each gRNA to target existing locations in a promoter, and identical sites, which typically require modification of the promoter. Here, we investigate the consequences of this choice for CRISPRi plant promoter regulation via simulation-based analysis, using model parameters based on single gRNA regulation and constitutive promoters in Nicotiana benthamiana and Arabidopsis thaliana. Using models of 2-6 gRNA target sites to compare heterogeneous versus identical sites for tunability, sensitivity to parameter values, and sensitivity to cell-to-cell variation, we find that identical gRNA target sites are predicted to yield far more effective transcriptional repression than heterogeneous sites.

模拟预测,在植物中,相同的gRNA靶位点比异质的gRNA靶位点更强的CRISPRi转录抑制。
植物合成生物学家一直致力于将CRISPRa和CRISPRi启动子调控方法应用于改良作物或安装其他有价值的途径。在其他生物体中,强转录控制通常需要多个gRNA靶点,这就在异质位点和相同位点之间提出了关键的工程选择,异质位点允许每个gRNA靶向启动子中的现有位置,而相同位点通常需要修饰启动子。在这里,我们通过基于模拟的分析,研究了这种选择对CRISPRi植物启动子调控的影响,使用了基于单gRNA调控和拟南芥组成启动子的模型参数。使用2-6个gRNA靶点的模型来比较异质和相同位点的可调性、对参数值的敏感性和对细胞间变异的敏感性,我们发现相同的gRNA靶点预计比异质位点产生更有效的转录抑制。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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