CN114736902B - Construction method and application of sgRNA sequence and editing system for NNT gene demethylation - Google Patents
Construction method and application of sgRNA sequence and editing system for NNT gene demethylation Download PDFInfo
- Publication number
- CN114736902B CN114736902B CN202210545516.9A CN202210545516A CN114736902B CN 114736902 B CN114736902 B CN 114736902B CN 202210545516 A CN202210545516 A CN 202210545516A CN 114736902 B CN114736902 B CN 114736902B
- Authority
- CN
- China
- Prior art keywords
- nnt
- gene
- sgrna
- sequence
- sense strand
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/713—Double-stranded nucleic acids or oligonucleotides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0688—Cells from the lungs or the respiratory tract
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2510/00—Genetically modified cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
Abstract
The invention discloses an NNT gene demethylated sgRNA sequence which is selected from one of sgRNA1, sgRNA4, sgRNA5, sgRNA6, sgRNA9 and sgRNA10, or is selected from the combination of sgRNA1 to sgRNA 10. The invention belongs to the technical field of genetic engineering, screens out a specific target site from an NNT gene promoter region, designs an sgRNA sequence, and constructs an NNT gene demethylation editing system by using a CRISPR/dCas9-TET1 technology, thereby realizing the demethylation of the specificity of the NNT gene promoter region, having small disturbance on the methylation of the whole gene and having good specificity, safety and reliability.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a construction method and application of an NNT gene demethylated sgRNA sequence and an editing system.
Background
The NNT gene is positioned on chromosome 5 of a human genome, has the length of 6421bp, contains 22 exons and 21 introns, encodes protein containing 1086 amino acids, and has a CpG island which has the length of 673bp and contains 67 CpG loci in a gene promoter region. The NNT protein is a mitochondrial inner membrane protein, is a symmetrical homodimer consisting of two identical polypeptide chains, is ubiquitous in various types of cells throughout the body, and is expressed in the heart and the liver in the highest abundance. The NNT protein produces a large amount of NADPH by using proton gradients, which is used in various biosynthetic processes and maintains the major antioxidant system in the mitochondrial matrix consisting of TXNRD2/TXN2/PRDX3 and PRDX5 to control peroxide levels.
The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas 9 system is an acquired immune defense system and is used for protecting bacteria or archaea from invasion of foreign plasmids or phages, the CRISPR sequences of genomes of the bacteria or archaea can express RNA (ribonucleic acid) recognized with genome sequences of invaders, exogenous genome DNA (deoxyribonucleic acid) is cut under the action of CRISPR-associated enzyme (CAS 9) to achieve the aim of resisting invasion, and endonuclease active sites of gene sequences for expressing Cas9 protein with endonuclease activity are inactivated through artificial modification and connected with coding sequences of TET1 protein (DNA demethylase) to construct a CRISPR/dCas9-TET1 demethylation system.
In the prior art, similar technologies to CRISPR/dCas9-DNA methylation editing technology include zinc finger nuclease (ZFP) and transcription activator-like effector (TALE), but the latter two technologies are complex in design and assembly, time-consuming and labor-consuming and have higher off-target effect. The CRISPR/dCas9-DNA methylation editing technology is expected to realize specific operation of targeting multiple genes by designing a specific sgRNA sequence, is not limited by the size of the genes, and has the limitations of large plasmid, low transfection efficiency or difficult stable transfection. Through retrieval, no report of a scheme for regulating and controlling NNT gene DNA methylation level by using a CRISPR (clustered regularly interspaced short palindromic repeats) related system is found, so that the sgRNA sequence for demethylating the NNT gene promoter region, the method and the application thereof have important significance.
Disclosure of Invention
In order to solve the problems in the prior art, the specific target site is screened from the NNT gene promoter region, the sgRNA sequence is designed, and the NNT gene demethylation editing system is constructed by using the CRISPR/dCas9-TET1 technology, so that the NNT gene promoter region specificity demethylation is realized, the disturbance on the methylation of the whole gene is small, and the specificity, the safety and the reliability are good.
The objects of the invention will be further illustrated by the following detailed description.
In one aspect, the invention provides an sgRNA sequence with an NNT gene demethylated, comprising one of sgRNA1, sgRNA4, sgRNA5, sgRNA6, sgRNA9, and sgRNA10, or a combination selected from sgRNA1 to sgRNA10; the sense strand of the sgRNA1 is TTGGGCCAGCGACGTCCTCG (SEQ ID NO: 1), and the antisense strand is AAACCGAGGACGTCGCTGGC (SEQ ID NO: 2); the sense strand of the sgRNA2 is TTGGCTTTTGGTTCGGAGAGCGCG (SEQ ID NO: 3), and the antisense strand is AAACCGCGCTCTCCGAACCAAAAAG (SEQ ID NO: 4); the sense strand of the sgRNA3 is TTGGCGCGCGCTCTCCGAACCAAA (SEQ ID NO: 5), and the antisense strand is AAACTTTGGTTCGGAGCGCGCGCGCG (SEQ ID NO: 6); the sense strand of the sgRNA4 is TTGGACGTCCTCTCGCCGGCCCTCC (SEQ ID NO: 7), and the antisense strand is AAACTGAGGCCGAGGAGGACGT (SEQ ID NO: 8); the sense strand of the sgRNA5 is TTGGGTTTCGCCTGTCCCGAGAG (SEQ ID NO: 9), and the antisense strand is AAACCTCTCTGGGGACAGGCGAAAC (SEQ ID NO: 10); the sense strand of the sgRNA6 is TTGGGACAGGTCTCTCTCCCGGCG (SEQ ID NO: 11), and the antisense strand is AAACCGCCGGGGAGAGACCTGTC (SEQ ID NO: 12); the sense strand of the sgRNA7 is TTGGTCAAAGGCTAGTTACCCCA (SEQ ID NO: 13), and the antisense strand is AAACTGGGGGTAACTAGCCTTTGA (SEQ ID NO: 14); the sense strand of the sgRNA8 is TTGGGAACAGCCCAAACTCCGCGG (SEQ ID NO: 15), and the antisense strand is AAACCCGCGCGGAGTTGGGCTGTTC (SEQ ID NO: 16); the sense strand of the sgRNA9 is TTGGCGCGCGCCTAGGCGCAGCT (SEQ ID NO: 17), and the antisense strand is AAACAGCTGCCCCTAGGCGCGCGCG (SEQ ID NO: 18); the sense strand of the sgRNA10 is TTGGTCCCGGGCGCAGGGTGCATT (SEQ ID NO: 19), and the antisense strand is AAACAATGCACCTGCGCCGGGA (SEQ ID NO: 20).
A combination of sgrnas 1 to 10, namely sgRNA1, sgRNA2, sgRNA3, sgRNA4, sgRNA5, sgRNA6, sgRNA7, sgRNA8, sgRNA9, and sgRNA10; the amount of sgrnas used may be the same or different.
Preferably, the sgRNA sequence of the NNT gene promoter region is demethylated, and is selected from one of sgRNA1, sgRNA6, sgRNA9, and sgRNA10, or is selected from a combination of sgRNA1 to sgRNA 10.
More preferably, the sgRNA sequence with demethylated promoter region of NNT gene is characterized by: a combination selected from gRNA1 through sgRNA 10.
Correspondingly, the invention also provides a construction method of the NNT gene demethylation editing system, which comprises the following steps:
1) Artificially synthesizing the sgRNA sequence;
2) Inserting the sgRNA into a multiple cloning site of a sgRNA framework expression plasmid vector, converting, selecting a monoclonal strain, performing sequencing identification to obtain a sgRNA strain with a correct target sequence, and extracting a sgRNA recombinant plasmid;
3) Transiently transfecting the sgRNA recombinant plasmid and the TETv4 plasmid into a human lung adenocarcinoma cisplatin-resistant cell A549/DDP;
4) After transfection for 46-50h, flow fluorescence sorting is carried out on the cells to obtain a double-fluorescence positive cell strain, namely the A549/DDP with the NNT gene promoter region demethylated.
Preferably, the sgRNA backbone expression plasmid vector is pgRNA-modified. The pgRNA-modified plasmid is an sgRNA backbone expression vector containing the U6 promoter, and carries mCherry red fluorescence, ampicillin resistance, and puromycin resistance.
Preferably, the double-fluorescence positive cell strain is a mCherry and tagBFP double-fluorescence positive cell strain.
In another aspect, the invention also provides application of the sgRNA sequence with the demethylated NNT gene promoter region in preparation of a medicine for treating diseases related to NNT gene DNA methylation.
In addition, the invention also provides a demethylation kit of the NNT gene promoter region, which comprises the sgRNA sequence of the NNT gene promoter region demethylation or the sgRNA recombinant plasmid.
Compared with the prior art, the invention has the beneficial effects that:
(1) According to the invention, a specific target site is screened from an NNT gene promoter region, and a high-efficiency sgRNA sequence is designed, particularly, sgRNA1, sgRNA6, sgRNA9, sgRNA10, or a combination of sgRNA1, sgRNA6, sgRNA9 and sgRNA10, or a combination of sgRNA1 to sgRNA10 can realize the specific demethylation of the NNT gene promoter region, and the methylation level of an NNT gene CpG island is obviously reduced.
(2) The invention utilizes CRISPR/dCas9-TET1 technology, can construct and obtain the NNT gene demethylation editing system through simple transient transfection, is easy to operate and control, has high transfection efficiency and good stability, realizes the activation of genes at the genome level through an epigenetic regulation mode, promotes the expression of related proteins, has small disturbance on the methylation of the whole genes, good specificity, safety and reliability, and overcomes the defect of poor gene specificity of the existing demethylation technology.
(3) The invention also provides application of the sgRNA sequence with the NNT gene promoter region demethylated in preparation of a medicine for treating NNT gene DNA methylation related diseases, and the constructed NNT gene demethylation editing system provides an effective platform for research on influence of NNT genes on intracellular redox balance, and provides a powerful tool for research on DNA methylation related chronic diseases and tumors.
Drawings
Fig. 1 is a schematic flow chart of a second embodiment of the present invention.
Fig. 2 is a map of sgRNA backbone expression plasmid vectors used in the present invention.
FIG. 3 spectrum of TETv4 plasmid used in the present invention.
FIG. 4 is a graph showing the result of mRNA expression level measurement of A549/DDP cell line edited by DNA methylation of NNT gene obtained in example two of the present invention; wherein A549/DDP on the horizontal axis is a cell strain without any treatment; NT is a cell transfected with TETv4 plasmid and sgRNA plasmid that does not target any position; sg1 to sg10 are cells transfected with TETv4 and the corresponding sgrnas, respectively; sgpool1 is a cell strain transfected with TETv4 and sgRNA1, sgRNA6, sgRNA9, and sgRNA10; sgpool2 is a cell line transfected with TETv4 and sgrnas 1 to 10.
FIG. 5 is a graph showing the results of measuring the protein expression levels of A549/DDP cell lines edited by DNA methylation of NNT genes obtained in example two of the present invention.
FIG. 6 is a DNA methylation level detection result chart of A549/DDP cell line edited by NNT gene DNA methylation obtained in example two of the present invention.
FIG. 7 is a graph of the results of testing the effect of the inventive NNT gene methylation editing on LINE-1 methylation.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and examples.
In the invention, the materials and reagents involved are conventional commercial products, or can be obtained by conventional technical means in the field, such as pgRNA-modified plasmid purchased from Addgene company with product number #84477; TETv4 plasmid was purchased from Addgene, inc. under product designation #167983; linear polyethyleneimine is available from Polyscience under the product designation 24765-1.
A549/DDP cell culture conditions: DMEM medium (containing 10% by volume fetal bovine serum), 5% CO 2 And culturing at 37 ℃.
Example sgRNA sequences with demethylated promoter region of the NNT Gene
Aiming at the gene sequence of the NNT gene promoter region CpG island, an sgRNA sequence is designed through an experimental design website (https:// zlab. Bio/guide-design-resources), and a group of sgRNAs with higher scores are selected as alternatives at about 100bp intervals in the CpG island to obtain 10 groups of sgRNAs in total. Removing a PAN sequence at the tail end of the sgRNA obtained by website design, adding enzyme cutting sites at two ends of the sgRNA, adding TTGG at the 5 'end of a sense strand of each sgRNA sequence, and adding AAAC at the 5' end of an antisense strand, thereby forming a cohesive tail end which is complementary to the pgRNA-modified plasmid after the digestion by AaRI. The designed sgRNA was sent to shanghai agility for primer synthesis, as shown in table 1.
TABLE 1 sgRNA sequences with demethylated promoter regions of the NNT genes
Example two NNT Gene demethylation editing System construction method
The construction method of the NNT gene demethylation editing system comprises the following steps:
1) Artificially synthesizing sgRNA sequences described in Table 1; phosphorylating and annealing the 10 sgRNA sequences in table 1 using T4-polyribonucleotide kinase (T4 PNK), respectively;
2) Carrying out enzyme digestion on the pgRNA-modified plasmid by using AaRI endonuclease, purifying and recovering a linearized vector; linking the linearized vector and the annealed sgRNA double-stranded sequence on a PCR instrument at 25 ℃ for 30min by using T4 ligase; transforming the link product into escherichia coli competent cells DH5 alpha, carrying out ice bath for 30min, carrying out water bath at 42 ℃ for 45s, keeping on ice for 2min, and screening clones on an ampicillin resistant LB plate; selecting a positive monoclonal strain, culturing by shaking, and sending to a sequencing mechanism for sequencing, wherein a sequencing primer is a forward primer sequence of a U6 promoter: CAGCACAAAGGAAACTCACC (SEQ ID NO: 21), extracting sgRNA recombinant plasmid from sgRNA strain with correct sequencing; sgRNA recombinant plasmids obtained from the sgRNA1 are abbreviated as sg1, sgRNA recombinant plasmids obtained from the sgRNA2 are abbreviated as sg2, and the rest can be done in the same way; an sgRNA recombinant plasmid obtained by simultaneously using sgRNA6, sgRNA9, and sgRNA10 is abbreviated as sgpool1, and an sgRNA recombinant plasmid obtained by simultaneously using sgRNA1 to sgRNA10 is abbreviated as sgpool2;
3) The 6-well plate is inoculated with target cells A549/DDP, and the inoculation number of the cells is 1 multiplied by 10 5 A/well, after 24h of culture, transient transfection of human lung adenocarcinoma cisplatin-resistant cells a549/DDP was performed using linear polyethyleneimine transfection reagent at a ratio of TETv4: sgRNA recombinant plasmid =2000 ng;
4) And (3) performing flow-type fluorescence sorting 48h after transfection, wherein channels comprise mCherry (excitation wavelength of 587nm and emission wavelength of 610 nm) and tagBFP (excitation wavelength of 400nm and emission wavelength of 450 nm), and obtaining a double-fluorescence positive cell strain, namely the A549/DDP with the NNT gene promoter region demethylated.
Example three NNT Gene demethylation editing-related Effect detection
(1) Expression assay for NNT Gene mRNA
The total RNA of A549/DDP cells demethylated in the promoter region of the NNT gene obtained in example two was extracted by TRIzol method, reverse transcription was performed using reverse transcription kit of TAKARA to produce cDNA, and TOYOBO was used
The Green qPCR kit was used for qRT-PCR, and the results are shown in FIG. 4. Compared with A549/DDP blank control and non-targeted NT control, the preferred 10 groups of sgRNAs of the invention, alone or in combination, enable the mRNA level of the NNT gene to be increased with statistical differenceMeaning. P < 0.05, P < 0.01, P < 0.001 compared to non-targeted NT controls. Particularly, when 1 group of sgrnas are used alone, the effect of sg1, sg6, sg9 and sg10 is relatively good, and although sgrnas of sg2, sg3, sg7 and sg8 are relatively high in score, the effect of improving relative expression level of NNT is not ideal when used alone, so that the biological significance is not great; through multiple tests, the system has better biological significance when the relative expression quantity of the NNT is improved to more than 5 times. When sgRNA1 to sgRNA10 are transfected at the same time, the NNT gene is increased to the highest level, which reaches about 39 times of that of a control. The total amount of sgRNA used for simultaneous transfection of sgRNA1 to sgRNA10 was the same as the amount of sgRNA used for sg1, sg6, sg9 or sg10 alone, and was 1200ng each.
(2) Protein expression assay for NNT genes
The protein expression of the NNT gene of the A549/DDP cells demethylated in the promoter region of the NNT gene obtained in example two (sgRNA 1 to sgRNA10 were transfected at the same time, and the amount of each sgRNA was 120 ng) was examined by using a western blot method, and the results are shown in FIG. 5. Simultaneous transfection of sgRNA1 to sgRNA10 significantly increased NNT protein expression levels compared to non-targeted NT controls.
(3) Methylation level detection of NNT genes
The A549/DDP cells obtained by demethylating the NNT gene promoter region obtained in the second embodiment are digested, and cell genome DNA is extracted by utilizing a TIANGEN blood/cell/tissue genome DNA extraction kit.
Performing bisulfite modification and sequencing, specifically comprising the following steps:
(1) performing Bisulfite modification of genomic DNA using the QIAGEN EpiTect Fast DNA bisufite Kit;
(2) using Promega Go
PCR amplification is carried out on CpG island region of NNT gene in DNA modified by bisulfite by MDx Hot Start Polymerase kit, and the sequence of forward primer is as follows: GGGAGAAGTAAATGTGTTTTGAAA (SEQ ID NO: 22), reverse primer sequence: ATACTTCTTCCCCCACCTAAACCTAC (SEQ ID NO: 23); />
(3) Subjecting the PCR product to agarose gel electrophoresis using MACHEREY-NAGEL
Gel and PCR clean-up kit is used for cutting and recovering the target fragment;
(4) connecting the target fragment with a T Vector by using an Optimus chinensis biological pClone007 Versatile Simple Vector Kit;
(5) the ligation product was transformed into competent E.coli cells DH 5. Alpha. By ice bath 30min, water bath at 42 ℃ for 45 sec, ice for 2min, colonies were selected on ampicillin resistant LB plates, 10 colonies per plate were selected and sequenced by Beijing Optimoku Biotechnology Ltd.
The result of the methylation level assay is shown in fig. 6, and each group comprises 67 columns of dots from left to right, which represent CpG sites of 67 from upstream to downstream on the CpG island of the NNT gene. From top to bottom, 10 rows represent 10 replicates of methylation assay results. The light dots indicate that the site is not methylated and the dark dots indicate that the site is methylated. The percentage of dark dots in each set of 670 dots to the total number of dots is the methylation level of that set. As can be seen from fig. 6, sg1 to sg10 transfected with specific sgrnas, and sgpool1 and sgpool2 both reduced the methylation level of the CpG island region of the NNT gene compared to the a549/DDP blank control and the non-targeted NT control. In particular, the CpG island DNA methylation level of the NNT promoter region of the sgpool2 group decreased most dramatically from 91.34% to 55.52% of the NT control.
The methylation level of LINE-1 is considered to be a good surrogate indicator of the methylation level of the whole genome, and in order to examine the specificity of the methylation level regulation, the invention uses a pyrosequencing mode to detect whether the methylation editing of the NNT gene can influence the methylation level of the whole genome, and the result is shown in FIG. 7. From fig. 7, it can be seen that the sgpool2 group simultaneously transfected with sgrnas 1 to 10 had little effect on the methylation level of 3 representative CpG sites of LINE-1 when the NNT gene was subjected to methylation editing, indicating that this methylation method for NNT gene has good specificity, small perturbation on methylation of the entire gene, safety and reliability.
The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.
Sequence listing
<110> Zhongshan university
<120> construction method and application of sgRNA sequence and editing system for NNT gene demethylation
<160> 23
<170> SIPOSequenceListing 1.0
<210> 1
<211> 24
<212> DNA
<213> Artificial sequence (RenGongXuLie)
<400> 1
ttgggccagc gcgacgtcct ctcg 24
<210> 2
<211> 24
<212> DNA
<213> Artificial sequence (RenGongXuLie)
<400> 2
aaaccgagag gacgtcgcgc tggc 24
<210> 3
<211> 24
<212> DNA
<213> Artificial sequence (RenGongXuLie)
<400> 3
ttggcttttg gttcggagag cgcg 24
<210> 4
<211> 24
<212> DNA
<213> Artificial sequence (RenGongXuLie)
<400> 4
aaaccgcgct ctccgaacca aaag 24
<210> 5
<211> 24
<212> DNA
<213> Artificial sequence (RenGongXuLie)
<400> 5
ttggcgcgcg ctctccgaac caaa 24
<210> 6
<211> 24
<212> DNA
<213> Artificial sequence (RenGongXuLie)
<400> 6
aaactttggt tcggagagcg cgcg 24
<210> 7
<211> 24
<212> DNA
<213> Artificial sequence (RenGongXuLie)
<400> 7
ttggacgtcc tctcgcggcc ctca 24
<210> 8
<211> 24
<212> DNA
<213> Artificial sequence (RenGongXuLie)
<400> 8
aaactgaggg ccgcgagagg acgt 24
<210> 9
<211> 24
<212> DNA
<213> Artificial sequence (RenGongXuLie)
<400> 9
ttgggtttcg cctgtccccg agag 24
<210> 10
<211> 24
<212> DNA
<213> Artificial sequence (RenGongXuLie)
<400> 10
aaacctctcg gggacaggcg aaac 24
<210> 11
<211> 24
<212> DNA
<213> Artificial sequence (RenGongXuLie)
<400> 11
ttgggacagg tctctctccc ggcg 24
<210> 12
<211> 24
<212> DNA
<213> Artificial sequence (RenGongXuLie)
<400> 12
aaaccgccgg gagagagacc tgtc 24
<210> 13
<211> 24
<212> DNA
<213> Artificial sequence (RenGongXuLie)
<400> 13
ttggtcaaag gctagttacc ccca 24
<210> 14
<211> 24
<212> DNA
<213> Artificial sequence (RenGongXuLie)
<400> 14
aaactggggg taactagcct ttga 24
<210> 15
<211> 24
<212> DNA
<213> Artificial sequence (RenGongXuLie)
<400> 15
ttgggaacag ccccaactcc gcgg 24
<210> 16
<211> 24
<212> DNA
<213> Artificial sequence (RenGongXuLie)
<400> 16
aaacccgcgg agttggggct gttc 24
<210> 17
<211> 24
<212> DNA
<213> Artificial sequence (RenGongXuLie)
<400> 17
ttggcgcgcg cctagggcgc agct 24
<210> 18
<211> 24
<212> DNA
<213> Artificial sequence (RenGongXuLie)
<400> 18
aaacagctgc gccctaggcg cgcg 24
<210> 19
<211> 24
<212> DNA
<213> Artificial sequence (RenGongXuLie)
<400> 19
ttggtcccgg gcgcagggtg catt 24
<210> 20
<211> 24
<212> DNA
<213> Artificial sequence (RenGongXuLie)
<400> 20
aaacaatgca ccctgcgccc ggga 24
<210> 21
<211> 21
<212> DNA
<213> Artificial sequence (RenGongXuLie)
<400> 21
cagcacaaaa ggaaactcac c 21
<210> 22
<211> 25
<212> DNA
<213> Artificial sequence (RenGongXuLie)
<400> 22
gggagaagta aatgtgtttt tgaaa 25
<210> 23
<211> 25
<212> DNA
<213> Artificial sequence (RenGongXuLie)
<400> 23
atacttcttc cccacctaaa cctac 25
Claims (7)
1. An sgRNA sequence that is demethylated from an NNT gene, comprising: the sequence is sgRNA1, sgRNA6, a combination of sgRNA9 and sgRNA10, or a combination of sgRNA1 to sgRNA10;
the sense strand of the sgRNA1 is TTGGGCCAGCGACGTCCTCG, and the antisense strand is AAACCGAGGACGTCGCTGGC; the sense strand of the sgRNA2 is TTGGCTTTTGGTTCGGAGAGCGCG, and the antisense strand is AAACCGCGCTCTCCGAACCAAAG; the sense strand of the sgRNA3 is TTGGCGCGCGCGCTCTCCGAACCAAA, and the antisense strand is AAACTTTGGTTCGGAGCGCGCGCG; the sense strand of the sgRNA4 is TTGGACGTCCTCTCGCCGGCCCTCC, and the antisense strand is AAACTGAGGCCGCGAGAGGACGT; the sense strand of the sgRNA5 is TTGGGTTTCGCCTGTCCCGAGA, and the antisense strand is AAACCTCTCTCCGGGGACAGGCGAAAC; the sense strand of the sgRNA6 is TTGGGACAGGTCTCTCTCCCGGCG, and the antisense strand is AAACCGCCGGGGAGAGACTGTC; the sense strand of the sgRNA7 is TTGGTCAAAGGCTAGTTACCCCA, and the antisense strand is AAACTGGGGGTAACTAGCCTTTGA; the sense strand of the sgRNA8 is TTGGGAACAGCCCAAACTCCGG, and the antisense strand is AAACCCGCGGGAGTTGGGCTGTTTC; the sense strand of the sgRNA9 is TTGGCGCGCGCCTAGGCGCAGCT, and the antisense strand is AAACAGCTGTCGCCCTAGGCGCGCGCG; the sense strand of the sgRNA10 is TTGGTCCCGGGCGCAGGGTGCATT, and the antisense strand is AAACAATGCACCTGCGGCCCGGGA.
2. The NNT gene demethylated sgRNA sequence of claim 1, wherein: the sequence is a combination of sgRNA1 to sgRNA 10.
A method for constructing a demethylation editing system of an NNT gene, the method comprising: the method comprises the following steps:
1) Artificially synthesizing the sgRNA sequence of any one of claims 1 to 2;
2) Inserting the sgRNA into a multiple cloning site of a sgRNA framework expression plasmid vector, converting, selecting a monoclonal strain, performing sequencing identification to obtain a sgRNA strain with a correct target sequence, and extracting a sgRNA recombinant plasmid;
3) Transiently transfecting the sgRNA recombinant plasmid and the TETv4 plasmid into a human lung adenocarcinoma cisplatin-resistant cell A549/DDP;
4) After transfection for 46-50h, flow fluorescence sorting is carried out on the cells to obtain a double-fluorescence positive cell strain, namely the A549/DDP with the NNT gene promoter region demethylated.
4. The method of claim 3, wherein the method of constructing an NNT gene demethylation editing system comprises: the sgRNA skeleton expression plasmid vector is pgRNA-modified.
5. The method of claim 3 for constructing a system for the demethylation editing of an NNT gene, in which: the double-fluorescence positive cell strain is a mCherry and tagBFP double-fluorescence positive cell strain.
6. Use of an NNT gene demethylated sgRNA sequence according to any of claims 1-2 in the preparation of a medicament for the treatment of a disease associated with NNT gene DNA methylation.
7. NNT gene demethylation kit, comprising an NNT gene demethylated sgRNA sequence of any of claims 1 to 2, or a sgRNA recombinant plasmid of claim 3.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210545516.9A CN114736902B (en) | 2022-05-19 | 2022-05-19 | Construction method and application of sgRNA sequence and editing system for NNT gene demethylation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210545516.9A CN114736902B (en) | 2022-05-19 | 2022-05-19 | Construction method and application of sgRNA sequence and editing system for NNT gene demethylation |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114736902A CN114736902A (en) | 2022-07-12 |
| CN114736902B true CN114736902B (en) | 2023-03-24 |
Family
ID=82287423
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210545516.9A Active CN114736902B (en) | 2022-05-19 | 2022-05-19 | Construction method and application of sgRNA sequence and editing system for NNT gene demethylation |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114736902B (en) |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017210600A1 (en) * | 2016-06-03 | 2017-12-07 | The Scripps Research Institute | Compositions and methods of modulating immune response |
| WO2019210268A2 (en) * | 2018-04-27 | 2019-10-31 | The Broad Institute, Inc. | Sequencing-based proteomics |
-
2022
- 2022-05-19 CN CN202210545516.9A patent/CN114736902B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN114736902A (en) | 2022-07-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US10927383B2 (en) | Cas9 mRNAs | |
| CN105646719B (en) | Efficient fixed-point transgenic tool and application thereof | |
| CN105518135B (en) | Method for specifically knocking out pig CMAH gene by CRISPR-Cas9 and sgRNA for specifically targeting CMAH gene | |
| Bock et al. | Identification of critical nucleotide positions for plastid RNA editing site recognition. | |
| CN113939591A (en) | Methods and compositions for editing RNA | |
| CN113373130A (en) | Cas12 protein, gene editing system containing Cas12 protein and application | |
| KR102626503B1 (en) | Target sequence-specific modification technology using nucleotide target recognition | |
| CN101993482A (en) | Protein associated with long grain foliaceous of paddy rice and coding gene and application thereof | |
| CN112159801B (en) | SlugCas9-HF protein, gene editing system containing SlugCas9-HF protein and application | |
| EP4085931A1 (en) | New method for targeted editing of rna | |
| CN114736902B (en) | Construction method and application of sgRNA sequence and editing system for NNT gene demethylation | |
| CN113583999A (en) | Cas9 protein, gene editing system containing Cas9 protein and application | |
| CN111051509A (en) | Composition for dielectric calibration containing C2CL endonuclease and method for dielectric calibration using the same | |
| EP0055742A4 (en) | System for amplification of eukaryotic genes. | |
| Kunnimalaiyaan et al. | Analysis of the tobacco chloroplast DNA replication origin (ori B) downstream of the 23 S rRNA gene | |
| KR102617424B1 (en) | Composition and kit for reducing methylation of target DNA and induction of expression of target gene in animal cell, and method using the same | |
| JP7454881B2 (en) | Target nucleotide sequence modification technology using CRISPR type ID system | |
| CN111235153B (en) | sgRNA for targeted knockout of human MC1R gene and cell strain constructed by same | |
| CN114990093A (en) | Protein sequence MINI RFX-CAS13D with small amino acid sequence | |
| CN110551763B (en) | CRISPR/SlutCas9 gene editing system and application thereof | |
| CN113174406A (en) | Preparation method of zebra fish LGP2 gene knockout homozygote | |
| Anderson et al. | Counter-selection facilitated plasmid construction by homologous recombination in Saccharomyces cerevisiae | |
| CN115261363B (en) | Method for measuring RNA deaminase activity of APOBEC3A and RNA high-activity APOBEC3A variant | |
| WO2023232024A1 (en) | System and methods for duplicating target fragments | |
| JP4373363B2 (en) | Long-chain DNA fragment-introduced chloroplast transformation vector |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |