The University of Tokyo establishes new technique for fluorescence visualization

One of the major differences in cell structure between eukaryotes and bacteria is the presence or absence of multiple types of organelles surrounded by biological membranes. However, the molecular mechanisms that control the various functions of these organelles in eukaryotes, including their division and proliferation, are still not fully understood. The research group consisting of Graduate Student Naoto Tanaka, Associate Professor Yamato Yoshida, and colleagues of the Graduate School of Science at the University of Tokyo aimed to develop a new molecular biology tool that enables the rapid functional analysis of organelles using the unicellular alga Cyanidioschyzon merolae (designated as CZON), an extremely simple eukaryotic organism whose cell contains only a nucleus, and a single mitochondrion, chloroplast, and peroxisome. The research group successfully established a new technique called "CZON-cutter," which not only is capable of freely modifying any target gene sequence but also allows the simultaneous visualization of the four types of organelles using fluorescent protein labels of different color wavelengths.

The research group constructed a CZON YMT1 strain, in which Cas9 fused with the fluorescent protein Venus and a nuclear localization signal was incorporated into the genomic DNA of CZON. Next, a sequence encoding a single-guide RNA (sgRNA) for cleaving the genomic DNA sequence of interest and a reporter gene cassette encoding the orange fluorescent protein mScarlet fused with a mitochondrial translocation signal were introduced into the cells of this CZON YMT1 strain. Mixing DNA fragments with arbitrary sequences during this process allowed them to visualize the cell nucleus, mitochondrion, and chloroplast while freely editing the genome sequence. They also successfully inserted a gene cassette encoding the blue fluorescent protein mCerulean3 fused with a peroxisomal translocation signal into the truncated genomic DNA. This allowed them to observe the four types of organelles via their fluorescent labels: cell nucleus (Venus; yellow-green), mitochondrion (mScarlet; orange), peroxisome (mCerulean3; blue), and chloroplast (chlorophyll autofluorescence; red).

With the CZON-cutter established in this study, organelle fluorescence imaging can be easily and efficiently achieved through a fully systematized process, regardless of the target gene, by simply changing the sgRNA of only 20 bases that specify the genomic region to be modified. This technique has made it possible to achieve genome-wide high-throughput analysis, which was previously not possible.

Assistant Professor Yoshida explained the developments, saying "By incorporating fluorescent protein-encoding genes into the plasmid DNA used for genome editing, it is now possible to perform genome editing and fluorescently label multiple types of organelles at the same time. The use of CZON-cutter is expected to lead to the clarification of the functions of genes controlling organelles and of the mechanisms of various diseases involving organelles, the details of which still need to be thoroughly investigated."

The journal article detailing these findings can be found at the following link:
Journal of Cell Science
CZON-cutter - a CRISPR-Cas9 system for multiplexed organelle imaging in a simple unicellular alga Naoto Tanaka, Yuko Mogi, Takayuki Fujiwara, Kannosuke Yabe, Yukiho Toyama, Tetsuya Higashiyama, and Yamato Yoshida*
https://doi.org/10.1242/jcs.258948