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. 2024 Dec 19;3(6):369–378. doi: 10.1021/cbmi.4c00089

Mini crRNA-Mediated CRISPR/Cas12a System Enhanced Imaging of Multiple MicroRNAs in Cells

Xiaolong Chen †, Qianyi You †, Yawen Guo †, Jing Zhang †, Qiao Hu †, Chaowang Huang †, Zhili Wu †, Junjiang Deng †, Jing Xu †, Peng Zhao †,*, Mingdong Hu †,‡,*
PMCID: PMC12188413  PMID: 40575085

Abstract

RNA imaging in live cells can provide comprehensive information on the expression, localization, degradation, storage, and regulation of RNA in cells, which is crucial for basic biology and clinical research. Our previous research finds that slicing the facilitated crRNA in the typical CRISPR/Cas12a system at a fitted site did not affect its trans-cleavage activity, which was previously reported to be triggered by targeted ssDNA or dsDNA, and a mini crRNA-mediated CRISPR/Cas12a (MCM-CRISPR/Cas12a) system was proposed. Here, we further apply it to enhanced imaging of MicroRNAs in cells by designing the activator in the system as a molecular beacon (MB), which can form a hybrid double-stranded structure of DNA/RNA with the targeted MicroRNA. When targeted MicroRNA is present, the hairpin structure of the MB is opened and the system emits fluorescence. Simultaneously, the DNA-RNA formed by the targeted MicroRNA and MB activates the trans-cleavage activity of LbCas12a, partially cleaving the single-stranded DNA extended from the MB and further enhancing the fluorescence intensity of the system. We designed the MCM-CRISPR/Cas12a system for miRNA-21, miRNA-155, and miRNA-10b and successfully applied it for sensitive and specific imaging of these MicroRNAs both inside and outside cells. This study provides a new idea for the sensitive and specific imaging of multiple MicroRNAs within cells, which is important for studying the distribution and dynamic changes of MicroRNAs in cells.

Keywords: MicroRNA, CRISPR/Cas12a, MCM-CRISPR/Cas12a, RNA imaging, molecular beacon

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1. Introduction

MicroRNAs are types of endogenous small RNAs with a length of approximately 18∼25 nucleotides; − they play an important role in gene expression regulation, cellular biology processes, disease occurrence and development, and immune regulation. − Due to the important role played by MicroRNAs in cells, achieving sensitive and specific imaging of MicroRNAs in cells is of great significance for a deeper understanding of the functions and regulatory mechanisms of MicroRNAs, improving the accuracy and early diagnosis of diseases, promoting drug development and innovation, and advancing the development of cell biology and regenerative medicine. − Nowadays, the main methods for imaging MicroRNAs in cells include fluorescence in situ hybridization (FISH), , locked nucleic acid (LNA) modified probe technology, , microarray technology, and nanoparticle and molecular imaging technology. , These methods have their own advantages and disadvantages in imaging MicroRNAs. For example, FISH can directly locate the expression location of MicroRNAs within cells, providing intuitive imaging results. However, its probe design and operations are relatively complex and require advanced technology. Besides, the sensitivity may not be sufficient to detect low abundance MicroRNAs. The probes used in LNA can enhance their binding ability with MicroRNAs, thereby improving the sensitivity of the detection. However, the high cost of these probes limits their application in large-scale experiments. In fact, compared to the technology that can image a single MicroRNA, the technology that can sensitively and specifically image multiple MicroRNAs at the same time is scarce because, in some cellular state changes, abnormal changes in multiple MicroRNAs are often involved. Micro-array technology can simultaneously detect the expression of multiple MicroRNAs, making it suitable for large-scale screening. Unfortunately, this method may have lower sensitivity compared to that of other methods. For MicroRNAs with high homology, there may be a problem of insufficient specificity. Overall, developing a new technology that can sensitively and specifically image multiple MicroRNAs in cells is still urgently needed.

The CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated proteins) system is an adaptive immune system derived from bacteria that protects bacteria from invading pathogens such as viruses by cleaving foreign DNA. Recently, this system has been developed into a powerful gene editing tool and is widely used in basic research and clinical treatment. With the deepening of research, the imaging application of this system in cells is also receiving increasing attention. , Compared to traditional imaging techniques, the CRISPR/Cas system has higher specificity and sensitivity and can achieve multicolor imaging, thus revealing molecular interactions and dynamic changes within cells more comprehensively. Nowadays, the application of the CRISPR/Cas system in cell imaging mainly focuses on the following aspects: (1) Genomic DNA imaging. By utilizing the specific recognition ability of the CRISPR/Cas system, Cas (such as dCas9, dCas12a, etc.) that has lost nuclease activity can be combined with fluorescent proteins or dyes to form fluorescent probes. These probes can target and bind to specific DNA or RNA sequences within cells, enabling real-time imaging of these sequences. Researchers have modified dCas9 or sgRNA to bind to multiple fluorescent proteins, enabling the real-time in situ imaging of intracellular nucleic acids. In addition, multisite imaging can also be achieved by modifying sgRNA, for example, by connecting sgRNA to different RNA adapters (such as MS2 or PP7 RNA adapters) to recruit adapter binding proteins (such as MS2 or PP7 coat proteins) fused with different fluorescent proteins to specific genomic sites. − At present, up to 6 colors of fluorescence imaging can be achieved by designing 6 orthogonal sgRNA scaffolds to simultaneously target 6 chromosomal loci, also known as CRISPRainbow. (2) Intracellular RNA imaging. Cas9 can use PAM to recognize and cleave target RNAs. Nelles developed a dCas9-GFP system for tracking mRNA in live cells. By designing sgRNAs that specifically target ACTB, CCNA2, and TFRC mRNA, researchers observed the accumulation of target RNA in RNA particles using dCas9-GFP, which is highly consistent with the results of RNA-FISH. In addition, due to the targeting and cleavage of RNA targets by the type VI CRISPR related protein Cas13 (formerly known as C2c2), its cleavage activity inactivated dCas13 has also been successfully applied for tracking and imaging target RNA. Although the CRISPR/Cas system has been applied in nucleic acid imaging in cells, sensitive and specific imaging for multiple MicroRNAs in cells still needs to be developed.

Here, three molecular beacons (MBs) targeting miRNA-21, miRNA-155, and miRNA-10b were designed for their imaging in vitro and in vivo. Results show that the fluorescence intensity emitted by MB in the mini crRNA-mediated CRISPR/Cas12a (MCM-CRISPR/Cas12a) system was significantly stronger than that of the MB opened by the targeted MicroRNAs. Interestingly, when two or more MBs target different MicroRNAs in one MCM-CRISPR/Cas12a system, they have a positive interaction in vitro. Based on this, we achieved enhanced imaging of miRNA-21, miRNA-155, and miRNA-10b in RAW 264.7 and A549 cells. This study provides a new idea for the sensitive and specific imaging of multiple MicroRNAs within cells, which is important for studying the distribution and dynamic changes of MicroRNAs in cells.

2. Experimental Section

2.1. Reagents and Materials

All nucleic acids used in this article, including DNA and RNA, were purchased from Shanghai Biotechnology Co., Ltd. (Shanghai, China). Their detailed information is shown in S1. All MBs undergo the following steps of annealing before use to form a stable hairpin structure: dilute all hairpins to a certain concentration with TE buffer and then maintain them at 95 °C for 5 min; after that, reduce them to 25 °C at a rate of 0.1 °C/s and maintain at 25 °C for 30 min. LbCas12a and its reaction solution were provided by New England Biolabs (Beijing, China). RNase inhibitor was also provided by New England Biolabs. Lipofectamin CRISPRMAX Cas9 was purchased from Thermo Fisher Scientific (America). The fluorescence detection of the system was carried out on an F-7000 FL Spectrophotometer (HITACHI, Japan). RAW 264.7 and A549 cells were fed a high sugar DMEM medium with 10% fetal bovine serum and 1.0% antibiotics (penicillin/streptomycin). When the density of cells reached 70–80%, the cells were seeded.

2.2. Verification of trans-Cleavage Activity of the MCM-CRISPR/Cas12a System

First, the trans-cleavage activity of the MCM-CRISPR/Cas12a system was validated. We designed one typical CRISPR/Cas12a system and two MCM-CRISPR/Cas12a systems targeting miRNA-21 and miRNA-155, respectively. In the typical CRISPR/Cas12a system, 2 μM LbCas12a, 2 μM crRNA, 10 U RNase inhibitor, 2 μM DNA activator, 5 μM reporter, and 2 μL of 10× NEB buffer r2.1 were added in an enzyme-free centrifuge tube and incubated at 37 °C for 60 min. After incubation, the fluorescence intensity of the system is detected using an F-7000 FL Spectrophotometer. During detection, the excitation wavelength was set at 470 nm, and the slot widths of excitation and emission wavelength were set at 10 nm and 10 nm, respectively. In the MCM-CRISPR/Cas12a system for miRNA-21 or miRNA-155, 2 μM LbCas12a, 2 μM precursor of crRNA, 10 U RNase inhibitor, 1 μM MB for miRNA-21 or miRNA-155, and 2 μL of 10× NEB buffer r2.1 were added in an enzyme-free centrifuge tube and incubated at 37 °C for 60 min. When detecting MB for miRNA-21, the excitation wavelength was set at 404 nm, and the slot widths of excitation and emission wavelength were set at 10 nm and 5 nm, respectively. When detecting MB for miRNA-155, the excitation wavelength was set at 518 nm, and the slot widths of excitation and emission wavelength were set at 10 nm and 10 nm, respectively. In the joint MCM-CRISPR/Cas12a system for miRNA-21 and miRNA-155, 2 μM LbCas12a, 2 μM precursor of crRNA, 10 U RNase inhibitor, 2 μM MB for miRNA-21, 1 μM MB for miRNA-155, and 2 μL of 10× NEB buffer r2.1 were added to an enzyme-free centrifuge tube and incubated at 37 °C for 60 min. Then, the fluorescence intensity of the system was detected separately in PB (for miRNA-21) and CY3 (for miRNA-155) channels.

2.3. Combining MCM-CRISPR/Cas12a with a Novel Molecular Beacon for MicroRNA Imaging in Vivo

Here, the MCM-CRISPR/Cas12a system containing LbCas12a (1500 ng), precursor of crRNA (250 ng), RNase inhibitor (10 U), and MBs (1 μM) for miRNA-21, miRNA-155, or miRNA-10b was subjected to cells through the Lipofectamin CRISPRMAX Cas9 kit. Its detailed formula is shown in S2. First, seed cells were 30–70% confluent at transfection. LbCas12a (1500 ng), precursor of crRNA (250 ng), RNase inhibitor (10 U), and MBs (1 μM) for miRNA-21, miRNA-155, or miRNA-10b were mixed (Tube 1). CRISPRMAX Reagent was diluted in Opti-MEM I Medium (Tube 2) and mixed well. The solution from Tube 1 was immediately added to Tube 2, and then, it was mixed well. The complex was incubated for 10 min at room temperature. Finally, the complex was added to the cells. The cells were incubated at 37 °C for 24 h. After incubation, the culture medium was removed and the cells were rinsed with 50–500 μL of PBS and lysed with 20–250 μL of lysis buffer; fluorescence microscopy (Olympus) and confocal microscopy (ZEISS 880) were performed.

For the MCM-CRISPR/Cas12a system without LbCas12a, the above steps during transfection were followed, except LbCas12a was not added to the MCM-CRISPR/Cas12a system. For the joint MCM-CRISPR/Cas12a system to detect two or more MicroRNAs, the other components in the MCM-CRISPR/Cas12a system were unchanged and the type and number of MBs were changed.

3. Results and Discussion

3.1. Comparison of DNase Activity between MCM-CRISPR/Cas12a System and Typical CRISPR/Cas12a System

Our previous research prospectively found that slicing the facilitated crRNA in the typical CRISPR/Cas12a system (Figure a) at a fitted site did not affect its trans-cleavage activity (DNase activity), and a mini crRNA-mediated CRISPR/Cas12a system (MCM-CRISPR/Cas12a) (Figure b) was proposed based on this. Firstly, we compared the DNase activity of the MCM-CRISPR/Cas12a system and typical CRISPR/Cas12a system in detecting microRNAs. As shown in Figure c, the designed MCM-CRISPR/Cas12a systems targeting miRNA-21 or miRNA-155 only emit fluorescence in the presence of miRNA-21 or miRNA-155, and they have nearly equal DNase activity to the typical CRISPR/Cas12a system. These results indicate that the designed MCM-CRISPR/Cas12a system have the potential for the highly sensitive and specific detection of MicroRNAs.

1.

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Principle and comparison of MCM-CRISPR/Cas12a system and the typical CRISPR/Cas12a for detecting microRNAs. (a) Schematic of the typical CRISPR/Cas12a system. (b) Schematic of the MCM-CRISPR/Cas12a system. (c) Comparison of trans-cleavage activity between the typical CRISPR/Cas12a and MCM-CRISPR/Cas12a system.

3.2. Combining MCM-CRISPR/Cas12a with a Novel Molecular Beacon for MicroRNA Detection in Vitro

To make the MCM-CRISPR/Cas12a system work, the precursor of crRNA, targeted MicroRNA, LbCas12a, single-stranded DNA activator, and molecular beacon (single-stranded DNA with a fluorescent group and fluorescence quenching group modified at both ends) are essential elements (Figure b). Here, we have designed a novel molecular beacon (abbreviated as MB afterward) that combines a single-stranded DNA activator with a traditional molecular beacon. It was designed as a hairpin, and the fluorescent group and its corresponding quenching groups were modified on its tail and middle (Figure a). The targeted MicroRNA can open the hairpin structure of MB and form RNA/DNA complexes with it, causing the fluorescent group and quenching group modified on the MB to move away and letting the system emit fluorescence. Moreover, the RNA/DNA complexes can activate the trans-cleavage activity of the MCM-CRISPR/Cas12a system, arbitrarily cleaving the single-stranded DNA extended from the MB, further enhancing the fluorescence intensity of the system (Figure a). To verify the progressiveness of this system, we compared the detection performance of this system with that of a traditional MB in detecting microRNAs. As seen in Figure b, the designed system can detect targeted RNA at low to 5 nM, which is 1 order of magnitude higher than traditional MB. As single-base mismatches can significantly impact the experimental results in RNA imaging and detection, we designed targeted RNAs containing 1 to 2 mutation points for the specificity validation of this system. Figure c shows that the single-base mutation of the targeted RNA has a significant impact on the trans-cleavage activity of the system, indicating that the designed system can specifically detect MicroRNAs. Then, we compared the detection performance of the complete MCM-CRISPR/Cas12a system (with LbCas12a) and the incomplete MCM-CRISPR/Cas12a system (without LbCas12a) in detecting targeted MicroRNAs. As shown in Figure a, the targeted miRNA-21 and miRNA-155 can open the hairpin structures of their corresponding MB through chain replacement reaction, causing the system to emit fluorescence. Further, when they were placed in the MCM-CRISPR/Cas12a system, the fluorescence intensity emitted by the MB is stronger. Compared to the fluorescence intensity with and without the addition of a MCM-CRISPR/Cas12a system, the fluorescence intensity with the addition of the MCM-CRISPR/Cas12a system is approximately 1.27 times higher than that without the addition of the MCM-CRISPR/Cas12a system (Figure f,g). This is because the binding of the targeted MicroRNA and MB can activate the trans-cleavage activity of LbCas12a in the MCM-CRISPR/Cas12a system, allowing for arbitrary cleavage of single-stranded DNA extending from MB and further enhancing the fluorescence intensity of the system.

2.

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Principle and performance study of the MCM-CRISPR/Cas12a system in detecting MicroRNAs. (a) The principle of the MCM-CRISPR/Cas12a system in detecting MicroRNAs. (b) Comparison of sensitivity between the MCM-CRISPR/Cas12a system and traditional MB in detecting MicroRNAs. (c) The specificity of the MCM-CRISPR/Cas12a system in detecting MicroRNAs. Comparison of the complete MCM-CRISPR/Cas12a system (+LbCas12a) and incomplete MCM-CRISPR/Cas12a system (-LbCas12a) in detecting miRNA-155 (d and f) and miRNA-21 (e and g).

Since the precursor of crRNA remains unchanged in the MCM-CRISPR/Cas12a system, we attempted to blend the two MCM-CRISPR/Cas12a systems for the detection of miRNA-21 and miRNA-155 together. When only miRNA-21 or miRNA-155 is present in the combined system, the MCM-CRISPR/Cas12a system designed for miRNA-21 or miRNA-155 comes into play. Only when both miRNA-21 and miRNA-155 are present does the designed combined detection system work, and the imaging effect is better than that of the individual system. This is because there is a mutually reinforcing effect in the combined system; that is, the trans-cleavage activity of the MCM-CRISPR/Cas12a system activated by miRNA-21 can cleave the MB in the MCM-CRISPR/Cas12a system designed for miRNA-155, thereby achieving a signal enhancement effect (Figure a). To verify this hypothesis, we conducted a series of relevant experiments. Figure b–d illustrates that the fluorescence intensity of channels in detecting miRNA-21 and miRNA-155 in a combined system is higher than channels in testing miRNA-21 or miRNA-155 separately, confirming our hypothesis.

3.

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Simultaneous detection of miRNA-21 and miRNA-155 using the MCM-CRISPR/Cas12a system. (a) Schematic of the joint MCM-CRISPR/Cas12a system in miRNA-21 and miRNA-155 detection. (b–d) Comparison of the performance between the joint MCM-CRISPR/Cas12a system and the separate MCM-CRISPR/Cas12a system in miRNA-21 and miRNA-155 detection.

3.3. Combining the MCM-CRISPR/Cas12a System with a Novel Molecular Beacon for MicroRNAs Imaging in Vivo

To achieve imaging of MicroRNAs in cells, we import the MCM-CRISPR/Cas12a system with or without LbCas12a into cells though liposomes. S3 shows that the imaging intensity of MicroRNAs in cells gradually increases with their coincubation time, but their fluorescence intensity does not significantly increase after 6 h of coincubation. In addition, the imaged subcellular organelles are mainly concentrated in the cytoplasm. Our previous research found that miRNA-155 was abnormally expressed in RAW264.7 cells induced by LPS, and it shows a time-dependent pattern that reached its highest expression at 24 h (S4). We imported the MCM-CRISPR/Cas12a system, which is designed for miRNA-155, into the RAW264.7 cells that have or have not been induced by LPS. The results show that the imaging intensity of the group in which the RAW264.7 cells have been induced by LPS was higher than that of the group in which the RAW264.7 cells have not been induced by LPS, and the imaging intensity of the group in which the MCM-CRISPR/Cas12a system contained LbCas12a was stronger than the group in which the MCM-CRISPR/Cas12a system did not have LbCas12a (Figures and S5). These results indicate that the MCM-CRISPR/Cas12a system designed for miRNA-155 can be used for miRNA-155 imaging in cells, and its imaging intensity depends on the expression level of miRNA-155.

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Application of the MCM-CRISPR/Cas12a system in miRNA-155 imaging in RAW264.7 cells.

3.4. Application of the MCM-CRISPR/Cas12a System in the Multiple MicroRNAs Imaging in Cells

We transport MCM-CRISPR/Cas12a systems designed for miRNA-21, miRNA-155, or their combination into A549 cells via liposomes (Figure a). The results find that the MCM-CRISPR/Cas12a system with LbCas12a is more advantageous than the MCM-CRISPR/Cas12a system without LbCas12a in miRNA-21 and miRNA-155 imaging in A549 cells (Figure b–d), further proving that the unabridged MCM-CRISPR/Cas12a system can arbitrarily cleave the partial single-stranded DNA modified with fluorescent groups derived from MB extension. Compared with the MCM-CRISPR/Cas12a system that images miRNA-21 or miRNA-155 separately, their combined system can image both miRNA-21 and miRNA-155, and there is a positive regulation between the two during the imaging process; that is, the trans-cleavage activity of the MCM-CRISPR/Cas12a system activated by miRNA-21 can act on the MCM-CRISPR/Cas12a system targeting miRNA-155 and vice versa (Figure b–d).

5.

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Application of the MCM-CRISPR/Cas12a system in miRNA-21 and miRNA-155 imaging in A549 cells. (a) Using the joint MCM-CRISPR/Cas12a system to image miRNA-21 and miRNA-155 in A549 cells. (b) Comparison of the imaging performance of the joint MCM-CRISPR/Cas12a system to the separate MCM-CRISPR/Cas12a system in miRNA-21 and miRNA-155 imaging in A549 cells. (c, d) Corresponding results of flow cytometry.

Subsequently, to achieve sensitive imaging of multiple MicroRNAs in cells, we designed the joint MCM-CRISPR/Cas12a system for miRNA-21, miRNA-155, and miRNA-10b imaging (Figure a). We first compared the imaging performance of the joint MCM-CRISPR/Cas12a system and the individual MCM-CRISPR/Cas12a system in imaging targeted MicroRNAs in vitro. As illustrated in Figure , when imaging a targeted MicroRNA, the joint MCM-CRISPR/Cas12a system capable of imaging three types of MicroRNAs is stronger than the joint MCM-CRISPR/Cas12a system capable of imaging two types of MicroRNAs, while the alone MCM-CRISPR/Cas12a system that can only image the targeted MicroRNA performs the worst in imaging. Then, we transport the designed joint MCM-CRISPR/Cas12a system into cells (Figure e). From Figure f, it can be seen that the joint MCM-CRISPR/Cas12a system designed for miRNA-21 and miRNA-10b can achieve sensitive imaging of miRNA-21 and miRNA-10b in A549 cells, while the joint MCM-CRISPR/Cas12a system designed for miRNA-155 and miRNA-10b can achieve sensitive imaging of miRNA-155 and miRNA-10b in A549 cells. Furthermore, the joint MCM-CRISPR/Cas12a system designed for miRNA-21, miRNA-155, and miRNA-10b can achieve imaging of these three MicroRNAs with the best imaging effect (Figure f). In addition, we conducted corresponding flow cytometry experiments, and the results were consistent with those of CLSM (S6), further verifying the above results.

6.

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Application of the joint MCM-CRISPR/Cas12a system in miRNA-21, miRNA-155, and miRNA-10b imaging in vitro or in A549 cells. (a) Schematic of miRNA-21, miRNA-155, and miRNA-10b imaging in vitro using the MCM-CRISPR/Cas12a system. (b–d) Comparison of the imaging performance of the trigeminal MCM-CRISPR/Cas12a system to the bigeminal MCM-CRISPR/Cas12a system in miRNA-21, miRNA-155, and miRNA-10b imaging in vitro. (e) Schematic of miRNA-21, miRNA-155, and miRNA-10b imaging in A549 cells using the MCM-CRISPR/Cas12a system. (f) Comparison of the imaging performance of the trigeminal MCM-CRISPR/Cas12a system to the bigeminal MCM-CRISPR/Cas12a system in miRNA-21, miRNA-155, and miRNA-10b imaging in A549 cells.

In addition to validating our hypothesis in A549 cells, we further validated it using RAW264.7 cells. As seen in Figure , the groups with the MCM-CRISPR/Cas12a system with LbCas12a showed stronger imaging performance in targeted MicroRNA compared to the groups with the MCM-CRISPR/Cas12a system without LbCas12a. In addition, the joint MCM-CRISPR/Cas12a system designed for two or more types of MicroRNAs can achieve imaging of the corresponding targeted MicroRNAs in RAW264.7 cells, and the imaging effect shows an increasing trend with the number of MicroRNAs presented (Figure ). In RAW264.7 cells, we observed a weak fluorescence intensity in the miRNA-21 imaging, which may be due to the low expression level of miRNA-21 in RAW264.7 cells. To confirm this hypothesis, we used RT-qPCR to detect the expression levels of these three MicroRNAs in the RAW264.7 cells. The results showed that the MicroRNA with the highest expression level in RAW264.7 cells was miRNA-155, followed by miRNA-10b, and the lowest expression level was miRNA-21 (S7). These results are consistent with our imaging results, further confirming the reliability of our designed method for imaging multiple MicroRNAs in cells.

7.

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Imaging performance of the separate MCM-CRISPR/Cas12a system in miRNA-21, miRNA-155, and miRNA-10b imaging in RAW264.7 cells.

8.

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Comparison of the imaging performance of the trigeminal MCM-CRISPR/Cas12a system to the bigeminal MCM-CRISPR/Cas12a system in miRNA-21, miRNA-155, and miRNA-10b imaging in RAW264.7 cells.

4. Conclusion

Here, we design a novel MB that can serve as both a DNA activator and a molecular beacon in the MCM-CRISPR/Cas12a system. When there is a targeted MicroRNA present, the targeted MicroRNA can open the hairpin structure of the designed MB, causing the fluorescent and quenching groups modified on the hairpin to move away, and the system emits fluorescence. Besides, the binding complex between the targeted MicroRNA and the designed MB can activate the trans-cleavage activity of LbCas12a, arbitrarily cleaving surrounding single-stranded DNA and further causing the system to emit fluorescence. Based on this, we designed the MCM-CRISPR/Cas12a system for multiple MicroRNAs imaging in vivo and in vitro. The results show that the designed MCM-CRISPR/Cas12a system can sensitively and specifically image multiple targeted MicroRNAs both inside and outside cells, and there is a synergistic reaction in imaging multiple MicroRNAs, further enhancing the imaging effect of targeted MicroRNAs. Overall, we utilized the MCM-CRISPR/Cas12a system and the newly designed MB to achieve enhanced imaging of multiple MicroRNAs inside cells, which is important for studying the distribution and dynamic changes of MicroRNAs in cells.

Supplementary Material

im4c00089_si_001.pdf (438.8KB, pdf)

Acknowledgments

This work was supported by the Young Scientists Fund of the National Natural Science Foundation of China (No. 32301155), Natural Science Foundation of Chongqing, China (CSTB2023NSCQ-MSX0388), Science and Technology Innovation Enhancement Project of Army Medical University (2022XQN33), and Young Doctoral Talent Incubation Program of the Xinqiao Hospital, Army Military Medical University (2022YQB065).

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/cbmi.4c00089.

  • S1: nucleic acids involved in this article; S2: detailed formula of MCM-CRISPR/Cas12a used in cell imaging; S3: subcellular organelles of MicroRNAs and their temporal dependence; S4: changes of miR-155 content in RAW264.7 cells after LPS induction; S5: application of the MCM-CRISPR/Cas12a system in miRNA-155 imaging in RAW264.7 cells (corresponding results of flow cytometry); S6: application of the joint MCM-CRISPR/Cas12a system in miRNA-21, miRNA-155, and miRNA-10b imaging in vitro or in A549 cells (corresponding results of flow cytometry); S7: Ct value of miRNA-21, miRNA-155, and miRNA-10b in RAW264.7 cells (PDF)

#.

X.C. and Q.Y. contributed equally.

The authors declare no competing financial interest.

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