School of Chemical and Biological Engineering, 1 Gwanak-ro, Gwanak-Gu, Seoul 08826, Korea.
Department of Chemical Engineering, Jeju National University, 102, Jejudaehak-ro, Jeju-si, Jeju-do 63243, Korea.
Nucleic Acids Res. 2023 May 22;51(9):4650-4659. doi: 10.1093/nar/gkad234.
Precise control of gene expression is essential for flux redistribution in metabolic pathways. Although the CRISPR interference (CRISPRi) system can effectively repress gene expression at the transcriptional level, it has still been difficult to precisely control the level without loss of specificity or an increase in cell toxicity. In this study, we developed a tunable CRISPRi system that performs transcriptional regulation at various levels. We constructed a single-guide RNA (sgRNA) library targeting repeat, tetraloop, and anti-repeat regions to modulate the binding affinity against dCas9. Each screened sgRNA could regulate the gene expression at a certain level between fully-repressing and non-repressing states (>45-fold). These sgRNAs also enabled modular regulation with various target DNA sequences. We applied this system to redistribute the metabolic flux to produce violacein derivatives in a predictable ratio and optimize lycopene production. This system would help accelerate the flux optimization processes in metabolic engineering and synthetic biology.
精确控制基因表达对于代谢途径中的通量重分配至关重要。虽然 CRISPR 干扰（CRISPRi）系统可以有效地在转录水平上抑制基因表达，但仍然难以在不损失特异性或增加细胞毒性的情况下精确控制水平。在这项研究中，我们开发了一种可调的 CRISPRi 系统，可在不同水平上进行转录调控。我们构建了一个靶向重复、四环和反重复区的单指导 RNA（sgRNA）文库，以调节 dCas9 的结合亲和力。每个筛选出的 sgRNA 可以在完全抑制和非抑制状态之间（>45 倍）调节特定水平的基因表达。这些 sgRNA 还可以与各种靶 DNA 序列进行模块化调节。我们将该系统应用于重分配代谢通量，以可预测的比例生产紫罗烯衍生物，并优化番茄红素的生产。该系统将有助于加速代谢工程和合成生物学中的通量优化过程。
