RT Journal Article
SR Electronic
T1 Multiplexed sgRNA Expression Allows Versatile Single Non-repetitive DNA Labeling and Endogenous Gene Regulation
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 121905
DO 10.1101/121905
A1 Shipeng Shao
A1 Lei Chang
A1 Yuao Sun
A1 Yingping Hou
A1 Xiaoying Fan
A1 Yujie Sun
YR 2017
UL http://biorxiv.org/content/early/2017/03/29/121905.abstract
AB The CRISPR/Cas9 system has made significant contribution to genome editing, gene regulation and chromatin studies in recent years. High-throughput and systematic investigations into the multiplexed biological systems and disease conditions require simultaneous expression and coordinated functioning of multiple sgRNAs. However, current co-transfection based sgRNA co-expression systems remain poorly efficient 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 non-repetitive genomic locus imaging as well as coordinated gene regulation in live cells. With its strong potency, our method will greatly facilitate the understandings in genome structure, function and dynamics, and will contribute to the systemic investigations into complex physiological and pathological conditions.