Crispr-cas system using molecular chaperone to improve trans-cleavage activity and use thereof
By adding molecular chaperones ClpB, HSP70, Spy, or HSP20 to the CRISPR-Cas system, the problem of low trans-cleavage activity of the CRISPR-Cas system was solved, and the sensitivity of nucleic acid detection was improved.
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- INNOVATION ACAD FOR PRECISION MEASUREMENT SCI & TECH CAS
- Filing Date
- 2025-03-24
- Publication Date
- 2026-07-30
AI Technical Summary
The existing CRISPR-Cas system has low trans-cleavage activity, resulting in low sensitivity for detecting unamplified nucleic acids. It needs to be combined with isothermal amplification technology to achieve the best results, and the existing research is quite complicated.
Adding molecular chaperones such as ClpB, HSP70, Spy, or HSP20 to the CRISPR-Cas system can enhance the trans-cleavage activity of the Cas enzyme.
It significantly improved the trans-cleavage activity of the CRISPR-Cas system and enhanced the sensitivity of detecting unamplified nucleic acids.
Smart Images
Figure CN2025084421_30072026_PF_FP_ABST
Abstract
Description
CRISPR-Cas systems using molecular chaperones to enhance trans-cleavage activity and their applications Technical Field
[0001] This invention belongs to the field of gene editing and molecular diagnostics technology, specifically relating to a CRISPR-Cas system that uses molecular chaperones to enhance trans-cleavage activity and its applications. Background Technology
[0002] CRISPR-Cas systems hold great promise for gene editing and molecular diagnostics. The CRISPR-Cas12 system can be used for the detection of double-stranded DNA and, upon activation, possesses the ability to trans-cleave single-stranded DNA. The CRISPR-Cas13 system can be used for RNA detection and, upon activation, possesses the ability to trans-cleave single-stranded RNA. Cas enzymes themselves exhibit low trans-cleavage activity and low sensitivity for detecting unamplified nucleic acids, often requiring isothermal amplification techniques to achieve optimal results. Therefore, enhancing the trans-cleavage activity of CRISPR-Cas systems is crucial for molecular diagnostics. However, current research has only optimized Cas detection systems in a few cases, and these are relatively complex. Therefore, there is an urgent need to develop a simple, feasible CRISPR-Cas system with high trans-cleavage activity to enhance detection sensitivity.
[0003] Molecular chaperones are a class of helper proteins that assist in the folding, assembly, and transport of intracellular proteins. They have functions such as assisting in the folding of unfolded proteins or nascent polypeptide chains, participating in protein transport and localization, preventing protein misfolding, preventing protein aggregation, and repairing heat-denatured proteins. Current research on molecular chaperones mainly focuses on their role in regulating folded proteins; no literature reports on their involvement in regulating the activity of folded enzyme molecules. Summary of the Invention
[0004] The purpose of this invention is to address the aforementioned problems in the prior art by providing a CRISPR-Cas system that uses molecular chaperones to enhance trans-cleavage activity and its applications.
[0005] To achieve the above objectives, the technical solution of the present invention is as follows:
[0006] In a first aspect, the present invention provides a CRISPR-Cas system for enhancing trans-cleavage activity using a molecular chaperone, the CRISPR-Cas system comprising: 1 pM-1 mM Cas enzyme, 1 pM-1 mM CrRNA, 1× reaction buffer, 0.1 pM-1 mM target substrate, 1 nM-1 mM ssDNA reporter gene, and 1 pM-1 mM molecular chaperone.
[0007] The CRISPR-Cas system comprises: 1 nM-1 μM Cas enzyme, 2 nM-2 μM CrRNA, 1× reaction buffer, 1 pM-1 μM target substrate, 1 μM-100 μM ssDNA reporter gene, and 1 nM-100 μM molecular chaperone.
[0008] The Cas enzyme is the Cas12a enzyme.
[0009] The molecular chaperone is ClpB, HSP70, Spy, or HSP20.
[0010] The Cas enzyme is the Cas13a enzyme.
[0011] The molecular chaperone is Spy or HSP20.
[0012] The molecular chaperones are derived from prokaryotes, eukaryotes, or archaea.
[0013] The molecular chaperone is a recombinantly expressed protein or polypeptide fragment.
[0014] Secondly, this invention provides the application of the aforementioned CRISPR-Cas system in molecular diagnostics.
[0015] Thirdly, the present invention provides a kit comprising the aforementioned CRISPR-Cas system that uses a molecular chaperone to enhance trans-cleavage activity.
[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0017] The present invention describes a CRISPR-Cas system that uses molecular chaperones to enhance trans-cleavage activity. Adding molecular chaperones such as ClpB, HSP70, Spy, or HSP20 to the CRISPR-Cas system can enhance the trans-cleavage activity of Cas enzymes to varying degrees, thereby enhancing detection sensitivity. Attached Figure Description
[0018] Figure 1 shows the relative NMR signal intensity of CRISPR-Cas12a without the addition of molecular chaperones and with the addition of different molecular chaperones.
[0019] Figure 2 shows the relative NMR signal intensity of CRISPR-Cas13a without the addition of molecular chaperones and with the addition of different molecular chaperones.
[0020] Figure 3 shows the fluorescence curves of CRISPR-Cas12a without the addition of molecular chaperones and with the addition of different molecular chaperones.
[0021] Figure 4 shows the fluorescence curves of CRISPR-Cas13a without the addition of molecular chaperones and with the addition of different molecular chaperones.
[0022] Figure 5 shows the trans-cleavage rate of CRISPR-Cas12a without the addition of molecular chaperones and with the addition of different molecular chaperones.
[0023] Figure 6 shows the trans-cleavage rate of CRISPR-Cas13a without the addition of molecular chaperones and with the addition of different molecular chaperones.
[0024] Figure 7 shows the detection limits of CRISPR-Cas12a without the addition of the molecular chaperone and with the addition of the molecular chaperone HSP20.
[0025] Figure 8 shows the detection limits of CRISPR-Cas12a without the addition of the molecular chaperone and with the addition of the molecular chaperone HSP20. Detailed Implementation
[0026] The present invention will now be described in further detail with reference to specific embodiments and accompanying drawings.
[0027] Main materials and instruments:
[0028] Table 1 Main Materials and Instruments
[0029] Example 1: Comparison of Relative NMR Signal Intensities
[0030] The relative NMR signal intensity of Cas enzymes was detected without and with different molecular chaperones. The NMR reaction system for the group without molecular chaperones consisted of: 50 μM Cas12a or Cas13a enzyme, 20 mM NaP; pH 7.4, 10% D2O. The NMR reaction system for the group with molecular chaperones consisted of: 50 μM Cas12a or Cas13a enzyme, 100 μM molecular chaperone, 20 mM NaP; pH 7.4, 10% D2O. The molecular chaperones were selected from ClpB (derived from thermophilic bacteria), HSP70 (derived from mammals), Spy (derived from bacteria), and HSP20 (derived from archaea). Molecular chaperone acquisition steps: Expression vectors containing molecular chaperones ClpB, HSP70, Spy, and HSP20 (synthesized by Sangon Biotech Co., Ltd., NCBI accession numbers: ClpB WP_011228712.1, HSP70 KFO35963.1, Spy HCO1701554.1, HSP20 WP_048053707.1) were transformed into E. coli cells. After lysing the recombinant cells, the recombinant proteins ClpB, HSP70, Spy, and HSP20 were crudely purified by affinity chromatography, and then finely purified by molecular sieve to obtain purified ClpB, HSP70, Spy, and HSP20 proteins.
[0031] The relative NMR signal intensity results of the Cas12a enzyme are shown in Figure 1, and those of the Cas13a enzyme are shown in Figure 2. The Apo group in the figures represents the group without added molecular chaperones. As can be seen from Figure 1, the relative NMR signal intensity of the Cas12a enzyme with different added molecular chaperones is significantly lower than that without added molecular chaperones. This is because molecular chaperones ClpB, HSP70, Spy, and HSP20 can all bind to the Cas12a enzyme to form larger complexes, resulting in a decrease in NMR signal intensity. As shown in Figure 2, the relative NMR signal intensity of the Cas13a enzyme after adding molecular chaperones HSP70, Spy, and HSP20 was significantly lower than that of the Cas13a enzyme without molecular chaperones. However, the relative NMR signal intensity of the CRISPR-Cas13a system after adding molecular chaperone ClpB was not much different from that of the CRISPR-Cas13a system without molecular chaperones. This indicates that molecular chaperones HSP70, Spy, and HSP20 can bind to Cas13a to form a larger complex, resulting in a decrease in NMR signal intensity, while ClpB may not interact with Cas13a.
[0032] Example 2: Comparison of Reverse Cutting Rates
[0033] Fluorescence curves of the CRISPR-Cas12a / Cas13a system were detected with and without the addition of molecular chaperones and with the addition of different molecular chaperones.
[0034] Cas12a reaction procedure: In a 200 μL PCR tube, add 2 μL of 100 nM Cas12a, 2 μL of 200 nM crRNA, 2 μL of 10× reaction buffer, and 8 μL of RNase-free H2O sequentially, and incubate at 37 °C for 15 min. Then add 2 μL of 1 μM molecular chaperone, 2 μL of 10 nM dsDNA target substrate, and 2 μL of 2.5 μM ssDNA reporter gene, bringing the final volume to 20 μL. The molecular chaperone was selected from ClpB, HSP70, Spy, and HSP20. Use a real-time quantitative PCR instrument to record fluorescence intensity every 30 seconds at 37 °C.
[0035] Cas13a reaction procedure: In a 200 μL PCR tube, add 1 μL of 1 μM Cas13a, 1 μL of 1 μM crRNA, 1 μL of 40 U / μL RNase inhibitor, 5 μL of 10× reaction buffer, and 12 μL of RNase-free H2O sequentially, and incubate at 37°C for 10 min. Then add 5 μL of 1 μM molecular chaperone, 5 μL of 10 nM RNA target substrate, and 20 μL of 1 μM ssRNA reporter gene, bringing the final volume to 50 μL. The molecular chaperone was selected from ClpB, HSP70, Spy, and HSP20. Use a real-time quantitative PCR instrument to record fluorescence intensity every 30 seconds at 37°C.
[0036] Table 2 Sequence Information
[0037] The fluorescence curves of the Cas12a system are shown in Figure 3, and those of the Cas13a system are shown in Figure 4. The Ctl group in the figures represents the control group without a molecular chaperone. Based on Figures 3 and 4, the trans-cleavage rates of the CRISPR-Cas12a / Cas13a systems with and without different molecular chaperones were calculated. The calculated trans-cleavage rate of the Cas12a system is shown in Figure 5, and that of the Cas13a system is shown in Figure 6. The Ctl group in the figures represents the control group without a molecular chaperone. Figure 5 shows that the trans-cleavage rate of the Cas12a system with different molecular chaperones was significantly increased compared to the Cas12a system without a molecular chaperone. Figure 6 shows that the trans-cleavage rate of the Cas12a system with the addition of the molecular chaperones HSP20 and Spy was significantly increased compared to the Cas12a system without a molecular chaperone, while the fluorescence curves of the Cas12a system with the addition of the molecular chaperones ClpB and HSP70 were basically the same as those of the Cas13a system without a molecular chaperone.
[0038] Example 3: Detection Limit Comparison
[0039] Using CRISPR-Cas12a / Cas13a systems with and without the addition of molecular chaperone HSP20, the trans-cleavage rate was detected at target substrate concentrations of 0 pM, 3 pM, 10 pM, 30 pM, 100 pM, 300 pM, and 1000 pM. The limit of detection (LOD) was calculated using LOD = 3σ / K.
[0040] Cas12a reaction procedure: Add 2 μL of 100 nM Cas12a, 2 μL of 200 nM crRNA, 2 μL of 10× reaction buffer, and 8 μL of RNase-free H2O sequentially to a 200 μL PCR tube, and incubate at 37 °C for 15 min. Add 2 μL of 1 μM HSP20. For each system, add 2 μL of the above-mentioned different concentrations of dsDN target substrate and 2 μL of 2.5 μM ssDNA reporter gene, respectively, to a final system volume of 20 μL. Record fluorescence intensity every 30 seconds using a real-time quantitative PCR instrument at 37 °C.
[0041] Cas13a reaction procedure: In a 200 μL PCR tube, add 1 μL of 1 μM Cas13a, 1 μL of 1 μM crRNA, 1 μL of 40 U / μL RNase inhibitor, 5 μL of 10× reaction buffer, and 12 μL of RNase-free H2O sequentially, and incubate at 37°C for 10 min. Add 5 μL of 1 μM HSP20, and then add 5 μL of the different concentrations of RNA target substrate and 20 μL of 1 μM ssRNA reporter gene to each system, bringing the final volume to 50 μL. Record the fluorescence intensity every 30 seconds using a real-time quantitative PCR instrument at 37°C.
[0042] The detection limits of the Cas12a system, calculated based on the Cas12a fluorescence curve, are shown in Figure 7. The detection limits of the Cas13a system, calculated based on the Cas13a fluorescence curve, are shown in Figure 8. The Ctl group in the figures represents the control group without the molecular chaperone. Figure 7 shows that the addition of the molecular chaperone HSP20 increased the detection limit of the Cas12a system by 1.8 times, indicating that HSP20 can enhance the sensitivity of the CRISPR-Cas12a system in molecular diagnostics. Figure 8 shows that the addition of HSP20 increased the detection limit of the Cas13a system by 1.2 times, indicating that HSP20 can enhance the sensitivity of the CRISPR-Cas12a system in molecular diagnostics.
[0043] Example 4
[0044] A kit comprising the CRISPR-Cas system as described in Example 2.
Claims
1. A CRISPR-Cas system that uses molecular chaperones to enhance trans-cleavage activity, characterized in that: The CRISPR-Cas system comprises: 1 pM-1 mM Cas enzyme, 1 pM-1 mM CrRNA, 1× reaction buffer, 0.1 pM-1 mM target substrate, 1 nM-1 mM ssDNA reporter gene, and 1 pM-1 mM molecular chaperone.
2. The CRISPR-Cas system for enhancing trans-cleavage activity using molecular chaperones according to claim 1, characterized in that: The CRISPR-Cas system comprises: 1 nM-1 μM Cas enzyme, 2 nM-2 μM CrRNA, 1× reaction buffer, 1 pM-1 μM target substrate, 1 μM-100 μM ssDNA reporter gene, and 1 nM-100 μM molecular chaperone.
3. The CRISPR-Cas system for enhancing trans-cleavage activity using molecular chaperones according to claim 1, characterized in that: The Cas enzyme is the Cas12a enzyme.
4. The CRISPR-Cas system for enhancing trans-cleavage activity using molecular chaperones according to claim 3, characterized in that: The molecular chaperone is ClpB, HSP70, Spy, or HSP20.
5. The CRISPR-Cas system for enhancing trans-cleavage activity using molecular chaperones according to claim 1, characterized in that: The Cas enzyme is the Cas13a enzyme.
6. A CRISPR-Cas system for enhancing trans-cleavage activity using molecular chaperones according to claim 5, characterized in that: The molecular chaperone is Spy or HSP20.
7. A CRISPR-Cas system for enhancing trans-cleavage activity using a molecular chaperone according to claim 4 or 6, wherein the molecular chaperone is derived from prokaryotes, eukaryotes, or archaea.
8. A CRISPR-Cas system for enhancing trans-cleavage activity using a molecular chaperone according to claim 4 or 6, wherein the molecular chaperone is a recombinantly expressed protein or polypeptide fragment.
9. The application of a CRISPR-Cas system using molecular chaperones to enhance trans-cleavage activity as described in claim 1 or 2 in molecular diagnostics.
10. A kit comprising the CRISPR-Cas system for enhancing trans-cleavage activity using a molecular chaperone as described in claim 1.