Construction And Application Of DLwaCas13a-SunTag-BiFC Live-cell Imaging System

Previous studies have shown that RNA molecules are responsible for a variety of cellular functions,including transcription and translation of m RNA,structural support of biomacromolecules,regulation of gene expression or silencing,and catalytic functions.Understanding the dynamic transport of RNA,including real-time localization,origin,and destination,would help us study the behavior and function of cells in health and disease states and deepen our understanding of RNA.A variety of RNA imaging techniques have been developed for subcellular imaging of RNA.As the mainstream RNA imaging system,RNA FISH has achieved high-resolution RNA subcellular imaging,but RNA FISH requires fixed cells for imaging,which made it difficult to track RNA in cells.In recent years,with the development of CRISPR technology,a class of Cas13 family proteins specifically targeting RNA has been discovered.Because RNA nuclease inactivated Cas13 protein only bound to RNA,which can be used for RNA imaging in living cells.Based on Cas13 protein,RNA living cell imaging systems such as d Lwa Cas13a-NF and d Psp Cas13b-FP were developed.However,the presence of free fluorescent proteins resulted in high fluorescence background and low imaging resolution.Therefore,based on the principle of Cas13 protein targeting RNA,this study used d Lwa Cas13 a protein as a tracker of targeted RNA.d Lwa Cas13 a which fused 24×Sun Tag polypeptide is responsible for recruiting amounts of Venus fluorescent proteins.The recruited Venus fluorescent protein was divided into two parts by the bimolecular fluorescence complementation technology,which reduced the free fluorescent proteins when the two parts were combined to re-stimulate fluorescence.We have designed a new tool for RNA imaging with higher resolution and lower fluorescence background in living cells.This study includes the following three experimental contents.In the first part of the experiment,d Lwa Cas13a-Sun Tag-Bi FC system was designed and constructed.First,we conducted a co-localization analysis of Ppib m RNA in the same cell with the d Lwa Cas13a-Sun Tag-Bi FC system,and the results showed that d Lwa Cas13a-Sun Tag-Bi FC system could accurately locate Ppib m RNA in living cells without affecting its m RNA transcription.Furthermore,this system was compared with the earlier d Lwa Cas13a-NF system,and the results showed that d Lwa Cas13a-NF under sg RNA guidance did not significantly improve the efficiency of its labeled m RNA transport from the nucleus to the cytoplasm compared with the control group without sg RNA,which may lead to false-positive judgment of RNA imaging.In contrast,the d Lwa Cas13a-Sun Tag-Bi FC system under sg RNA guidance increased the efficiency about 2 times of labeled m RNA transport from the nucleus to the cytoplasm compared to the control group without sg RNA.In the second part of the experiment,d Lwa Cas13a-Sun Tag-Bi FC system was optimized for the fluorescence background,imaging resolution,and nuclear RNA localization.In addition,d Lwa Cas13a-Sun Tag-Bi FC system for cytoplasmic and nuclear RNA was successfully constructed,and cell lines for imaging cytoplasmic and nuclear RNA were also constructed.Experimental results show that the imaging resolution of 24×Sun Tag is 5 times compared to 2×Sun Tag.In addition,four nuclear localization signals were used to accurately locate Xist RNA in d Lwa Cas13a-Sun TagBi FC system.In the third part of the experiment,d Lwa Cas13a-Sun Tag-Bi FC system was applied in living cells.Among them,d Lwa Cas13a-Sun Tag-Bi FC system was used to label endogenous Ppib m RNA and image the co-localization of Ppib m RNA with endoplasmic reticulum and stress particles,which proved that Ppib m RNA could interact with er and stress particles.In another application,we combined d Lwa Cas13 aSun Tag-Bi FC system with MS2 system to image premature termination mutation(PTC)inducing Oxt m RNA exon skipping in the nucleus.In addition,we compared the signalto-noise ratio of d Lwa Cas13a-Sun Tag-Bi FC system with that of 12×MS2 system,and the results showed that the signal-to-noise ratio of d Lwa Cas13a-Sun Tag-Bi FC system was 1.5 times higher than that of 12×MS2 tag.In summary,this study successfully constructed a living cell RNA imaging system with a high resolution based on CRISPR-Cas13 system,Sun Tag,and bimolecular fluorescence complementation technology,providing a new tool for RNA localization and alternative splicing imaging in living cells.Meanwhile,the transport of Ppib m RNA to the endoplasmic reticulum and stress granules were imaged,providing evidence for the regulation of the endoplasmic reticulum to m RNA localization and function.