CN114561429A - Treatment method for inhibiting HBV surface antigen based on base editing ATG initiation codon - Google Patents
Treatment method for inhibiting HBV surface antigen based on base editing ATG initiation codon Download PDFInfo
- Publication number
- CN114561429A CN114561429A CN202210283122.0A CN202210283122A CN114561429A CN 114561429 A CN114561429 A CN 114561429A CN 202210283122 A CN202210283122 A CN 202210283122A CN 114561429 A CN114561429 A CN 114561429A
- Authority
- CN
- China
- Prior art keywords
- editing
- atg
- hbv
- sgrna
- surface antigen
- 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.)
- Pending
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
- C12N15/63—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
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- 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
- C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
-
- 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
- C12N2730/00—Reverse transcribing DNA viruses
- C12N2730/00011—Details
- C12N2730/10011—Hepadnaviridae
- C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
- C12N2730/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Virology (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of pathogenic biology treatment, and particularly relates to a treatment method for inhibiting HBV surface antigen based on base editing ATG initiation codon, which comprises the steps of analyzing the positive chain of HBV genome contained in HepG2.215 and HepAD38, determining the target point of base editing, designing and synthesizing universal sgRNA primers of targeted editing inactivation PreS1, PreS2 and ATG codon of S gene matched with ABE8e and BE4-max, connecting the universal sgRNA primers to a vector recovered by BsaI enzyme digestion, transforming Escherichia coli DH5 alpha, selecting positive colony clone, extracting plasmid, carrying out enzyme digestion and sequencing identification, and then obtaining large-quality-improved particles for later use. The invention prevents viral protein synthesis to block virus replication by changing HBV genetic information, explores a method and a strategy for thoroughly curing and removing HBV surface antigen, and has great theoretical significance and clinical practice value.
Description
Technical Field
The invention belongs to the technical field of pathogenic biology treatment, and particularly relates to a treatment method for inhibiting HBV surface antigen based on base editing ATG initiation codon.
Background
About half of the global population is in a high-incidence Hepatitis B Virus (HBV) region, 20 million people are infected once, about 3.5 million people are chronic infected, about 100 million people die from HBV infection every year, and the seventh of ten global death diseases is listed. The phenomenon of HBV cryptic infection is very common, and the content of HBV cryptic infection in Asian population is as high as 7.5-16%. The HBV vaccine coverage rate in China is more than 90%, the successful blocking rate of mothers and babies is as high as 95%, and the number of infected people is obviously reduced. The 2016 world health organization first proposed a far reaching goal of eliminating viral hepatitis worldwide by 2030. China has high HBV infection rate and large base number. The standardization of treatment is the key to the improvement of diagnosis rate and treatment rate, and the development of more effective anti-HBV treatment methods is important and is an important prerequisite for realizing the goal of hepatitis B elimination in 2030, which is proposed by WHO.
HBV infection is an important cause of chronic hepatitis, cirrhosis and hepatocellular carcinoma, and seriously threatens human health. In recent years, the research on the anti-HBV of nucleoside analogues is rapidly advanced, the clinical inhibition of HBV has definite curative effect, and the generation of drug-resistant mutation is the main problem of the current drugs. The increase of the selectable drugs helps the drug resistance problem to a certain extent, but the drugs only can inhibit HBV replication, have no effect on covalently closed circular DNA (cccDNA), and cannot completely eliminate and cure HBV. At present, no medicine and method for effectively removing cccDNA exist. The exploration of a treatment strategy for completely eliminating HBV is significant.
The CRISPR/Cas9 can edit genomes of any species, is simple and efficient, and brings eosin for the treatment of viral infection, genetic disease, chromosome defects and the like which are difficult to cure radically. Many research groups have attempted to edit the HBV genome with CRISPR/Cas9 to achieve antiviral effects, but there is an uncontrollable occurrence of unknown mutations that cause viral DNA double strand breaks. A base editing enzyme CBE developed by fusing Cas9 and cytosine deaminase APOBEC1 can induce the transition from C-conductive E to T.A; the base editing enzyme ABE is developed by fusing dCas9 and TadA of Escherichia coli, and can induce A.T-G.C conversion. The ABE-8e and the BE4-max used by the method are updated versions of the ABE and the CBE respectively, and the editing efficiency is higher, the accuracy is higher and the miss rate is lower.
In view of this, the present invention is proposed.
Disclosure of Invention
Aiming at the problems in the prior art, the invention is based on HepG2.215 and HepAD38 which stably carry HBV genome and can support the continuous replication of HBV, can complete the fixed-point editing of the base editing technology without causing DNA double-strand break, can change the HBV genetic information to prevent the synthesis of viral proteins and block the replication of viruses and explore the method and strategy for thoroughly curing and eliminating HBV surface antigen, and has great theoretical significance and clinical practice value for the fixed-point editing of ATG initiation codons of PreS1, PreS2 and S which are crucial to the infection, replication and pathogenesis of viruses.
In order to achieve the purpose, the invention is solved by the following technical scheme:
a treatment method for inhibiting HBV surface antigen based on base editing ATG initiation codon comprises the steps of analyzing the positive strand of HBV genome contained in HepG2.215 (human liver cancer cells) and HepAD38 (human liver cancer cells), determining base editing target points, designing and synthesizing universal sRNA of ATG codon of targeted editing inactivation PreS1, PreS2 and S genes matched with ABE8e (base editing enzyme) and BE4-max (base editing enzyme), connecting to pGL3-U6-sgRNA-PGK-EGFP (vector) or pGL3-U6 sgRNA-acg-puro (vector) after BsaI enzyme digestion recovery, transforming escherichia coli DH5 alpha, picking positive colony clone, small extracting plasmid, enzyme digestion and sequencing to identify correct large extracting particles for later use.
The method for constructing the vector comprises the following steps:
a: preparation of sgRNA plasmid
(1) The positive strand of HBV genome contained in HepG2.215 and HepAD38 was analyzed using default parameters of DNAstar to locate the ATG of PreS1, PreS2 and S gene;
(2) evaluating the editability of the ATG initiation codon and the downstream nearby region thereof and the PAM editing fitness of the 3' end of the target region;
(3) after editing targets of PreS1, PreS2 and S gene ORF are selected, copying 50-70nt sequences and inputting the sequences into a BE-Hive machine model, clicking a CRISPR protospacer window to adjust according to the Target position, selecting a primer sequence with the highest predicted editing efficiency as a final primer sequence, taking a 20nt sequence displayed by a Target genomic DNA window as a designed sgRNA upstream primer, taking a trans-complementary strand as a downstream primer, and adding a joint ACCG and AAAC at the 5 'end of the upstream primer and the 5' end of the downstream primer respectively;
(4) designing and synthesizing primers, diluting to 10uM, adding 10ul of upstream and downstream primers into a centrifuge tube, placing in boiling water for denaturation, and naturally cooling to room temperature (ddH for synthesized primer powder)2Preparing working concentration by O, mixing an upstream primer centrifugal tube and a downstream primer centrifugal tube, placing the mixture in boiling water for denaturation, namely colorless and transparent, breaking hydrogen bonds between double DNA chains formed by a primer pair to form two single chains, naturally cooling the mixture in boiling water, taking out the centrifugal tube for next connection), connecting the correctly paired primer pair to a BsaI enzyme-digested pGL3-U6-sgRNA-PGK-EGFP or pGL3-U6sgRNA-accg-puro vector by using T4 ligase, transforming escherichia coli DH5 alpha, picking positive clones, small upgraded particles, and amplifying the large upgraded particles for later use after enzyme digestion and sequencing are identified to be correct;
b: editing and screening positive cells
(1) HepG2.215/HepAD 38-based HBV stable transfer cell line, and paired cotransfection with universal sgRNA expression vectors paired with ABE8e and BE4-max for targeted editing of inactivated PreS1, PreS2 and S ATG codons;
(2) carrying out flow sorting or purine pyrimidine screening on transfected cells through EGFP labels, collecting cell culture supernatant after transfection for 72-96h, extracting total DNA (deoxyribonucleic acid) for sequencing analysis, and analyzing base editing condition, off-target incidence and editing efficiency according to a sequencing map;
c: detecting the inhibitory effect of hepatitis B surface antigen
And (c) detecting the surface antigen expression level of the positive cells (HBV) obtained by screening in the step b by ELISA, and researching the inhibition efficiency of the base editing on the HBV antigen expression.
The reaction system for BsaI enzyme digestion is BsaI 1ul, NEBuffer 32 ul, Plasmid 1ul and ddH2O 16ul。
The sequencing map analysis method in editing and screening positive cells comprises the following steps: the sgRNA vector is matched with ABEmax and BE4-max plasmids to transfect HepG2.215, sequencing analysis is carried out to analyze the deamination modification condition of ATG initiation codon target points of PreS1, PreS2 and S (PreS 1, PreS and S all belong to S-ORF), and double peaks appear at A position of ATG (positions indicated by arrows in figures 4, 5 and 6), which indicates that ATG is successfully modified into GTG; or the double peak of the G bit of the ATG (indicated by the arrow in fig. 4, 5 and 6) indicates that the ATG was successfully modified to ATA.
The invention has the following beneficial effects:
(1) a new version base editing system ABE8e and BE4-max constructed on the basis of transformation of Cas9 are adopted, the base A is modified into the base G through deamination conversion in the former, so that A.T-G.C conversion is realized, and the G.C-A.T conversion is realized in the latter, so that the PAM dependency is small, the off-target rate is low, double-strand break of DNA is not caused, the safety is strong, and the editing efficiency and the accuracy are high;
(2) by means of currently recognized HBV in-vitro research systems HepG2.215 and HepAD38, ABE8e and BE4-max are used for targeting ATG initiation codons of key genes PreS1, PreS2 and S genes closely related to HBV infection, replication and pathogenesis, target gene site-specific inactivation is adopted, translation synthesis of target protein is closed, surface antigen expression and secretion are inhibited, HBV life cycle is blocked, and HBV replication is inhibited; HBV cccDNA, rcDNA and integrated DNA are ideal targets for base editing; the invention is expected to thoroughly eliminate HBV, focuses on the leading edge, develops a new way and provides a new idea for thoroughly overcoming HBV for human beings.
Drawings
FIG. 1 shows the ATG conservation analysis in HBV genome and the effect of S-ORF ATG point mutation on HBV replication;
FIG. 2 is a plasmid map of sgRNA designed using Be-Hive along with the existing base editing enzymes ABE8e and BE4 max;
FIG. 3 shows strategy, efficiency calculation and sequencing verification primer design for base editing ATG shutdown;
FIG. 4 shows that ATG of ABEmax and BE4-max modified S inhibits HBV replication and surface antigen expression;
FIG. 5 is a graph showing that ABEmax and BE4-max modify ATG of PreS1 to inhibit HBV replication and surface antigen expression;
FIG. 6 shows that ABEmax and BE4-max modify ATG of PreS2 to inhibit viral replication and surface antigen expression.
Detailed Description
The invention will be further described with reference to specific embodiments.
As shown in FIGS. 1-6, firstly, targets for treating HBV by base editing are determined, and the specific steps are as follows: the biological influence of pBSK-HBV1.3-P-null (ATG point mutation of P-ORF initiation codon), -S5, -S6, -S5/S6 (ATG initiation codon fifth and sixth of S-ORF) mutation on the life cycle of HBV is studied, and the study provides a basis for the treatment of HBV through base editing. On the positive strand of HBV genome, there are a total of 31 ATG-initiated ORFs. As shown in FIG. 1A, the S-ORF has 9 fusion-framed ATGs, sequentially named S1-S9, S1, S3 and S4 initiating translation of L, M and S proteins, respectively. Designing primers, constructing mutant viruses such as pBSK-HBV1.3-P-null, -S5, -S6, -S5/S6 and the like by utilizing a fusion PCR method, and sequencing, analyzing and identifying; ELISA and western-blot were used to detect HBs/c/e expression, and Realtime-PCR, Southern-blot and Northern-blot were used to detect HBV DNA, RNA and cccDNA. The result shows that the ATG initiation codon can close the transcription and translation of genes, the replication and antigen expression level of the virus are obviously reduced after the ATG mutation of HBV P and S genes, and the mutant ATG can be theoretically used as a target spot for treating HBV through base editing.
The method for constructing the vector comprises the following steps:
a: preparation of sgRNA plasmid
(1) The positive strand of HBV genome contained in HepG2.215 and HepAD38 was analyzed using default parameters of DNAstar to locate the ATG of PreS1, PreS2 and S gene;
(2) evaluating the editability of the ATG initiation codon and the downstream nearby region thereof and the PAM editing fitness of the 3' end of the target region;
(3) after editing targets of PreS1, PreS2 and S gene ORF are selected, 50-70nt (25-35 nt before and after the Target) sequences are copied and input into a BE-Hive machine model (ttps:// www.crisprbehive.design /), according to the Target position, a CRISPR protospacer window is clicked to adjust, the sequence with the highest predicted editing efficiency is selected as a final primer sequence (as shown in table one), a 20nt sequence displayed by a Target genomic DNA window is a designed sgRNA upstream primer, a reverse complementary chain is a downstream primer, and a joint ACCG and AAAC are added to the 5' end of the upstream primer and the downstream primer respectively;
(4) designing and synthesizing primers, diluting to a working concentration of 10uM, taking 10ul upstream and downstream primers to a centrifugal tube, placing in boiling water for denaturation, naturally cooling the boiling water to room temperature (about 2-4 hours), then connecting correctly paired primer pairs to BsaI enzyme-digested pGL3-U6-sgRNA-PGK-EGFP or pGL3-U6-sgRNA-accg-puro vectors by utilizing T4 ligase, transforming escherichia coli DH5 alpha, selecting positive clones, small upgraded grains, and amplifying bacteria for later use after enzyme digestion and sequencing are identified as correct; at this time, the latest version of the base editing enzymes ABE8e and BE4-max used in the present invention have been constructed;
TABLE I design sgRNA to turn off the ATG of the start codon of HBV S, Pre-S1, PreS2
b: editing and screening Positive cells
(1) HepG2.215/HepAD 38-based HBV stable transfer cell line, and paired cotransfection with universal sgRNA expression vectors paired with ABE8e and BE4-max for targeted editing of inactivated PreS1, PreS2 and S ATG codons;
(2) subjecting transfected cells to EGFP label flow sorting or purine pyrimidine screening, collecting cell culture supernatant after transfection for 72-96h, extracting total DNA for sequencing analysis, and analyzing base editing condition, off-target incidence and editing efficiency according to a sequencing map (implementing the base editing process, sgRNA and editing enzyme double plasmid cotransfection HepG2.215, wherein the culture supernatant contains secreted HBV virus, and analyzing the base editing condition by extracting DNA and sequencing);
c: detection of surface antigen inhibition Effect
And (c) detecting the surface antigen expression level of the positive cells obtained by screening in the step b by using ELISA, and researching the HBV antigen expression inhibition efficiency by base editing. The ELISA is used for detecting the expression of the surface antigen, and the result shows that the expression of the HBsAg can be inhibited by closing ATG of PreS1, PreS2 and S; cell wells co-transfected with ABE8e/BE4-max plasmid and blank plasmid pGL3-U6-sgRNA-PGK-EGFP/pGL3-U6-sgRNA-accg-puro were used as controls. The average value of the OD values of the surface antigens of the three multiple-well control groups is set as 100%, the ratio of the OD value of the base editing group to the OD value of the control group is a vertical coordinate, and the surface antigen inhibition rate is obtained by subtracting the ratio by 100%. As shown in fig. 4, 5 and 6, expression and secretion with the surface antigen were significantly reduced after base editing of the start codons ATG of PreS1, PreS2 and S, compared to the control group.
BsaI digestion reaction system is BsaI 1ul, NEBuffer 32 ul, Plasmid 1ul and ddH2O 16ul。
The sequencing map analysis method in editing and screening positive cells comprises the following steps: the sgRNA vector is paired with ABEmax and BE4-max plasmids to transfect HepG2.215, sequencing analysis is carried out on the deamination modification condition of the ATG initiation codon target point of S-ORF, the sequencing map result is shown in figure 4-figure 6, and the A site of ATG has double peaks (namely the positions indicated by arrows in figure 4, figure 5 and figure 6), which indicates that ATG is successfully modified into GTG; or the G bit of the ATG appears double-peaked (i.e., the position indicated by the arrow in fig. 4, 5, and 6), indicating that the ATG was successfully modified to ATA.
From the clinical applicability, the ABE8e and BE4-max based on the Cas9 can modify the base A into the base G or convert C into T through deamination conversion, and has the advantages of small PAM dependence, low off-target rate, no DNA double-strand break, strong safety, high editing efficiency and high precision. The invention edits the ATG initiation codon of S, PreS1 and PreS2 of HBV by ABE8e and BE4-max base at cellular level, so that the ATG initiation codon loses the transcription initiation function, and the target gene is closed, thereby realizing the inhibition and elimination of HBV surface antigen. With the optimization of the in vivo delivery system, gene therapy has important value and application prospect in the anti-HBV treatment field.
The above-mentioned embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solution of the present invention by those skilled in the art should fall within the protection scope defined by the claims of the present invention without departing from the spirit of the present invention.
Claims (4)
1. A therapeutic method for suppressing HBV surface antigen based on base editing ATG initiation codon, characterized in that: analyzing positive chains of HBV genomes contained in HepG2.215 and HepAD38, determining a target point of base editing, designing and synthesizing a universal sgRNA primer which is matched with ABE8e and BE4-max and can target and edit the ATG codon of inactivated PreS1, PreS2 and S genes, connecting the universal sgRNA primer to pGL3-U6-sgRNA-PGK-EGFP or pGL3-U6-sgRNA-accg-puro vectors recovered by BsaI enzyme digestion, transforming Escherichia coli DH5 alpha, picking out positive colony clone, extracting plasmids, and carrying out enzyme digestion and sequencing to identify correct large-quality particles for later use.
2. The method for treating HBV surface antigen suppression based on base editing ATG initiation codon according to claim 1, wherein said method for constructing the vector comprises the following steps:
a: preparation of sgRNA plasmid
(1) The positive strand of HBV genome contained in HepG2.215 and HepAD38 was analyzed using default parameters of DNAstar to locate the ATG of PreS1, PreS2 and S gene;
(2) evaluating the editability of the ATG initiation codon and the area nearby the downstream of the ATG initiation codon and the PAM editing fitness at the 3' end of the target area;
(3) after editing targets of PreS1, PreS2 and S gene ORF are selected, copying 50-70nt sequences and inputting the sequences into a BE-Hive machine model, clicking a CRISPR protospacer window to adjust according to the positions of the targets, selecting a sequence with the highest predicted editing efficiency as a final primer sequence, wherein a 20nt sequence displayed in a Target genomic DNA window is a designed sgRNA upstream primer, a reverse complementary chain is a downstream primer, and joints ACCG and AAAC are added to the 5' ends of the upstream and downstream primers respectively;
(4) designing and synthesizing primers, diluting to a working concentration of 10uM, taking 10ul upstream and downstream primers to a centrifugal tube, placing in boiling water for denaturation, naturally cooling the boiling water to room temperature, then connecting correctly paired primer pairs to BsaI enzyme-digested pGL3-U6-sgRNA-PGK-EGFP or pGL3-U6-sgRNA-accg-puro vectors by using T4 ligase, transforming escherichia coli DH5 alpha, selecting positive clones, small upgraded grains, and amplifying large upgraded grains for later use after enzyme digestion and sequencing are identified to be correct;
b: editing and screening positive cells
HepG2.215/HepAD 38-based HBV stable transfer cell line, and paired cotransfection with universal sgRNA expression vectors paired with ABE8e and BE4-max for targeted editing of inactivated PreS1, PreS2 and S ATG codons;
carrying out flow sorting or purine pyrimidine screening on transfected cells through EGFP labels, collecting cell culture supernatant after transfection for 72-96h, extracting total DNA (deoxyribonucleic acid) for sequencing analysis, and analyzing base editing condition, off-target incidence and editing efficiency according to a sequencing map;
c: detecting the inhibitory effect of hepatitis B surface antigen
And (c) detecting the surface antigen expression level of the positive cells obtained by screening in the step b by using ELISA, and researching the HBV antigen expression inhibition efficiency by base editing.
3. The method of treatment for suppressing HBV surface antigen based on the base-editing ATG initiation codon according to claim 1 or 2, wherein: the reaction system for BsaI enzyme digestion is BsaI 1ul, NEBuffer 32 ul, Plasmid 1ul and ddH2O 16ul。
4. The method according to claim 2, wherein the sequencing analysis in editing and screening positive cells is performed by the method of base editing ATG initiation codon inhibition of HBV surface antigen: the sgRNA vector is matched with ABEmax and BE4-max plasmids to transfect HepG2.215, sequencing analysis is carried out on the deamination modification condition of ATG initiation codon targets of PreS1, PreS and S, and A position of ATG has double peaks, which indicates that ATG is successfully modified into GTG; or a double peak in the G bit of the ATG indicating that the ATG was successfully modified to ATA.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210283122.0A CN114561429A (en) | 2022-03-22 | 2022-03-22 | Treatment method for inhibiting HBV surface antigen based on base editing ATG initiation codon |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210283122.0A CN114561429A (en) | 2022-03-22 | 2022-03-22 | Treatment method for inhibiting HBV surface antigen based on base editing ATG initiation codon |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114561429A true CN114561429A (en) | 2022-05-31 |
Family
ID=81719950
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210283122.0A Pending CN114561429A (en) | 2022-03-22 | 2022-03-22 | Treatment method for inhibiting HBV surface antigen based on base editing ATG initiation codon |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114561429A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114561392A (en) * | 2022-03-22 | 2022-05-31 | 绍兴市妇幼保健院 | Method for removing HBV e antigen by closing target gene based on base editing technology |
| CN117126818A (en) * | 2023-10-25 | 2023-11-28 | 江西农业大学 | Method for constructing gE gene deletion PRV strain by utilizing ABE and application |
Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1523113A (en) * | 2003-02-20 | 2004-08-25 | 重庆医科大学 | Construction of hepatitis B virus siRNA expression vector and its application in antivirus treatment |
| CN104498493A (en) * | 2014-12-30 | 2015-04-08 | 武汉大学 | Method for specifically knocking out hepatitis B virus by CRISPR/Cas9 and gRNA applied to specific targeting HBV DNA |
| CN109652439A (en) * | 2018-12-27 | 2019-04-19 | 宜春学院 | Utilize the method for the CRISPR/Cas9 adenine base editing system improvement rice blast resistance of wide spectrum mediated |
| CN109706185A (en) * | 2019-02-01 | 2019-05-03 | 国家卫生计生委科学技术研究所 | The method and application of gene knockout are realized based on base editing system mutation initiation codon |
| CN110734900A (en) * | 2019-11-06 | 2020-01-31 | 上海科技大学 | cytosine base editing tool and application thereof |
| CN110904155A (en) * | 2019-11-27 | 2020-03-24 | 佛山科学技术学院 | Base editor and preparation method and application thereof |
| WO2020241869A1 (en) * | 2019-05-30 | 2020-12-03 | 国立大学法人東京大学 | GENOME EDITING SYSTEM USING Cas PROTEIN HAVING TWO TYPES OF NUCLEIC ACID BASE-CONVERTING ENZYMES FUSED THERETO |
| CN112292139A (en) * | 2018-03-13 | 2021-01-29 | 明尼苏达大学董事会 | Lymphohematopoietic engineering using Cas9 base editor |
| CN112746083A (en) * | 2020-12-11 | 2021-05-04 | 中山大学 | Method for editing target gene promoter inactivated gene through single base |
| CN114107377A (en) * | 2020-08-31 | 2022-03-01 | 复旦大学附属华山医院 | Hepatitis B virus vector and application thereof |
| CN114561392A (en) * | 2022-03-22 | 2022-05-31 | 绍兴市妇幼保健院 | Method for removing HBV e antigen by closing target gene based on base editing technology |
-
2022
- 2022-03-22 CN CN202210283122.0A patent/CN114561429A/en active Pending
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1523113A (en) * | 2003-02-20 | 2004-08-25 | 重庆医科大学 | Construction of hepatitis B virus siRNA expression vector and its application in antivirus treatment |
| CN104498493A (en) * | 2014-12-30 | 2015-04-08 | 武汉大学 | Method for specifically knocking out hepatitis B virus by CRISPR/Cas9 and gRNA applied to specific targeting HBV DNA |
| CN112292139A (en) * | 2018-03-13 | 2021-01-29 | 明尼苏达大学董事会 | Lymphohematopoietic engineering using Cas9 base editor |
| CN109652439A (en) * | 2018-12-27 | 2019-04-19 | 宜春学院 | Utilize the method for the CRISPR/Cas9 adenine base editing system improvement rice blast resistance of wide spectrum mediated |
| CN109706185A (en) * | 2019-02-01 | 2019-05-03 | 国家卫生计生委科学技术研究所 | The method and application of gene knockout are realized based on base editing system mutation initiation codon |
| WO2020241869A1 (en) * | 2019-05-30 | 2020-12-03 | 国立大学法人東京大学 | GENOME EDITING SYSTEM USING Cas PROTEIN HAVING TWO TYPES OF NUCLEIC ACID BASE-CONVERTING ENZYMES FUSED THERETO |
| CN110734900A (en) * | 2019-11-06 | 2020-01-31 | 上海科技大学 | cytosine base editing tool and application thereof |
| CN110904155A (en) * | 2019-11-27 | 2020-03-24 | 佛山科学技术学院 | Base editor and preparation method and application thereof |
| CN114107377A (en) * | 2020-08-31 | 2022-03-01 | 复旦大学附属华山医院 | Hepatitis B virus vector and application thereof |
| CN112746083A (en) * | 2020-12-11 | 2021-05-04 | 中山大学 | Method for editing target gene promoter inactivated gene through single base |
| CN114561392A (en) * | 2022-03-22 | 2022-05-31 | 绍兴市妇幼保健院 | Method for removing HBV e antigen by closing target gene based on base editing technology |
Non-Patent Citations (7)
| Title |
|---|
| SIYU CHEN等: "CRISPR Start-Loss: A Novel and Practical Alternative for Gene Silencing through Base-Editing-Induced Start Codon Mutations", MOLECULAR THERAPY: NUCLEIC ACIDS, pages 1062 - 1073 * |
| XINJIE WANG等: "Efficient Gene Silencing by Adenine Base Editor-Mediated Start Codon Mutation", MOLECULAR THERAPY, vol. 28, no. 2, pages 431 - 440, XP093064044, DOI: 10.1016/j.ymthe.2019.11.022 * |
| 周豪: "碱基编辑抑制HBV基因表达及PD-L1抗体恢复HBV特异性T细胞功能的研究", 中国博士学位论文全文数据库 医药卫生科技辑（电子期刊）, pages 061 - 4 * |
| 易韬;: "乙型肝炎病毒PreS1蛋白研究进展", 四川解剖学杂志, no. 01, pages 28 - 31 * |
| 朱甜甜;李毓雯;胡平平;李军;朱传龙;: "功能性受体钠离子牛磺胆酸共转运蛋白-Huh7细胞株的构建及其HBV易感性", 医学研究生学报, no. 11, pages 28 - 33 * |
| 李俊维等: "谷氨酸棒杆菌碱基编辑的条件优化", 生物工程学报, vol. 36, pages 143 - 151 * |
| 王小强;张立营;: "乙型肝炎病毒基因组结构及功能研究进展", 热带医学杂志, no. 11, pages 1435 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114561392A (en) * | 2022-03-22 | 2022-05-31 | 绍兴市妇幼保健院 | Method for removing HBV e antigen by closing target gene based on base editing technology |
| CN117126818A (en) * | 2023-10-25 | 2023-11-28 | 江西农业大学 | Method for constructing gE gene deletion PRV strain by utilizing ABE and application |
| CN117126818B (en) * | 2023-10-25 | 2024-02-02 | 江西农业大学 | Method for constructing gE gene deletion PRV strain by utilizing ABE and application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN114561429A (en) | Treatment method for inhibiting HBV surface antigen based on base editing ATG initiation codon | |
| CN107446923B (en) | rAAV8-CRISPR-SaCas9 system and application thereof in preparation of hepatitis B treatment drug | |
| Jun-Bin et al. | A quantitative method to detect HBV cccDNA by chimeric primer and real-time polymerase chain reaction | |
| Pourcel et al. | Transcription of the hepatitis B surface antigen gene in mouse cells transformed with cloned viral DNA | |
| CN105176981B (en) | DNA sequence and expression vector and its application for circular rna expression | |
| Kramvis et al. | Hepatitis B virus precore mutants in serum and liver of Southern African Blacks with hepatocellular carcinoma | |
| CN107893074A (en) | A kind of gRNA, expression vector, knockout system, kit for being used to knock out CXCR4 genes | |
| US5024938A (en) | Recombinant DNA inserted with hepatitis B virus gene, mammalian cells transformed with cloned viral DNA, and production of hepatitis B virus proteins | |
| Sureau et al. | Cloned hepatitis delta virus cDNA is infectious in the chimpanzee | |
| McReynolds et al. | The ovalbumin gene: cloning of a complete ds-cDNA in a bacterial plasmid | |
| CN110343785B (en) | Kit for detecting hepatitis B virus covalent closed circular DNA based on PCR-CRISPR-cas13a | |
| CN110573628A (en) | probe combination for detecting cancer | |
| CN114561392A (en) | Method for removing HBV e antigen by closing target gene based on base editing technology | |
| Chen et al. | Pacbio sequencing of PLC/PRF/5 cell line and clearance of HBV integration through CRISPR/Cas-9 system | |
| Zhu et al. | HBV DNA replication mediated by cloned patient HBV reverse transcriptase genes from HBV genotypes A–H and its use in antiviral phenotyping assays | |
| CN110669735A (en) | System for inducing formation of HBV cccDNA and construction method | |
| Lin et al. | Full‐length genomic analysis of hepatitis B virus isolates in a patient progressing from hepatitis to hepatocellular carcinoma | |
| CN109897825A (en) | It is a kind of to be simple and efficient the cell system for generating hepatitis type B virus recombination cccDNA | |
| Wang et al. | In vitro resistance to interferon-alpha of hepatitis B virus with basic core promoter double mutation | |
| CN111057791A (en) | Kit for detecting HBV pgRNA in blood | |
| CN112251515A (en) | Primer and kit for detecting thyroid cancer targeted medication related gene variation | |
| CN106893732A (en) | A kind of rescue method of Goose Parvovirus clone | |
| JPS58194897A (en) | Novel dna, its preparation and host transformed with the same | |
| CN113862287B (en) | 3a type hepatitis C virus full-length infectious clone mutant and application thereof | |
| Su et al. | Hepatitis B virus transcripts in a human hepatoma cell line, Hep 3B |
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 |