CN117187215A - CRISPR/CasRx system capable of being reassembled under induction of chemical small molecules and application thereof - Google Patents
CRISPR/CasRx system capable of being reassembled under induction of chemical small molecules and application thereof Download PDFInfo
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Abstract
The application belongs to the technical field of gene editing and gene therapy, and particularly relates to a CRISPR/CasRx system which is reassembled under the induction of chemical small molecules and application thereof. The CRISPR/CasRx system comprises NLS-CasRx (N) -XTEN-FRB-NLS-FLAG fusion protein and NLS-FKBP-XTEN-CasRx (C) -NLS-HA fusion protein, which can be polymerized into complete CasRx protein under the induction of chemical micromolecular substances. The application establishes a split-CasRx system with inducible regulation and control characteristics, which can knock down specific mRNA in human cells under the induction of rapamycin, achieves the aim of efficiently and controllably knocking down the expression level of the specific mRNA, and remarkably reduces the off-target rate of the traditional CRISPR/CasRx system.
Description
Technical Field
The application belongs to the technical field of gene editing and gene therapy, and particularly relates to a CRISPR/CasRx system which is reassembled under the induction of chemical small molecules and application thereof.
Background
The CRISPR/Cas system is an adaptive immune system for bacteria to resist external virus invasion, and various gene editing technologies developed based on the system greatly promote research progress in various fields in biomedicine. From the safety perspective, the direct in vivo knockout of a particular gene using the CRISPR/Cas9 system permanently disrupts the downstream biological function of the gene, and its off-target at the genomic level is also continuously transmitted to daughter cells, leading to serious safety implications for clinical use of the CRISPR/Cas9 system.
The CRISPR/Cas13 system is a class II VI CRISPR/Cas system that specifically targets single stranded RNA molecules. Because it targets RNA without affecting DNA, the CRISPR/Cas13 system does not affect the integrity of the cell genome. Among the Cas13 protein family, cas13d protein of ruminococcus flavus XPD3002 (RfxCas 13d, abbreviated as CasRx) exhibits high targeted silencing efficiency on mRNA in mammalian cells. Compared with the traditional RNA interference technology, the CasRx has the advantages of high knocking-down efficiency and low off-target rate. Currently, casRx has been widely used to degrade and silence target RNAs within cell lines, animal embryo tissues, and animal adult tissues. Because the CRISPR/CasRx system has the advantages of high efficiency, low off-target rate, high safety and the like, the CRISPR/CasRx system has good clinical application prospect for delivering the CRISPR/CasRx system into a body and targeting the silencing target RNA. However, CRISPR/Cas systems still face a number of potential safety hazards in vivo applications, including immunogenicity of Cas proteins and high off-target rates due to overexpression, among others. Therefore, achieving accurate expression of CasRx proteins within a specific type of cell and being able to control their expression time and intensity is of great importance for increasing the safety of their in vivo applications.
Disclosure of Invention
The application aims to provide a CRISPR/CasRx system which is reassembled by chemical small molecule induction based on the prior art, and a preparation method and application thereof. The system provided by the application can be matched with corresponding gRNA to target down specific mRNA efficiently and controllably under the induction of chemical small molecules, and the off-target rate is reduced.
The aim of the application can be achieved by the following measures:
a chemically small molecule induced reassembly CRISPR/CasRx system, abbreviated as sCasRx.v1 system, comprising NLS-CasRx (N) -XTEN-FRB-NLS-FLAG fusion protein and NLS-FKBP-XTEN-CasRx (C) -NLS-HA fusion protein, both capable of polymerizing into a complete CasRx protein under the induction of a chemically small molecule substance; wherein:
the DNA sequence of the NLS-CasRx (N) -XTEN-FRB-NLS-FLAG fusion protein is shown as SEQ NO.1, the amino acid sequence of the NLS-CasRx (N) -FRB fusion protein is shown as SEQ NO.5, and the NLS-CasRx (N) -FRB fusion protein or CasRx (N) -FRB fusion protein is called as the abbreviation;
the DNA sequence of NLS-FKBP-XTEN-CasRx (C) -NLS-HA fusion protein is shown in SEQ NO.2, the amino acid sequence of NLS-FKBP-CasRx (C) fusion protein or FKBP-CasRx (C) fusion protein is shown in SEQ NO. 6.
The application provides a preparation method of a CRISPR/CasRx system which is reassembled by chemical small molecule induction, comprising the following steps: dividing the HEPN of two RNA endonuclease domains in the CasRx protein into an N end and a C end equally, designing an N/C end cutting combination mode, and dividing a site into a non-structural region and a structural region to obtain a CasRx (N) fragment and a CasRx (C) fragment; synthesizing DNA sequences of rapamycin response elements FRB and FKBP, and recombining a CasRx (N) fragment and an FRB fragment, a CasRx (C) fragment and an FKBP fragment into a pCDNA3.1 vector respectively by using a Clonexpress kit, wherein the gene fragments are connected by an XTEN linker with the length of 18 amino acids; the NLS-CasRx (N) -XTEN-FRB-NLS-FLAG and NLS-FKBP-XTEN-CasRx (C) -NLS-HA plasmids were constructed.
The application provides another CRISPR/CasRx system which is reassembled under the induction of chemical small molecules, namely a sCasRx.v2 system for short, comprising NLS-CasRx (N) #3-XTEN-FRB-NLS-FLAG fusion protein and FKBP-XTEN-CasRx (C) #3-NES-HA fusion protein which can be polymerized into complete CasRx protein under the induction of chemical small molecule substances; wherein:
the DNA sequence of NLS-CasRx (N) #3-XTEN-FRB-NLS-FLAG fusion protein is shown in SEQ NO.3, the amino acid sequence of the NLS-CasRx (N) #3-FRB fusion protein or CasRx (N) #3-FRB is shown in SEQ NO.7, and the NLS-CasRx (N) #3-FRB fusion protein can be also called CasRx (N) -FRB fusion protein or CasRx (N) -FRB in the case of being clearly indicated as a sCasRx.v2 system;
the DNA sequence of FKBP-XTEN-CasRx (C) #3-NES-HA fusion protein is shown in SEQ NO.4, the amino acid sequence is shown in SEQ NO.8, the FKBP-CasRx (C) #3 fusion protein or FKBP-CasRx (C) #3 is also called FKBP-CasRx (C) fusion protein or FKBP-CasRx (C) in case of the sCasRx.v2 system being explicitly specified.
The chemical small molecule substance of the present application may be a chemical small molecule substance capable of fusion expression in the presence of the response elements FRB and FKBP, each carrying a nuclear localization sequence NLS, preferably rapamycin.
The application provides an application method of a CRISPR/CasRx system which is reassembled by chemical small molecule induction, comprising the following steps: after transfection of the CRISPR/CasRx system with gRNA into cells, the chemical small molecule species are used to induce the CRISPR/CasRx system to knock down mRNA expression levels within the cell or cell line.
In the application method, preferably, the chemical small molecule substance is rapamycin with 200 mu M, and the gRNA is NT gRNA, EGFR gRNA, VHL gRNA or KRAS gRNA; the cell or cell line is an EGFR cell, KRAS cell, vhlβ cell, HEK293T cell line or human lung fibroblast.
The application also includes a recombinant expression vector comprising the DNA sequences of the NLS-CasRx (N) -XTEN-FRB-NLS-FLAG fusion protein and the NLS-FKBP-XTEN-CasRx (C) -NLS-HA fusion protein, or the DNA sequences of the NLS-CasRx (N) #3-XTEN-FRB-NLS-FLAG fusion protein and the FKBP-XTEN-CasRx (C) #3-NES-HA fusion protein,
the DNA sequence of the NLS-CasRx (N) -XTEN-FRB-NLS-FLAG fusion protein is shown as SEQ NO. 1;
the DNA sequence of the NLS-FKBP-XTEN-CasRx (C) -NLS-HA fusion protein is shown as SEQ NO. 2;
the DNA sequence of NLS-CasRx (N) #3-XTEN-FRB-NLS-FLAG fusion protein is shown as SEQ NO.3
The DNA sequence of FKBP-XTEN-CasRx (C) #3-NES-HA fusion protein is shown in SEQ NO. 4.
In the recombinant expression vector, the vector is preferably a plasmid, a lentivirus, or a stem cell.
The application firstly screens and obtains a specific site for dividing the CasRx protein into an N-end part polypeptide segment (1-539 amino acids) and a C-end part polypeptide segment (540-967 amino acids). In order to further realize the controllable and accurate target mRNA knocking down target of the CasRx protein in human cells, the application fuses the CasRx (N) and FRB elements at the N end part of the CasRx protein through an XTEN connector, adds a nuclear localization sequence NLS at two ends, and is also connected with a FLAG tag at the 3' end for indicating protein localization, thus obtaining NLS-CasRx (N) -XTEN-FRB-NLS-FLAG fusion protein, and the whole fusion protein is driven to be expressed by an EF1 alpha promoter. Meanwhile, FKBP and CasRx protein C end part-CasRx (C) passes through an XTEN linker, nuclear localization sequences NLS are added at two ends, and an HA tag is connected at the 3' end for indicating protein localization, so that NLS-FKBP-XTEN-CasRx (C) -NLS-HA fusion proteins are obtained. Because FRB and FKBP dimerize under rapamycin induction, the CasRx (N) -FRB and FKBP-CasRx (C) fusion proteins also repolymerize in response to induction by an inducible chemical small molecule (e.g., rapamycin) to form the intact CasRx protein, the system is designated sCasRx.v1.
Further, after transfection of the sCasRx.v1 system and specific gRNAs into human HEK293T cells, rapamycin at a concentration of 200. Mu.M was able to induce the sCasRx.v1 system to effectively knock down the intracellular mRNA expression levels of EGFR, KRAS and VHL.
Further, the application optimizes the sCasRx.v1 system: the sCasRx.v2 system was obtained by replacing two NLS sequences in the NLS-FKBP-XTEN-CasRx (C) -NLS-HA fusion protein with one out-core sequence NES. The combined molecular cell biology experiment verifies that sCasRx.v2 can be matched with the induction of specific gRNA response rapamycin to target and knock down the expression level of specific mRNA in a human HEK293T cell line and a human lung fibroblast.
Furthermore, the simple addition of inducible chemical small molecules (such as rapamycin) does not affect the expression levels of EGFR, KRAS and VHL mRNA in HEK293T cells. Experiments prove that the sCasRx.v2 system can respond to rapamycin induction to knock down mRNA in human cells with high efficiency and specificity.
Importantly, compared with the traditional CRISPR/CasRx system, the off-target rate of the sCasRx.v2 system is reduced by 81%, and the application safety is remarkably improved.
Advantages of the present application compared to the prior art include:
1. the application establishes a novel split-CasRx system with inducible regulation and control characteristics, and the optimized sCasRx.v2 system can knock down specific mRNA in human cells under the induction of rapamycin, thereby realizing the aim of efficiently and controllably knocking down the expression level of the specific mRNA.
2. The sCasRx.v2 system established by the application obviously reduces the off-target rate of the traditional CRISPR/CasRx system and widens the clinical application prospect of the CRISPR/CasRx related system.
Drawings
FIG. 1 is a diagram of a working model of a Split-CasRx system;
under the induction of rapamycin, the fusion proteins of CasRx (N) -FRB and FKBP-CasRx (C) dimerize to form complete CasRx proteins, and target binding and target mRNA cleavage are performed under the guidance of gRNA, so that the specific mRNA knockdown is realized.
FIG. 2 is a functional verification of split site screening and knockdown mRNA for the sCasRx.v1 system;
in the figure, a: in each domain of the CasRx protein, the split site No.1 is located in the helil 1 domain, the split site No.2 is located in the HEPN I-II domain, and the split site No.3 is located between the HEPN I-II domain and the helil 2 domain;
b: predicting the protein structure of CasRx by using alpha fold software, and marking a segmentation site, wherein No.1 and No.2 are positioned in a structure region with higher rigidity, and No.3 is positioned in a loose non-structure region;
c: 3 pairs of split-CasRx plasmids are transfected in human HEK293T cells, gRNA targeting EGFR mRNA is co-transfected, a rapamycin induction system with the concentration of 200 mu M is added for working after 24 hours, and qRT-PCR experiment results show that three pairs of split-CasRx plasmids can mediate the knockdown of EGFR mRNA, but the knockdown efficiency of split-CasRx#3 is the highest, namely sCasRx.v1. (G) The efficiency of the gRNA complex split-CasRx#3 targeting VHL and KRAS mRNAs to knock down the corresponding mRNAs was verified in HEK293T cells, and the results indicate that the sCasRx.v1 system was able to knock down the corresponding mRNA expression levels with VHL and KRAS gRNA complexes.
FIG. 3 is a schematic diagram of the molecular architecture of the sCasRx.v1 system;
in the figure, casRx (N) -FRB fusion protein portion: the N end of CasRx is connected with FRB through an XTEN linker, two ends are respectively provided with an NLS nuclear positioning sequence, the 3' end is also connected with a FLAG tag for indicating protein positioning, and the whole fusion protein is driven to be expressed by an EF1 alpha promoter; FKBP-CasRx (C) fusion protein portion: the C ends of FKBP and CasRx are connected through an XTEN linker, two ends are respectively provided with an NLS nuclear positioning sequence, the 3' end is also connected with an HA tag for indicating protein positioning, and the whole fusion protein is driven to be expressed by an EF1 alpha promoter.
FIG. 4 is a graph of the effect of substrate knockdown caused by the presence of self-assembly in the sCasRx.v1 system;
in the figure, a: the positioning initially constructed and the basal knockdown efficiency of the sCasRx.v1 system in the nucleus are detected by qRT-PCR, and the result shows that the sCasRx.v1 system can mediate partial knockdown effect of EGFR mRNA under the condition of not adding rapamycin for induction, and the result proves that partial CasRx (N) and CasRx (C) proteins can self-assemble to form complete CasRx protein and show the basal knockdown activity of knockdown target RNA;
b: subcellular localization of the two fusion proteins NLS-CasRx (N) -FRB-NLS-FLAG and NLS-FKBP-CasRx (C) -NLS-HA in the sCasRx.v1 system was examined using immunofluorescence, which indicated that both CasRx (N) and CasRx (C) were successfully expressed and localized to the nucleus, which resulted in the possibility of mediating basal knockdown of target RNA after self-assembly of the two fusion proteins. Scale = 50 μm.
FIG. 5 is a graph of the effect of the optimized sCasRx.v2 system in significantly reducing self-assembly induced basal knockdown;
in the figure, a: schematic construction of sCasRx.v2 System molecular cloning vector. CasRx (N) -FRB fusion protein portion: the N end of CasRx is connected with FRB through XTEN linker, two ends are respectively provided with an NLS nuclear positioning sequence, the 3' end is also connected with a FLAG label, and the whole fusion protein is driven to express by EF1 alpha promoter. FKBP-CasRx (C) fusion protein portion: the C ends of FKBP and CasRx are connected through an XTEN linker, the 3 'end is connected with a NES nuclear-out sequence instead, the 3' end is also connected with an HA tag, and the whole fusion protein is driven to be expressed by an EF1 alpha promoter;
b: work model prediction of the scasrx.v2 system: under the condition that rapamycin is not added, the modified CasRx (N) and CasRx (C) fusion proteins cannot be assembled spontaneously due to different subcellular localization, so that complete CasRx proteins cannot be formed, and therefore, a baseline knockdown effect cannot be caused; under the induction of rapamycin, the translated CasRx (N) and CasRx (C) fusion proteins in cytoplasm are recombined in cytoplasm to form complete CasRx protein, and enter nucleus after balancing NLS and NES signals to play a role of knocking down target mRNA;
c: immunofluorescence was used to examine the expression of NLS-CasRx (N) -XTEN-FRB-NLS-FLAG and FKBP-XTEN-CasRx (C) -NES-HA fusion proteins in the cells, which indicated that CasRx (N) was localized in the nucleus and most CasRx (C) was localized in the cytoplasm, indicating that the optimized sCasRx.v2 system successfully defined CasRx (N) and CasRx (C) expressed in different regions in the cells. Scale = 50 μm;
d: the use of qRT-PCR technology verifies that the sCasRx.v2 system can efficiently mediate EGFR mRNA knockdown in human HEK293T cells in response to rapamycin induction at a concentration of 200. Mu.M.
FIG. 6 is a graph of the results of rapamycin treatment without significantly affecting mRNA expression levels in HEK293T cells;
in the figure, a: RNA expression levels of the DMSO control group and the rapamycin treatment group with the concentration of 200 mu M are not obviously different by using RNA-seq second-generation sequencing;
b: verification using qRT-PCR techniques confirmed that rapamycin treatment had no significant effect on mRNA expression levels of EGFR, PNPLA2, KRAS and VHL.
FIG. 7 is a graph of the results of sCasRx.v2 being able to mediate mRNA knockdown in primary cells;
in the figure, a: the qRT-PCR technology is utilized to verify that the sCasRx.v2 system can efficiently and specifically knock down the expression level of EGFR, KRAS and VHL mRNA in human lung epithelial cells in response to rapamycin induction with the concentration of 200 mu M;
b: verification using qRT-PCR technique confirmed that rapamycin treatment had no significant effect on mRNA expression levels of EGFR, KRAS and VHL in human lung epithelial cells.
FIG. 8 is a graph of the effect of sCasRx.v2 on the ability to mediate mRNA in primary cells;
the use of RNA-seq second generation sequencing to verify that the scasrx.v2 system significantly reduces the off-target rate of the traditional CRISPR/CasRx system for targeted knockdown EGFR mRNA in human HEK293T cells.
Detailed Description
The application will be better understood from the following examples. However, it will be readily appreciated by those skilled in the art that the description of the embodiments is provided for illustration only and should not limit the application as described in detail in the claims.
The gene sequences of specific grnas used in the description and examples of the present application are shown in table 1.
TABLE 1
| Name of the name | Sequence (5 '-3') |
| NT gRNA | TCACCAGAAGCGTACCATACTCG |
| EGFR gRNA1 | CACTGCTTTGTGGCGCGACCCTT |
| EGFR gRNA2 | CTATCCTCCGTGGTCATGCTCC |
| VHL gRNA | CAACAAAAATAGAGGGCAGAACCT |
| KRAS gRNA | ACCATAGGTACATCTTCAGAGTCCTTAA |
The gene sequences of specific qPCR primers used in the description and examples of the present application are shown in table 2.
TABLE 2
Example 1, construction of an inducible split-CasRx System sCasRx.v1 and verification of its effectiveness
1) The molecular structure of CasRx protein is predicted by using alpha Fold, compared with the analyzed EsCas13d protein structure (PDB: 6E 9E), the N/C end cutting combination mode is designed according to the principle that HEPN of two RNA endonuclease domains in the CasRx protein is equally divided into an N end and a C end, and a cutting site comprises a non-structural region and a structural region (figures 2A and 2B).
2) DNA sequences of the rapamycin response elements FRB and FKBP were obtained synthetically, and the CasRx (N) fragment and FRB fragment, casRx (C) fragment and FKBP fragment were recombined into the pCDNA3.1 vector, respectively, using the ClonExpress kit, with the gene fragments being linked by an XTEN linker of 18 amino acids in length (FIG. 3).
3) After constructing NLS-CasRx (N) -XTEN-FRB-NLS-FLAG and NLS-FKBP-XTEN-CasRx (C) -NLS-HA plasmids, transfecting the plasmids into HEK293T cells in a ratio of 1:1, and simultaneously co-transfecting plasmids expressing EGFR gRNA; after 24h transfection, 200 mu M rapamycin is added to induce the split-CasRx system to work, cells are collected after 48h induction, and then the mRNA level of EGFR in the cells is detected by using qRT-PCR technology, and the result shows that the N/C end segmentation mode of the CasRx protein with the highest knockdown efficiency is split-CasRx#3 (figure 3C), namely the sCasRx.v1 system.
4) Co-transfecting sCasRx.v1 system and gRNA plasmid targeting KRAS or VHL mRNA in HEK293T cells, adding 200 mu M rapamycin after 24 hours to induce the split-CasRx system to work, collecting cells after 48 hours of induction, and detecting the levels of KRAS and VHL mRNA in the cells by using qRT-PCR technology, thus indicating that the sCasRx.v1 system can cooperate with corresponding gRNA to target down specific mRNA under the induction of rapamycin.
5) Co-transfection of sCasRx.v1 system and EGFR mRNA targeting gRNA plasmids in HEK293T cells, EGFR mRNA levels were detected by qRT-PCR without rapamycin induction, and the results indicated that the sCasRx.v1 system had mRNA knockdown effects due to self-assembly of the CasRx (N) and CasRx (C) fusion proteins (FIG. 4A). Subcellular distribution of the CasRx (N) and CasRx (C) fusion proteins were separately spiked with FLAG and HA antibodies using immunofluorescence techniques, and the results indicated that both CasRx (N) and CasRx (C) fusion proteins were localized in the nuclei and self-assembly was readily occurred (fig. 4B).
Example 2 optimization of sCasRx.v2 System and reduction of off-target Rate
1) And removing two NLS sequences in the NLS-FKBP-XTEN-CasRx (C) #3-NLS-HA plasmid in the sCasRx.v1 system by using a molecular cloning high-fidelity PCR technology, and adding NES sequences after CasRx (C) #3 to obtain the FKBP-CasRx (C) #3-NES-HA fusion protein plasmid. NLS-CasRx (N) -XTEN-FRB-NLS-FLAG and FKBP-CasRx (C) #3-NES-HA fusion proteins together constitute the sCasRx.v2 system (FIG. 5A).
2) Subcellular distribution of the CasRx (N) and CasRx (C) fusion proteins is respectively tracked by FLAG and HA antibodies by immunofluorescence technology, and the results show that the CasRx (N) and CasRx (C) fusion proteins in the sCasRx.v2 system are mainly positioned in cell nuclei and cytoplasm respectively, and are not easy to self-assemble (figure 5C).
3) Co-transfection of the sCasRx.v2 system and EGFR mRNA targeting gRNA plasmids in HEK293T cells, EGFR mRNA levels were detected by qRT-PCR without rapamycin induction, and the results indicated that the sCasRx.v2 system did not cause basal knockdown effects of EGFR mRNA (FIG. 5D).
4) Co-transfecting sCasRx.v2 system and EGFR mRNA targeting gRNA plasmid in HEK293T cells, adding 200 μM rapamycin after 24h induced sCasRx.v2 system work, and collecting cells after 48h induction, and then detecting the intracellular EGFR mRNA level using qRT-PCR technique, the result shows that sCasRx.v2 system can cooperate with corresponding gRNA to target specific mRNA in knockdown human HEK293T cells under rapamycin induction (FIG. 5E).
5) Furthermore, the RNA-seq and qRT-PCR validation showed that rapamycin did not significantly affect mRNA expression levels in HEK293T cells (fig. 6).
6) Co-transfecting the sCasRx.v2 system and the gRNA plasmid targeting EGFR, KRAS or VHL mRNA in human lung epithelial cells, working with 200. Mu.M rapamycin added 24h later, and harvesting the cells 48h later after induction, and then detecting intracellular EGFR, KRAS or VHL mRNA levels using qRT-PCR technology, indicating that the sCasRx.v2 system is able to target specific mRNA in knockdown human primary cells under rapamycin induction in coordination with the corresponding gRNA (FIG. 7A). Verification using RNA-seq and qRT-PCR showed that rapamycin did not significantly affect mRNA expression levels in HEK293T cells (fig. 7B).
7) Co-transfecting sCasRx.v2 system and EGFR mRNA targeting gRNA plasmid in HEK293T cells was performed 24h later with 200. Mu.M rapamycin induction of sCasRx.v2 system and cells were harvested 48h later after induction to extract total RNA. In parallel, casRx and EGFR mRNA-targeted gRNA plasmids were co-transfected in HEK293T cells and total RNA was extracted from the cells after 48 h. RNA-seq analysis of the sCasRx.v2 system and the traditional CRISPR/CasRx system resulted in the amount of off-target mRNA in HEK293T cells when EGFR mRNA was knocked down. The results indicate that the sCasRx.v2 system caused a knockdown of 30 mRNA with the exception of EGFR, whereas the traditional CRISPR/CasRx system had a corresponding number of 158, with a 81% reduction in off-target rate (128/158) (FIG. 8).
The above embodiments are only for illustrating the technical solution of the present application, and are not limiting; although the application has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments may be modified or some technical features may be replaced equivalently; such modifications and substitutions do not depart from the spirit of the application.
Claims (10)
1. A chemically small molecule induced reassembly CRISPR/CasRx system characterized in that it comprises an NLS-CasRx (N) -XTEN-FRB-NLS-FLAG fusion protein and an NLS-FKBP-XTEN-CasRx (C) -NLS-HA fusion protein, both capable of polymerizing to a complete CasRx protein under the induction of a chemical small molecule substance; wherein:
the DNA sequence of the NLS-CasRx (N) -XTEN-FRB-NLS-FLAG fusion protein is shown as SEQ NO. 1; the DNA sequence of the NLS-FKBP-XTEN-CasRx (C) -NLS-HA fusion protein is shown as SEQ NO. 2.
2. The chemically small molecule induced reassembly CRISPR/CasRx system according to claim 1, characterized in that the amino acid sequence of the NLS-CasRx (N) -XTEN-FRB-NLS-FLAG fusion protein is shown in SEQ No. 5; the amino acid sequence of the NLS-FKBP-XTEN-CasRx (C) -NLS-HA fusion protein is shown in SEQ NO. 6.
3. The method for preparing the chemical small molecule induced reassembled CRISPR/CasRx system, as claimed in claim 1, is characterized in that a principle of equally dividing HEPN of two RNA endonuclease domains in CasRx protein into N end and C end is designed, an N/C end cutting combination mode is designed, and a cutting site comprises a non-structural region and a structural region, so as to obtain CasRx (N) fragments and CasRx (C) fragments; synthesizing DNA sequences of rapamycin response elements FRB and FKBP, and recombining a CasRx (N) fragment and an FRB fragment, a CasRx (C) fragment and an FKBP fragment into a pCDNA3.1 vector respectively by using a Clonexpress kit, wherein the gene fragments are connected by an XTEN linker with the length of 18 amino acids; the NLS-CasRx (N) -XTEN-FRB-NLS-FLAG and NLS-FKBP-XTEN-CasRx (C) -NLS-HA plasmids were constructed.
4. A chemically small molecule induced reassembly CRISPR/CasRx system characterized in that it comprises an NLS-CasRx (N) #3-XTEN-FRB-NLS-FLAG fusion protein and an FKBP-XTEN-CasRx (C) #3-NES-HA fusion protein, both capable of polymerizing to a complete CasRx protein under the induction of a chemical small molecule substance; wherein:
the DNA sequence of the NLS-CasRx (N) #3-XTEN-FRB-NLS-FLAG fusion protein is shown in SEQ NO. 3;
the DNA sequence of the FKBP-XTEN-CasRx (C) #3-NES-HA fusion protein is shown in SEQ NO. 4.
5. A chemically small molecule induced reassembly CRISPR/CasRx system as claimed in claim 3, characterized in that the amino acid sequence of the NLS-CasRx (N) #3-XTEN-FRB-NLS-FLAG fusion protein is shown in SEQ No. 7; the amino acid sequence of the FKBP-XTEN-CasRx (C) #3-NES-HA fusion protein is shown in SEQ NO. 8.
6. The chemically small molecule induced reassembly CRISPR/CasRx system according to claim 1 or 4, characterized in that said chemical small molecule substance is rapamycin.
7. The method of using the chemically small molecule induced reassembly of the CRISPR/CasRx system of claim 1 or 4, wherein after transfection of the CRISPR/CasRx system and gRNA into cells, the chemically small molecule substance is used to induce knockdown of mRNA expression levels within the cells or cell lines.
8. The method of claim 8, wherein the chemical small molecule substance is 200 μΜ rapamycin and the gRNA is NT gRNA, EGFR gRNA, VHL gRNA or KRAS gRNA; the cell or cell line is an EGFR cell, KRAS cell, vhlβ cell, HEK293T cell line or human lung fibroblast.
9. A recombinant expression vector comprising the DNA sequences of NLS-CasRx (N) -XTEN-FRB-NLS-FLAG fusion protein and NLS-FKBP-XTEN-CasRx (C) -NLS-HA fusion protein or the DNA sequences of NLS-CasRx (N) #3-XTEN-FRB-NLS-FLAG fusion protein and FKBP-XTEN-CasRx (C) #3-NES-HA fusion protein,
the DNA sequence of the NLS-CasRx (N) -XTEN-FRB-NLS-FLAG fusion protein is shown as SEQ NO. 1;
the DNA sequence of the NLS-FKBP-XTEN-CasRx (C) -NLS-HA fusion protein is shown as SEQ NO. 2;
the DNA sequence of the NLS-CasRx (N) #3-XTEN-FRB-NLS-FLAG fusion protein is shown as SEQ NO.3
The DNA sequence of the FKBP-XTEN-CasRx (C) #3-NES-HA fusion protein is shown in SEQ NO. 4.
10. Recombinant expression vector according to claim 9, characterized in that the vector is a plasmid, a lentivirus or a stem cell.
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