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.

中文翻译：

---

多重sgRNA表达可实现通用的单个非重复性DNA标记和内源基因调节

近年来，CRISPR / Cas9系统为基因组编辑，基因调控和染色质研究做出了重要贡献。对多重生物学系统的高通量和系统研究要求同时表达和协调多个sgRNA的功能。但是，当前基于共转染的sgRNA共表达系统仍然效率低下，并且基于病毒的转染方法相对昂贵且劳动强度大。在这里，我们建立了一个独立于载体的方法，允许将多个sgRNA表达盒串联组装成一个质粒。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。利用这种多合一sgRNA表达系统的灵活性优势，我们进一步探讨了其在单个非重复性基因组基因座成像以及活细胞中的协调基因调控中的应用。凭借其强大的功能，我们的方法将有助于研究了解基因组的结构，功能和动力学。