CN114561392A - Method for removing HBV e antigen by closing target gene based on base editing technology - Google Patents
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Abstract
The invention belongs to the technical field of pathogenic biology treatment, and particularly relates to a method for eliminating HBV e antigen by closing a target gene based on a base editing technology, which comprises the steps of analyzing a positive chain of HBV genomes contained in HepG2.215 and HepAD38, determining a target point of base editing, designing and synthesizing a universal sgRNA primer of an ATG codon of a targeted editing inactivation PreC matched with ABE8e and BE4-max, connecting the universal sgRNA primer to a vector after BsaI enzyme digestion recovery, transforming Escherichia coli DH5 alpha, selecting a positive bacterial colony clone, extracting a small plasmid, and extracting a large-size plasmid for later use after enzyme digestion and sequencing identification are correct. The invention prevents the synthesis of viral protein, blocks the replication of virus and explores a method and a strategy for thoroughly curing and removing HBV e antigen by changing HBV genetic information, thereby having 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 method for removing HBV e antigen by closing a target gene based on a base editing technology.
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. The HBV infection rate is high, the base number is large, the treatment rate is low, the treatment effect is not good, the current diagnosis and treatment situation is not optimistic, and heavy diseases and economic burden are caused. 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, research on the anti-HBV activity of nucleoside analogues has progressed rapidly, and clinical inhibition of HBV has definite curative effect, and the generation of drug-resistant mutation is a major problem of the drugs at present. The increase of the selectable drugs helps the drug resistance problem to some extent, but the drugs only can inhibit HBV replication, are ineffective in covalently closed circular DNA (cccDNA), and cannot completely cure HBV. The discovery of a treatment which can completely eliminate HBV e antigen has great significance.
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 base editing technology of fixed-point editing without causing DNA double-strand break, can edit the ATG initiation codon of the viral protein PreC which is crucial to the infection, replication and pathogenesis of the virus, change the genetic information of the HBV, and prevent the synthesis and secretion of HBV e antigen, and has great theoretical significance and clinical practice value.
In order to achieve the purpose, the invention is solved by the following technical scheme:
a method for eliminating HBV e antigen by closing target genes based on a base editing technology comprises the steps of analyzing the positive chain of HBV genomes contained in HepG2.215 (human liver cancer cells) and HepAD38 (human liver cancer cells), determining base editing targets, designing and synthesizing a universal sgRNA primer of ATG codons of targeted editing inactivation PreC matched with ABE8e (base editing enzyme) and BE4-max (base editing enzyme), connecting the universal sgRNA primer to pGL3-U6-sgRNA-PGK-EGFP (vector) or pGL3-U6sgRNA-accg-puro (vector) after BsaI enzyme digestion recovery, converting Escherichia coli DH5 alpha, selecting positive colony clone, extracting small plasmids, and carrying out enzyme digestion and sequencing identification to obtain large-size upgraded 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 ATG of PreC;
(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 an editing Target of the PreC is selected, copying a 50-70nt sequence and inputting the sequence into a BE-Hive machine model, clicking a CRISPR protospacer window to adjust according to the position of the Target, selecting a sequence with the highest predicted editing efficiency as a final primer sequence, taking a 20nt sequence displayed in 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 an upstream primer and a downstream primer respectively;
(4) designing and synthesizing primers, diluting to 10uM, adding 10 ul of primers to centrifugal tube, placing in boiling water for denaturation, and naturally cooling to room temperature (ddH for synthesized primer powder)2Preparing working concentration by using O, mixing the centrifugal tubes of the upstream primer and the downstream primer, placing the centrifugal tubes into boiling water for denaturation, namely colorless and transparent, breaking hydrogen bonds between double DNA chains formed by the primer pair to form two single chains, naturally cooling the centrifugal tubes by using the boiling water, taking out the centrifugal tubes for the next connection), connecting the correctly paired primer pair to a BsaI enzyme-digested pGL3-U6-sgRNA-PGK-EGFP or pGL3-U6-sgRNA-accg-puro vector by using T4 ligase, transforming escherichia coli DH5 alpha, picking positive clones, small upgraded grains, and amplifying the large upgraded grains for later use after enzyme digestion and sequencing are identified to be correct;
b: editing and screening Positive cells
(1) Based on HepG2.215/HepAD38 HBV stable cell line, and matched and co-transfected with ABE8e and BE4-max matched universal sgRNA expression vector pair of target editing inactivation PreC codon;
(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: detection of e antigen inhibitory Effect
And (c) detecting the e antigen expression level of the positive cells (HBV) obtained by screening in the step b by ELISA, and researching the HBV e antigen expression efficiency eliminated by base editing.
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 the ATG initiation codon target point of the PreC, and the A site of ATG has double peaks (indicated by an arrow in figure 3), which indicates that ATG is successfully modified into GTG; or a double peak in the G bit of the ATG (indicated by the arrow in fig. 3), indicating that the ATG was successfully modified to ATA.
The invention has the following beneficial effects:
1. the new version base editing systems ABE8e and BE4-max constructed on the basis of transformation of Cas9 are characterized in that base A is modified into base G through deamination conversion, A.T-G.C conversion is achieved, G.C-A.T conversion is achieved, PAM dependency is small, off-target rate is low, double-strand break of DNA is not caused, safety is high, editing efficiency is high, and editing accuracy is 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 PreC closely related to HBV infection, replication and pathogenesis, target genes are inactivated, translation and synthesis of core antigens and e antigens are closed, and expression and secretion of the e antigens are inhibited.
Drawings
FIG. 1 shows ABE8e/BE4-max mediated deamination of adenine/cytosine to guanine/uracil and editing target;
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 that base editing modification of PreC ATG inhibits the expression and secretion of e antigen.
Detailed Description
The invention will be further described with reference to specific embodiments.
As shown in FIGS. 1-3, a method for eliminating HBV e antigen by closing target gene based on base editing technology, comprising the steps of analyzing the positive strand of HBV genome contained in HepG2.215 and HepAD38, determining the target point of base editing, designing and synthesizing a universal sgRNA primer of target editing inactivation PreC ATG codon paired with ABE8e and BE4-max, connecting the universal sgRNA primer to pGL3-U6-sgRNA-PGK-EGFP or pGL3-U6sgRNA-accg-puro vector recovered by BsaI enzyme digestion, transforming Escherichia coli DH5 alpha, picking positive colony clone, extracting small plasmid, and preparing large-quality-improved particles for later use after enzyme digestion and sequencing are correct.
The method for constructing the vector comprises the following steps:
a: preparation of sgRNA plasmid
(1) Analyzing the positive strand of HBV genomes contained in HepG2.215 and HepAD38 using default parameters of DNAstar, and mapping the PreC ATG;
(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 a PreC ATG initiation codon is selected as an editing Target, copying a 50-70nt (25-35 nt before and after the Target) sequence to BE input into a BE-Hive machine model (ttps:// www.crisprbehive.design /), clicking a CRISPR protospacer window to adjust according to the position of the Target, selecting a final primer sequence (shown as table one) with the highest predicted editing efficiency, taking a 20nt sequence displayed in a Target genomic DNA window as a designed sgRNA upstream primer, taking a reverse complementary strand as a downstream primer, and adding a joint ACCG and AAAC at the 5' end of the upstream and downstream primers respectively;
(4) designing and synthesizing primers, diluting to a working concentration of 10uM, taking 10 ul 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 of sgRNA editing of the ATG of the inactivated HBV Pre-C initiation codon
b: editing and screening positive cells
(3) Based on HepG2.215/HepAD38 HBV stable cell line, and matched and co-transfected with ABE8e and BE4-max matched universal sgRNA expression vector pair of target editing inactivation PreC codon;
(4) performing EGFP label flow sorting or purine pyrimidine screening on transfected cells, collecting cell culture supernatant after transfection for 72-96h, extracting total DNA (deoxyribonucleic acid) for sequencing analysis, and analyzing base editing condition, off-target occurrence rate and editing efficiency according to a sequencing map (a base editing process is implemented, sgRNA and editing enzyme double-plasmid cotransfection HepG2.215 is performed, the culture supernatant contains secreted HBV (hepatitis B virus), and the base editing condition is analyzed by DNA extraction and sequencing);
c: detection of the inhibitory Effect of e antigen
And (d) detecting the e antigen expression level of the positive cells screened in the step b by using enzyme-linked immunosorbent assay (ELISA). The elimination of e antigen expression efficiency by base editing was investigated by increasing or decreasing the antigen expression level. A cell well co-transfected with the ABE8e/BE4-max plasmid and pGL3-U6-sgRNA-PGK-EGFP/pGL3-U6-sgRNA-accg-puro blank plasmid was used as a negative control. The average value of the e antigen OD values of the three multi-well control groups is set as 100%, the ratio of the OD value of the base editing group to the control group is the ordinate, and the e antigen inhibition rate is obtained by subtracting the ratio from 100%. As shown in fig. 3, e antigen expression and secretion were significantly reduced after base editing the start codon ATG of PreC compared to the blank plasmid 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 matched with ABEmax and BE4-max plasmids to transfect HepG2.215, sequencing analysis is carried out to analyze the deamination modification condition of the ATG initiation codon target point of the PreC, and the A site of ATG has double peaks (indicated by an arrow in figure 3), which indicates that ATG is successfully modified into GTG; or a double peak in the G bit of the ATG (indicated by the arrow in fig. 3), 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 PreC of HBV by using ABE8e and BE4-max bases 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 e antigen. With the optimization of the in vivo delivery system, gene therapy has important value and application prospect in the field of anti-HBV treatment.
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 method for eliminating HBV e antigen by closing target gene based on base editing technology is 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 is used for target editing and inactivating ATG codon of PreC, connecting the universal sgRNA primer to pGL3-U6-sgRNA-PGK-EGFP or pGL3-U6sgRNA-accg-puro vector recovered by BsaI enzyme digestion, transforming escherichia coli DH5 alpha, picking out positive colony clone, extracting plasmid, and preparing large-quality plasmid for later use after enzyme digestion and sequencing identification are correct.
2. The method for eliminating HBV e antigen based on base editing technology closing target gene as claimed in claim 1, wherein said method for constructing 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 ATG of PreC;
(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 an editing Target of the PreC is selected, copying a 50-70nt sequence and inputting the sequence into a BE-Hive machine model, clicking a CRISPR protospacer window to adjust according to the position of the Target, selecting a sequence with the highest predicted editing efficiency as a final primer sequence, taking a 20nt sequence displayed in 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 an upstream primer and a downstream primer respectively;
(4) designing and synthesizing primers, diluting to a working concentration of 10uM, taking 10 ul 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
Based on HepG2.215/HepAD38 HBV stable cell line, and matched and co-transfected with ABE8e and BE4-max matched universal sgRNA expression vector pair with target editing and inactivation of PreC initiation codon;
performing 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: detection of inhibitory Effect of HBV e antigen
And (c) detecting the e antigen expression level of the positive cells obtained by screening in the step b by using ELISA, and researching the HBV e antigen expression efficiency eliminated by base editing.
3. The method for eliminating HBV e antigen based on base editing technology closing target gene as claimed in 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 for eliminating HBV e antigen by closing target gene based on base editing technology as claimed in claim 2, wherein the sequencing map analysis method in editing and screening positive cells is as follows: the sgRNA vector is matched with ABEmax and BE4-max plasmids to transfect HepG2.215, sequencing analysis is carried out on the editing condition of the ATG initiation codon target spot of PreC, and double peaks appear at the A site of ATG, which indicates that ATG is successfully modified into GTG; or double peaks in the G bit of the ATG, indicating that the ATG was successfully modified to ATA.
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