Abstract
RNAs have important and diverse functions. Visualizing an isolated RNA in living cells provide us essential information of its roles. By now, there are two kinds of live RNA imaging systems invented, one is the MS2 system and the other is the Cas13a system. In this study, we show that when fused with split-Fp, CasE can be engineered into a live RNA tracking tool.
Introduction
CasE is a core component of type I-E CRISPR complex, which solely processes pre-crRNA by binding specific stem-loop region, which we called CasE Binding Site (CBS). Even with restriction digest, it still tightly binds to the 3’-terminus of mature crRNA1 (Figure 1A). The conserved His20 of CasE is involved in the catalysis activity2; The identical mutation, “ΔHis26-TtCse3, lost the catalytic activity but still tightly binds to its target3. In this study, by conjugating the split Venus to the N-and C-terminus of dCasE (“ΔHis20), a live RNA tracking tool, VN-dCasE-VC, was constructed. This system emits florescence only when the target RNAs are present, thus enhancing the signal to noise performance.
Results
Visualizing RNA in living cells requires the tracking tool without preference for specific sub-cellular distribution. The high-level expression and even distribution of CasE-GFP and dCasE-GFP in HEK293T cells indicated that CasE and dCasE are suitable for RNA manipulation in living cells (Figure 1B).
To test whether CasE has robust activity when expressed in mammalian cells, a “turn-off” reporter, plasmid CBS-GFP-N1, was constructed. It consisted of a GFP mRNA and a CasE Binding Site (CBS) in its 5’-UTR. When CasE was introduced into the system, the GFP expression level was sharply reduced (Figures 1C and 1D). To further test the CasE activity, a “turn-on” reporter, plasmid RED-16×CBS-Lin28-C1, was also constructed, in which the CBS was inserted in the 3’-UTR region of RED monomer gene and upstream of Lin28. Lin28 is an RNA nuclear retain signal, the RED monomer mRNA with a lin28 signal in its 3’-UTR can hardly be translated into protein4. The CBS-CasE dependent restriction cuts off the lin28 signal and release the target mRNA from nuclear for translation (Figure 1E and Figure 1F). These indicated that CasE can bind and cleave the CBS in mammalian cells.
Aiming at engineering the dCasE-CBS interaction into a RNA tracking system, we tried many schemes by conjugating the split-FP5–7 to the dCasE protein. We found that one version, the VN-dCasE-VC can hardly emit fluorescent which may due to un-properly folding, or un-stable state3, but when the target RNA (CBS) was bound, the Venus signal can be clearly captured under fluorescent microscopy even when the transfection dosage of VN-dCasE-VC expression plasmid is very low (Figure 2A and Figure 2B).
The VN-dCasE-VC system was then used to track the overexpressed β-actin mRNA in mammalian cells, with the MS2 system8–11 as a control. As shown in Figure 2C, both systems indicated the same localization of β-actin mRNA. However, the MS2 system showed vague image. It might be ascribed to excessive MS2 Coat Protein (MCP) without target mRNA binding. In contrast, the VN-dCasE-VC system showed strong fluorescence in the cytoplasm (Figure 2C).
We also observed that, adding more CBS to the target mRNA improves signal, although 2×CBS per molecule is enough for imaging the overexpressed β-actin mRNA (Figure 2D). Thus low abundant mRNA can be detected by increasing the number of CBS to compensate the low concentration.
Applying the VN-dCasE-VC system in imaging RNAs displays some interestingresults: 2×CBS RNA transcribed from H1 promoter mainly distributed in nuclear, while 16×CBS RNA from CMV promoter mainly located in cytoplasm(Figure 2E). This preliminary result may reveal that the length of RNA or type of promoter might affect the subcellular localization of RNA.
Discussion
We invented an new RNA tracking tool, which named VN-dCasE-VC. This system is able to tracking specific RNA without background in living cells. Small Molecular Weight of dCasE make it fit for live RNA tracking: dCasE is only a little bit bigger than MCP but much smaller than Cas13a (13 kDa of MCP; 22 kDa of dCasE; more than 130 kDa of Cas13a). More than that, VN-dCasE-VC system needs much less target regions than that of MS2 system: 2×CBS is enough for VN-dCasE-VC to obtain high resolution image (Figure 2C) while at least 24×MCP Binding Site (MBS) is needed for MS2 system9, 11. To further improve the performance of VN-dCasE-VC system, optimization of the fluorescent protein segmentation or screening of more Cas orthologues will be applied.