Moon Soo Young, Kim Mi Ri, An Nan-Yeong, Noh Myung Hyun, Lee Ju Young
Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Republic of Korea.
Division of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology (POSTECH), Pohang, Gyeongbuk 37673, Republic of Korea.
Nucleic Acids Res. 2025 Aug 11;53(15). doi: 10.1093/nar/gkaf818.
CRISPR (clustered regularly interspaced palindromic repeats)-mediated transcriptional regulation is a powerful and programmable approach for controlling gene expression. While CRISPR-based gene repression is well established in bacteria, simultaneous activation and repression remain challenging due to the limited availability of effective bacterial activation domains. Here, we provide an efficient dual-mode CRISPR activation and interference (CRISPRa/i) system that integrates an evolved protospacer adjacent motif (PAM)-flexible dxCas9 with an engineered Escherichia coli cAMP receptor protein (CRP). Through systematic optimization of the CRP domains and linkers, we developed a versatile effector capable of precise gene expression control when combined with dxCas9. Our dxCas9-CRP system demonstrated robust activation of upstream regulatory regions and effective repression of coding sequences, enabling targeted and programmable gene regulation. Using dual-fluorescent reporters, we validated the ability of this system to concurrently regulate multiple genes. Furthermore, with pooled guide RNA libraries, we applied the dxCas9-CRP system to increase violacein production in E. coli via genome-scale activation and repression in a coordinated manner, successfully identifying key regulatory targets that significantly increase production. Overall, this dual-mode CRISPRa/i system advances the potential for bacterial metabolic pathway rewiring, providing precise and flexible control for a wide range of biotechnological applications.
CRISPR（成簇规律间隔短回文重复序列）介导的转录调控是一种用于控制基因表达的强大且可编程的方法。虽然基于CRISPR的基因抑制在细菌中已得到充分确立，但由于有效细菌激活结构域的可用性有限，同时激活和抑制仍然具有挑战性。在此，我们提供了一种高效的双模式CRISPR激活与干扰（CRISPRa/i）系统，该系统将经过进化的原间隔序列临近基序（PAM）灵活的dxCas9与工程化的大肠杆菌cAMP受体蛋白（CRP）整合在一起。通过对CRP结构域和接头进行系统优化，我们开发了一种多功能效应器，当与dxCas9结合时能够精确控制基因表达。我们的dxCas9-CRP系统展示了对上游调控区域的强大激活作用以及对编码序列的有效抑制作用，实现了靶向和可编程的基因调控。使用双荧光报告基因，我们验证了该系统同时调控多个基因的能力。此外，通过汇集的向导RNA文库，我们应用dxCas9-CRP系统通过全基因组规模的协同激活和抑制来提高大肠杆菌中紫菌素的产量，成功鉴定出显著提高产量的关键调控靶点。总体而言，这种双模式CRISPRa/i系统提升了细菌代谢途径重新布线的潜力，为广泛的生物技术应用提供了精确且灵活的控制。
