CN115820603B - DCasRx-NSUN6 single-gene-specific M5C modification editing method - Google Patents

Abstract

The invention provides an RNA-targeted 5-methylcytosine modification editing system, which comprises an inactivated CasRx nuclease fused 5-methylcytosine modification enzyme, a protein expression vector of an enzyme activity functional region of the enzyme, and a crRNA expression vector of at least one site of targeted RNA. Further the invention also targets crRNA expression vectors at a site in the CDS region of mRNA of RPSA. The invention also provides a preparation method and application of the RNA-targeted 5-methylcytosine modified vector system. The editing system provided by the invention can be used for carrying out targeted modification on the RNA 5-methylcytosine modification site to obtain the 5-methylcytosine modified RNA, and is accurate and efficient.

Description

DCasRx-NSUN6 single-gene-specific M5C modification editing method

Technical Field

The invention relates to the technical field of RNA editing, in particular to a dCasRx-m5C modified vector system targeting RNA methylation, and a construction method and application thereof.

Background

Cytosine 5-methylation (i.e., m 5C) is a relatively common modification in RNA, and studies have demonstrated that m5C modification occurs primarily in the nucleus, partially in the mitochondria, dynamically regulated by the NOL1/NOP2/SUN (NSUN) family and DNMT2 (m 5C 'Writer', m5C encoder) as methyltransferases and demethylases Tet2 (m 5C 'Eraser', m5C decomers). m5C can exert its biological effect by binding ALYREF and YBX1 (m 5C 'Reader', m5C Reader).

RPSA is an important transmembrane protein receptor, belonging to the family of non-integrins, also known as laminin receptor 1, consisting of 295 amino acids, including an intracellular, transmembrane and extracellular segment consisting of 85, 16, 194 amino acids, respectively. The study demonstrates that RPSA, as a type II transmembrane receptor, is a key node of a variety of signaling pathways, involved in a variety of biological processes including cell adhesion, differentiation, migration, signaling, neurite outgrowth and metastasis, plays an important role in the development of a variety of tumorigenesis, with a correlation between upregulation in cancer cells and their invasive and metastatic phenotypes. Our prior review of the data found that m5C methylation modification exists in the CDS of RPSA.

The CRISPR/Cas13 system is an adaptive immune system that bacteria and archaea evolve to protect against foreign virus or plasmid invasion. In the CRISPR/Cas13 system, after the crRNA forms a complex with the Cas13 protein, the Cas13 protein is activated, and can target and cleave the RNA, causing the RNA to break and damage. And Cas13 has two catalytic active centers, and after mutation, the Cas13 loses endonuclease activity, can only be combined with RNA, and can not cut RNA, so that endogenous RNA can be identified by dCasRx technology and can be used for targeted RNA tracing.

Disclosure of Invention

The invention provides an editing system for targeting RNA 5-methylcytosine modification, which comprises an inactivated CasRx nuclease system and a crRNA system, wherein the inactivated CasRx nuclease in the inactivated CasRx nuclease system is fused with a 5-methylcytosine modifying enzyme and/or a 5-methylcytosine modifying enzyme active functional region.

Preferably, the inactivated CasRx nuclease system comprises an inactivated CasRx nuclease protein expression vector, the inactivated CasRx nuclease protein expression vector comprises an inactivated CasRx nuclease fusion 5-methylcytosine modifying enzyme and/or a protein expression vector of a 5-methylcytosine modifying enzyme active functional region, and the crRNA system comprises a crRNA expression vector that targets an RNA site.

Preferably, any of the above is that the inactivated CasRx nuclease system and crRNA system is dCasRx-NSUN6 protein and crRNA complex and/or dCasRx-NSUN6 enzyme active functional region and crRNA complex. Can precisely locate the target RNA sequence and target the corresponding modification site. Preferably, the editing system is capable of m5C modification regulation of RNA either intracellularly or extracellularly.

Preferably, any of the above-mentioned inactivated CasRx nuclease protein expression vectors include PCS2-EF1 alpha-dCasRx-NSUN 6-T2A-EGFP vector and/or PCS2-EF1 alpha-dCasRx-NSUN 6 enzyme activity domain-T2A-EGFP vector. EF1 alpha is the name for the promoter and functions to initiate expression of the sequence.

Preferably, any of the above, the inactivated CasRx nuclease protein expression vector comprises the following sequence: (a) The EF1 alpha-SV 40-NLS-dCasRx sequence shown as SEQ ID NO. 1; (b) An NSUN6 enzyme sequence shown as SEQ ID NO.2 and/or an NSUN6 enzyme activity functional region sequence shown as SEQ ID NO. 3; (c) EGFP sequence shown in SEQ ID NO. 4.

Preferably, any of the above is that the editing system is capable of modulating 5-methylcytosine modification of RNA either intracellularly or extracellularly.

Preferably, any of the above cells include eukaryotic cells and prokaryotic cells; the eukaryotic cells include mammalian cells and plant cells; the mammalian cells include chinese hamster ovary cells, baby hamster kidney cells, mouse Sertoli cells, mouse mammary tumor cells, buffalo rat liver cells, rat liver tumor cells, monkey kidney CVI lines transformed by SV40, monkey kidney cells, canine kidney cells, human cervical cancer cells, human lung cells, human liver cells, HIH/3T3 cells, human U2-OS osteosarcoma cells, human a5413 cells, human K562 cells, human HEK2133T cells, human HCT116 cells or human MCF-7 cells or TRI cells.

Preferably, any of the above is that the crRNA sequence is altered based on changes in the target sequence, modifying and modulating RNA including mRNA, tRNA, rRNA, ncRNA for different targets. Preferably, the term "based on" means that the expressed sequence of the crRNA is fully complementary to the target.

Preferably, any of the above, the crRNA system targets at least one site in the CDS region of mRNA of RPSA.

Preferably, any of the above is that the crRNA system is a crRNA expression vector.

Preferably, any of the above-mentioned sequences of the crRNA expression vector are shown in SEQ ID NO. 15.

The invention also provides a preparation method of the targeted RNA 5-methylcytosine modified editing system, which comprises the following steps:

1) The EF1 alpha-dCasRx-T2A-EGFP sequence is amplified from a vector pXR002, EF1 alpha-dCasRx-T2A-EGFP, addgene #60954, and the EF1 alpha-dCasRx-T2A-EGFP sequence is connected to a pCS2+ vector through PCR amplification, recombination, connection and transformation to obtain an inactivated CasRx nuclease protein expression vector; the protein expression vector of the inactivated CasRx nuclease comprises an EF1 alpha-SV 40-NLS-dCasRx sequence shown as SEQ ID NO.1 and an EGFP sequence shown as SEQ ID NO. 4;

2) Amplifying NSUN6 enzyme and/or active region sequence thereof from HEK 293 cells, and connecting NSUN6 enzyme and/or active region sequence thereof to PCS2-EF1 alpha-dCasRx-T2A-EGFP vector by PCR amplification, recombination, connection and conversion to obtain an inactivated CasRx nuclease fusion 5-methylcytosine modified enzyme and/or protein expression vector of 5-methylcytosine modified enzyme active functional region; the NSUN6 enzyme sequence is shown as SEQ ID NO. 2; the sequence of the NSUN6 enzyme activity functional region is shown as SEQ ID NO. 3;

3) Constructing a crRNA scaffold structure comprising an enzyme cleavage site sequence and a crRNA scaffold sequence; synthesizing crRNA based on the target sequence; cloning the crRNA bracket structure and the crRNA based on the target sequence into an expression vector to obtain the expression vector comprising a promoter sequence, the crRNA based on the target sequence, the crRNA bracket sequence and an enzyme cleavage site sequence, wherein the crRNA based on the target sequence is shown as SEQ ID NO. 15; (the enzyme cutting site is the enzyme cutting site of the conventional enzyme cutting connection application)

The editing system for obtaining the target RNA 5-methylcytosine modification comprises a protein expression vector of the inactivated CasRx-methylcytosine modification enzyme and/or 5-methylcytosine modification enzyme active functional region fused by nuclease, and an expression vector comprising a target sequence-based crRNA, a crRNA bracket sequence and an enzyme cleavage site sequence of a promoter sequence.

Preferably, the expression vector in step 2) is a pcs2+ plasmid vector.

The invention also provides application of the editing system in preparation of medicines for treating diseases caused by RNA modification abnormality.

The invention also provides a medicament for treating diseases caused by RNA modification abnormality, which comprises the editing system.

Based on the prior art, in order to find a new idea for treating diseases caused by abnormal m5C modification of RNA, the invention connects m5C modification enzyme or an enzyme activity functional domain thereof to the C end of dCasRx for the first time, and specifically targets crRNA to RNA by designing crRNA so as to carry out m5C modification on a specific site (shown in figure 4); the invention provides a new way for preventing and treating human diseases caused by RNA m5C modification abnormality through a strategy of specifically targeting a substrate by engineering RNA modification enzyme to change RNA m5C modification.

As a first aspect of the invention, the invention provides an RNA-targeting 5-methylcytosine modified vector system comprising an inactivated CasRx nuclease fusion 5-methylcytosine modified enzyme or an enzymatically active functional region thereof protein expression vector, a crRNA expression vector targeting at least one site in the CDS region of mRNA of RPSA.

It should be noted that CasRx, also known as Cas13, a nuclease, in the present invention; CDS refers to a coding fragment of a messenger RNA (mRNA) molecule; crRNA refers to the guide RNA (CRISPR guide RNA) of nuclease CasRx. Wherein, the crRNA expression vector can target one site, two sites or three sites or even more sites in the CDS region of mRNA of RPSA, and can be selected according to the requirement.

Preferably, the nucleotide sequence corresponding to the 5-methylcytosine modifying enzyme or the enzymatically active functional region thereof is linked to the C-terminus of the nucleotide sequence corresponding to CasRx inactivated by nuclease activity, whereby the expressed fusion protein (i.e., the inactivated CasRx nuclease fused to the 5-methylcytosine modifying enzyme or the protein corresponding to the enzymatically active functional region thereof) can be subjected to 5-methylcytosine modification of a particular RNA under the guidance of a crRNA.

Preferably, the 5-methylcytosine modifying enzyme is an NSUN6 enzyme; the enzyme activity functional region of the 5-methylcytosine modifying enzyme is an NSUN6 enzyme activity region sequence.

Preferably, the inactivated CasRx nuclease system of the present invention comprises ribonucleic acid which is circular DNA, and comprises eukaryotic expression vector, and nuclear localization sequence (SV 40-NLS sequence, 250 th to 270 th base sequence in SEQ ID NO. 1) and NSUN6 enzyme or active region sequence thereof inserted into the eukaryotic expression vector. Thus, the expressed NSUN6 enzyme can methyl catalyze the first cytosine in the specific motif-CUCCA, i.e., cytosine 5-methyl (i.e., m 5C) modification in the present invention. Preferably, the inactivated CasRx nuclease system comprises circular DNA which is a protein expression vector of the inactivated CasRx nuclease fusion 5-methylcytosine modifying enzyme and an enzyme activity functional region thereof.

Preferably, the eukaryotic expression vector is inserted with an EGFP sequence. EGFP sequences act as tags for successful intracellular expression.

As a second aspect of the invention, the invention provides a method for preparing an mRNA-targeted 5-methylcytosine modified vector system comprising the steps of:

(1) The EF1 alpha-dCasRx-T2A-EGFP sequence is amplified from a vector pXR002, and the EF1 alpha-dCasRx-T2A-EGFP sequence is connected to a pCS2+ vector through PCR amplification, recombination, connection and transformation to obtain a protein expression vector of inactivated CasRx nuclease; the protein expression vector of the inactivated CasRx nuclease comprises: the EF1 alpha-SV 40-NLS-dCasRx sequence is shown as SEQ ID NO. 1; the EGFP sequence is shown as SEQ ID NO. 4;

(2) Amplifying NSUN6 enzyme and an active region sequence thereof from HEK 293 cells, and connecting the NSUN6 enzyme and/or the active region sequence thereof to a PCS2-EF1 alpha-dCasRx-T2A-EGFP vector through PCR amplification, recombination, connection and conversion to obtain an inactivated CasRx nuclease fused 5-methylcytosine modified enzyme protein expression vector and/or an inactivated CasRx nuclease fused 5-methylcytosine modified enzyme active functional region protein expression vector; the NSUN6 enzyme sequence is shown as SEQ ID NO. 2; the sequence of the NSUN6 enzyme active region is shown in SEQ ID NO. 3;

(3) Constructing a crRNA scaffold structure comprising an enzyme cleavage site sequence and a crRNA scaffold sequence; synthesizing crRNA based on the target sequence; cloning the crRNA bracket structure and the crRNA based on the target sequence into an expression vector to obtain the expression vector comprising a promoter sequence, the crRNA based on the target sequence, the crRNA bracket sequence and an enzyme cleavage site sequence, wherein the crRNA based on the target sequence is shown as SEQ ID NO. 15;

As a third aspect of the invention, the invention provides any of the editing systems described above and the use of the vector in the preparation of a medicament for treating a disease caused by an abnormality in RNA modification. The invention provides a preparation method and application of an accurate and efficient medicament for diseases caused by abnormal methylation modification of 5-methylcytosine.

As a fourth aspect of the present invention, there is provided a medicament for treating a disease caused by abnormal RNA modification, the medicament comprising the editing system described above and the vector provided. Among them, the patient corresponding to the disease caused by the abnormal RNA modification may be a human, or may be an animal, a plant, or the like, and is preferably a human.

The beneficial effects of the invention are as follows: the editing system can carry out targeted modification on the 5-methylcytosine caused by RNA modification abnormality, is accurate and efficient, and can fundamentally treat the diseases caused by RNA modification deficiency.

The CasRx nuclease is Cas13d nuclease, and dCasRx is inactivated CasRx nuclease or inactivated Cas13d nuclease.

The nucleotide sequences contained in the sequence table of the invention are all conventional nucleotide sequences, and do not comprise branched chain structures and nucleotide analogues.

Drawings

FIG. 1 is a map of the PCS2-EF1 alpha-dCasRx-NSUN 6-T2A-EGFP vector in preferred embodiment 1 of the present invention.

FIG. 2 is a map of the PCS2-EF1 alpha-dCasRx-NSUN 6 active region-T2A-EGFP vector of preferred embodiment 1 of the present invention.

FIG. 3 is a map of crRNA vector of preferred embodiment 2 of the present invention.

FIG. 4 is a schematic diagram of the modification of specific sites by m5C in accordance with the present invention.

FIG. 5 shows the results of enriched m5C modified RPSA mRNA after BSP experiments in preferred example 3 of the present invention.

FIG. 6 shows the mRNA expression levels of RPSA after cotransfection of plasmid PCS2-EF1 alpha-dCasRx-NSUN 6-T2A-EGFP and plasmid RPSA CRRNA back bone in preferred example 3 of the present invention.

FIG. 7 shows the mRNA expression levels of RPSA after cotransfection of plasmid PPCS, EF1 alpha-dCasRx-NSUN 6 enzyme activity domain-T2A-EGFP and plasmid RPSA CRRNA back in preferred example 3 of the present invention.

Detailed Description

The present invention will be more clearly and fully described by the following examples, which are intended to be illustrative of only some, but not all, of the examples. The examples are presented to aid in understanding the invention and should not be construed to limit the scope of the invention in any way.

Example 1

Construction of a target RNA dCasRx-2 XNLS (NLS is SV 40-NLS), NSUN6/NSUN6 enzyme activity region and EGFP fusion expression vector PCS2-EF1 alpha-dCasRx-NSUN 6-T2A-EGFP.

1. Primer design

The EF1 alpha-dCasRx-T2A-EGFP sequence is amplified from a vector pXR002:EF1 alpha-dCasRx-T2A-EGFP (addgene # 60954), wherein the EF1 alpha-SV 40 NLS-dCasRx sequence is shown in SEQ ID NO. 1.

The NSUN6 enzyme and the active region sequence thereof are amplified in HEK 293 cells, the NSUN6 enzyme sequence is shown as SEQ ID NO.2, and the NSUN6 enzyme active region sequence is shown as SEQ ID NO. 3.

The PCR primer of the three sequences is designed by using a seamless cloning primer design tool, and the primer sequences are synthesized by Shanghai biological company. The primer sequence table is shown in table 1 below:

TABLE 1 primer sequence listing

2. PCR amplification of related gene sequences

PCR reaction system, total 50ul:

PCR amplification conditions:

3. PCR product recovery

(1) After electrophoresis, a gel strip containing a target fragment is cut by a scalpel into a clean 1.5mlEP tube under the ultraviolet condition, and an equal volume of solution PN (Tiangen Biochemical technology (Beijing) Co.) is added into a centrifuge tube according to the proportion of 100mg gel to 100ul of solution PN.

(2) The centrifuge tube containing the glue block is placed in a water bath kettle with the temperature of 50 ℃, and the centrifuge tube is continuously and gently turned up and down to ensure that the glue block is fully dissolved.

(3) Column balancing: 500. Mu.l of the balance liquid BL was added to the adsorption column (the adsorption column was placed in the collection tube), and the mixture was centrifuged at 12,000rpm for 1min, and the waste liquid in the collection tube was discarded, and the adsorption column was replaced in the collection tube.

(4) The solution obtained in the second step was put into an adsorption column (the adsorption column was put into a collection tube), left at room temperature for 2min, centrifuged at 12,000rpm for 30-60sec, the waste liquid in the collection tube was poured out, and the adsorption column CA2 was put into the collection tube.

(5) 600. Mu.l of the rinse PW was added to the column, centrifuged at 12,000rpm for 30-60sec, the waste liquid in the collection tube was poured off, and the column was placed in the collection tube.

(6) And (5) repeating the operation step 5.

(7) The adsorption column was put back into the collection tube, and centrifuged at 12,000rpm for 2min to remove the rinse solution as much as possible. The adsorption column CA2 was left at room temperature for several minutes and dried thoroughly to prevent the residual rinse from affecting the next experiment.

(8) Placing the adsorption column into a clean centrifuge tube, suspending and dripping a proper amount of elution buffer EB into the middle position of the adsorption film, and standing at room temperature for 2min. The DNA solution was collected by centrifugation at 12,000rpm for 2min.

4. Recombinant recovery of fragments of interest

Two PCR fragments carrying homology arms at both ends were cloned into pCS2+ vector using ClonExpress technology, procedure same as ClonExpress Ultra One Step Cloning Kit (Vazyme C-01) kit instructions from Norpraise.

5. Conversion of ligation products

(1) The ligation product was added to 50. Mu.l DH 5. Alpha. Competent cells, gently swirled and mixed, and ice-bathed for 30min.

(2) The EP tube was rapidly transferred to an ice bath for 5min by heat shock in a water bath at 42℃for 90 s.

(3) 200 Μl of LB liquid medium was added, mixed well, and cultured with shaking at 37℃for 30min at 200 r/min.

(4) The bacterial solution in a 1.5ml EP tube was all applied to the surface of LB plate containing ampicillin (Amp) (100. Mu.g/ml). The plate was inverted and placed in a 37℃biochemical incubator overnight for incubation.

6. Sequencing to identify positive clones:

5 colonies were picked up from the plate and cultured in 10ml LB liquid medium containing ampicillin at 37℃and 220r/min with shaking for 8 hours, and 1ml of the bacterial liquid was poured into a 1.5ml centrifuge tube in an ultra clean bench and sent to Shanghai Biotechnology company for sequencing.

7. And (3) performing amplification culture on bacterial liquid with correct sequencing, and extracting the endotoxin-free plasmid.

8. The vector with correct sequencing is named as a PCS2-EF1 alpha-dCasRx-NSUN 6-T2A-EGFP vector, and the PCS2-EF1 alpha-dCasRx-NSUN 6 active region-T2A-EGFP vector, and the maps are shown in figures 1 and 2.

Example 2

Construction of crRNA expression vector targeting RPSA RNA

1. Design of crRNA sequences

The map of the crRNA vector is shown in figure 3, and the crRNA with higher targeting efficiency is designed by utilizing https:// cas13design.

Table 2 crRNA sequences

2. Adding enzyme cutting joint to the designed crRNA sequence, synthesizing by Shanghai biological company, the oligonucleotide chain is shown as SEQ ID NO. 16-17;

TABLE 3 oligonucleotide chains

RPSA-0-crRNA-F	AAACgaggatataacactgacatcagc SEQ ID NO.16
		RPSA-0-crRNA-R	AAAAgctgatgtcagtgttatatcctc SEQ ID NO.17

3. Linearization of crRNA backbone vectors

Plasmid pXR003: casRx gRNA cloning backbone ((addgene # 109053) was digested with BbsI endonuclease, and digested overnight at 37℃with the following cleavage system:

After enzyme digestion at 37 ℃, gel running is verified, and recovery and purification are performed (same as in step 3 of example 1).

4. Annealing

Annealing the synthesized oligonucleotide chain, placing the oligonucleotide chain in a PCR instrument, and carrying out 5min at 95 ℃ according to the annealing system as follows:

After the annealing was completed, it was taken out from the PCR and left at room temperature for 30min.

5. Connection

The annealed oligonucleotide strand was ligated to the digested vector pXR003: casRx gRNA cloning backbone, placed in a ligation apparatus at 16℃overnight, and the ligation system was as follows:

6. Conversion of ligation products

The ligated plasmid was transformed into competent cells DH 5. Alpha. In the same manner as in example 1, step 5.

7. Sequencing to identify positive clones:

3 colonies were picked up from the plate and cultured in 10ml LB liquid medium containing ampicillin at 37℃and 220r/min with shaking for 8 hours, and 1ml of the bacterial liquid was poured into a 1.5ml centrifuge tube in an ultra clean bench and sent to Shanghai Biotechnology company for sequencing.

8. And (3) performing amplification culture on bacterial liquid with correct sequencing, and extracting the endotoxin-free plasmid.

9. The vector with correct sequencing is named RPSA CRRNA backbone (namely the crRNA expression vector disclosed by the invention, namely the expression vector comprising a target sequence-based crRNA of a promoter sequence, a crRNA bracket sequence and an enzyme cutting site sequence).

EXAMPLE 3 RNA targeting 5-methylcytosine modified vector System

One embodiment of the mRNA targeting 5-methylcytosine modified vector system of the invention includes PCS2-EF1 alpha-dCasRx-NSUN 6-T2A-EGFP, the vector map is shown in FIG. 1; the PCS2-EF1 alpha-dCasRx-NSUN 6 enzyme active center-T2A-EGFP, and the vector diagram is shown in figure 2; RPSA CRRNA backbone, vector map is shown in FIG. 3.

RNA BSP sequencing and QPCR

1. Transiently transfected cells

(1) 293T cells were cultured and appropriate amount of cells were separated in 6-well plates, and cultured overnight to achieve a cell density of about 80%.

(2) The experiment was divided into three groups designated as a, B, C. Group A co-transfects plasmid PCS2-EF1 alpha-dCasRx-NSUN 6-T2A-EGFP and plasmid RPSA CRRNA back bone in a 2:1 ratio, wherein plasmid PCS2-EF1 alpha-dCasRx-NSUN 6-T2A-EGFP 2 mug; group B co-transfecting plasmid PCS2-EF1 alpha-dCasRx-NSUN 6 active center-T2A-EGFP and plasmid RPSA CRRNA back bone in a ratio of 2:1, wherein plasmid PCS2-EF1 alpha-dCasRx-NSUN 6 active center-T2A-EGFP 2 mug; group C co-transfected plasmid PCS2-EF1 alpha-dCasRx-EGFP and plasmid RPSA CRRNA backup in 2:1, wherein plasmid PCS2-EF1 alpha-dCasRx-EGFP 2 mug.

(3) Transfection was performed as per Yeasen company HIEFF TRANSTM Liposomal Transfection Reagent protocol.

(4) After 48h of cell culture following transfection, cells were washed with PBS.

2. Extraction of total RNA by Trizol method

1Ml Trizol was added to blow the cells, transferred to a 1.5ml centrifuge tube, and RNA was extracted according to the procedure instructions provided for the product.

Bisulphite treatment of RNA

RNA was processed according to the product instructions using EZ RNA Methylation kit kit from ZYMO RESEACH, to finally obtain BSP-post-RNA.

Synthesis of cDNA

Reverse transcription is carried out on RNA before and after treatment, reverse transcription is carried out by using a reverse transcription kit according to the steps provided by the product, and the reaction system is as follows:

The reaction conditions were 42℃for 15min and 95℃for 3min.

5.RT-PCR

And (3) performing RT-PCR by using a designed PCR primer, wherein the RT-PCR primer is shown as SEQ ID NO. 20-SEQ ID NO. 21, and the reaction system is as follows:

The reaction procedure was as follows:

TABLE 3RT-PCR primer sequences

RPSA-BSP-F	AAATTTTAAGAGGATTTGGGAGAAGTTTTTG SEQ ID NO.20
		RPSA-BSP-R	CAACCCTAAAATCAATAACCACAAAAAACCATA SEQ ID NO.21

6. Agarose gel electrophoresis

Electrophoresis is carried out on the PCR product by agarose gel with the concentration of 2%, the agarose gel is subjected to 120V 35min, a target strip is cut and put into a 1.5ml centrifuge tube for recovery of the PCR product, and the specific method is shown in the step 3 of the example 1.

7. Ligation of fragments of interest

The recovered target fragment is connected to a T vector, pGM-T cloning kit of Beijing Tiangen biochemical technology is used, the operation is carried out according to specific steps on the kit instruction, the connection is carried out at 16 ℃ overnight, and the connection system is as follows:

8. Conversion of ligation products

The ligated plasmid was transformed into competent cells DH 5. Alpha. And co-screened by adding x-Gal and IPTG, as in example 1, step 5.

9. Sequencing to identify positive clones:

20 white colonies were picked up from the plates respectively, cultured in 10ml LB liquid medium containing ampicillin at 37℃and 220r/min under shaking for 8 hours, and 1ml of the bacterial liquid was poured into a 1.5ml centrifuge tube in a super clean bench and sent to Shanghai Biotechnology company for sequencing.

10、RT-QPCR

The cDNA formed by reverse transcription of RNA which has not undergone BSP treatment is subjected to RT-QPCR, and the QPCR primers SEQ ID NO. 22-SEQ ID NO. 25 are shown in Table 4, and the reaction system is as follows:

The Q-RTPCR run was as follows:

TABLE 4 RT-QPCR primer sequences

RPSA-E23-89-F	GCAGCTCGTGCAATTGTT SEQ ID NO.22
		RPSA-E23-89-R	GTGGCAGCAGCAAACTTC SEQ ID NO.23
RPSA-E34-101-F	CGTGCAATTGTTGCCATTGA SEQ ID NO.24
		RPSA-E34-101-R	GCAGCAAACTTCAGCACAG SEQ ID NO.25

11. Analysis of experimental results:

As shown in FIG. 5, the experimental group of co-transfected plasmids PCS2-EF1 alpha-dCasRx-NSUN 6-T2A-EGFP and plasmid RPSA CRRNA back-bone and the experimental group of co-transfected plasmids PCS2-EF1 alpha-dCasRx-NSUN 6 enzyme activity center-T2A-EGFP and plasmid RPSA CRRNA back-bone were significantly higher than the control group (p < 0.05) after BSP experiments, which demonstrated that the vector system of example 1 constructed by us could effectively make m5C modifications to the RPSA-specific sites (where WT was the non-transfected wild-type cell control group, dCasRx-NSUN6 was the experimental group of co-transfected plasmids PCS2-EF1 alpha-dCasRx-NSUN 6-T2A-EGFP and plasmid RPSA CRRNA back-bone, dCasRx-NSUN6-PUA mtase was the experimental group of co-transfected plasmids PCS2-EF1 alpha-dCasRx-NSUN 6 enzyme activity center-T2A-EGFP and plasmid RPSA CRRNA back-bone). Meanwhile, as shown in FIG. 6, the RT-QPCR result shows that mRNA expression level of the experimental group RPSA of the cotransfected plasmid PCS2-EF1 alpha-dCasRx-NSUN 6-T2A-EGFP and the plasmid RPSA CRRNA backup is significantly increased (p < 0.05) compared with the control group (wherein WT is an untransfected wild type cell control group, RPSA-crRNA is a cotransfection experimental group, E23-89 is a detection result with primer RPSA-E23-89, and E34-101 is a detection result with primer RPSA-E34-101). As shown in FIG. 7, the RT-QPCR results showed that mRNA expression levels of the experimental group RPSA of the co-transfected plasmid PPCS-EF 1 alpha-dCasRx-NSUN 6 enzyme active center-T2A-EGFP and plasmid RPSA CRRNA backup were significantly increased (p < 0.05) compared to the control group (wherein WT is an untransfected wild-type cell control group, RPSA-crRNA is a co-transfected experimental group, E23-89 is a detection result with primer RPSA-E23-89, and E34-101 is a detection result with primer RPSA-E34-101).

Claims (2)

1. An editing system for targeting RNA 5-methylcytosine modification comprising an inactivated CasRx nuclease system and a crRNA system, wherein the inactivated CasRx nuclease in the inactivated CasRx nuclease system is fused to a 5-methylcytosine modifying enzyme and/or a 5-methylcytosine modifying enzyme active functional region, the inactivated CasRx nuclease system comprises an inactivated CasRx nuclease protein expression vector comprising a PCS2-EF1 a-dCasRx-NSUN 6-T2A-EGFP vector and/or a PCS2-EF1 a-dCasRx-NSUN 6 enzyme active functional region-T2A-EGFP vector, comprising the sequence: (a) The EF1 alpha-SV 40-NLS-dCasRx sequence shown as SEQ ID NO. 1; (b) A NSUN6 sequence shown as SEQ ID NO.2 and/or a NSUN6 enzyme activity functional region sequence shown as SEQ ID NO. 3; (c) EGFP sequence shown in SEQ ID NO. 4; the crRNA system is a crRNA expression vector with a sequence shown as SEQ ID NO. 15.

2. A method of preparing the RNA-targeted 5-methylcytosine modified editing system of claim 1, comprising the steps of:

1) The EF1 alpha-dCasRx-T2A-EGFP sequence is amplified from a vector pXR002, and is connected to a pCS2+ vector through PCR amplification, recombination, connection and transformation to obtain a protein expression vector of inactivated CasRx nuclease; the protein expression vector of the inactivated CasRx nuclease comprises an EF1 alpha-SV 40-NLS-dCasRx sequence shown as SEQ ID NO.1 and an EGFP sequence shown as SEQ ID NO. 4;

2) Amplifying NSUN6 enzyme and/or active region sequence thereof from HEK 293 cells, and connecting NSUN6 enzyme and/or active region sequence thereof to PCS2-EF1 alpha-dCasRx-T2A-EGFP vector by PCR amplification, recombination, connection and conversion to obtain an inactivated CasRx nuclease fusion 5-methylcytosine modified enzyme and/or protein expression vector of 5-methylcytosine modified enzyme active functional region; the NSUN6 enzyme sequence is shown as SEQ ID NO. 2; the sequence of the NSUN6 enzyme active region is shown in SEQ ID NO. 3;

3) Constructing a crRNA scaffold structure comprising an enzyme cleavage site sequence and a crRNA scaffold sequence; synthesizing crRNA based on the target sequence; cloning the crRNA bracket structure and the crRNA based on the target sequence into an expression vector to obtain the expression vector comprising a promoter sequence, the crRNA based on the target sequence, the crRNA bracket sequence and an enzyme cleavage site sequence, wherein the crRNA based on the target sequence is shown as SEQ ID NO. 15;

The editing system for obtaining the target RNA 5-methylcytosine modification comprises a protein expression vector of the inactivated CasRx-methylcytosine modification enzyme and/or 5-methylcytosine modification enzyme active functional region fused by nuclease, and an expression vector comprising a target sequence-based crRNA, a crRNA bracket sequence and an enzyme cleavage site sequence of a promoter sequence.