Shao Shipeng, Chang Lei, Sun Yuao, Hou Yingping, Fan Xiaoying, Sun Yujie
State Key Laboratory of Membrane Biology, Biodynamic Optical Imaging Center (BIOPIC), School of Life Sciences, Peking University , Beijing 100871, China.
ACS Synth Biol. 2018 Jan 19;7(1):176-186. doi: 10.1021/acssynbio.7b00268. Epub 2017 Sep 7.
The CRISPR/Cas9 system has made significant contributions to genome editing, gene regulation and chromatin studies in recent years. High-throughput and systematic investigations into the multiplexed biological systems require simultaneous expression and coordinated functioning of multiple sgRNAs. However, current cotransfection based sgRNA coexpression systems remain inefficient, and virus-based transfection approaches are relatively costly and labor intensive. Here we established a vector-independent method allowing multiple sgRNA expression cassettes to be assembled in series into a single plasmid. This synthetic biology-based strategy excels in its efficiency, controllability and scalability. Taking the flexibility advantage of this all-in-one sgRNA expressing system, we further explored its applications in single nonrepetitive genomic locus imaging as well as coordinated gene regulation in live cells. With its full potency, our method will facilitate the research in understanding genome structure, function and dynamics.
近年来，CRISPR/Cas9系统在基因组编辑、基因调控和染色质研究方面做出了重大贡献。对多重生物系统进行高通量和系统性研究需要多个sgRNA同时表达并协同发挥作用。然而，目前基于共转染的sgRNA共表达系统效率仍然较低，而基于病毒的转染方法相对昂贵且劳动强度大。在此，我们建立了一种不依赖载体的方法，使多个sgRNA表达盒能够串联组装到单个质粒中。这种基于合成生物学的策略在效率、可控性和可扩展性方面表现出色。利用这种一体化sgRNA表达系统的灵活性优势，我们进一步探索了其在单个非重复基因组位点成像以及活细胞中协同基因调控方面的应用。凭借其全部效能，我们的方法将有助于开展对基因组结构、功能和动态的研究。
