The aim of this project is to treat viral hepatitis B with gene editing technology. It is hoped that by designing special target sites, modifying and using high-precision and high-activity MAD7 nuclease, editing and clearing HBV DNA can be achieved. We plan to design on-target based on the HBsAg gene and establish a high-throughput screening system in Escherichia coli. The MAD7 mutation library was established by error-prone PCR, saturation mutation and other technologies, and the screening system was used for rapid screening to obtain high-precision and high-activity MAD7 nuclease variants targeting specific targets of hepatitis B virus for hepatitis B therapy.
The HBsAg gene sequences are contained in the rcDNA, cccDNA and DNA integrated into the host genome of hepatitis B virus. Elimination of HBsAg gene is the most effective means to eliminate the DNA of hepatitis B in various forms and reduce the indication of hepatitis B detection. Therefore, we plan to design an on-target locus with a length of about 20 bp in HBsAg gene according to the PAM locus of MAD7.The similarity of the on-target site to the human gene should be as low as possible to ensure that the human gene is not incorrectly edited during treatment.
Based on the determined on-target sequences, the spacer and gRNA of the MAD7 nuclease can be determined. In order to evaluate the off-target rate of MAD7 nucleases, we intend to introduce mutations in on-target designed as off-target to simulate DNA sequences prone to off- target. On-target and off-target were put into different plasmid vectors to express MAD7 for editing, and the off-target of MAD7 could be detected through plasmid characterization.
We plan to design and construct a new high-throughput screening system based on pBBR1MCS-2 and P15A plasmids, incorporating on-target and off-target sequences, and selecting sacB gene and ampicillin (Amp) resistance gene as the selection markers. We will also introduce MAD7 gene and gRNA expression sequence into pSC101 plasmid, and introduce temperature-regulated promoter and chloramphenicol resistance gene for transcription control and characterization. Ultimately, we will construct a new three-plasmid screening system, which will be named EP1-on, P15A-off, and EP7-wt (Figure 1).
During the screening, we need to control the temperature-induced expression of MAD7 nuclease in E. coli. When MAD7 nuclease specifically cuts the on-target sequence, the sacB gene cannot be expressed. On the other hand, when the MAD7 nuclease does not occur off-target cutting, the antibiotic resistance gene can be normally expressed. In the end, we can obtain high-precision MAD7 nuclease with low off-target rate in E. coli that can grow normally on chloramphenicol (Chl) + Amp + Sucrose medium.
Based on the above principles, we can reasonably design the structure of plasmids, and then use gibson assembly, CPEC, etc. to modify plasmids to complete the construction of the three-plasmid selection system.
Levansucrase expressed by sacB gene can catalyze the production of levan and cause the death of E. coli. To obtain different lethality rates of the sacB gene for selection, we plan to use molecular dynamics simulation and mutation scoring prediction to predict mutations that affect the lethality rate of sacB. Combining the predicted data, we will introduce single/multiple point mutations on the sacB gene using SDM and calculate the lethality rate of the sacB mutant. We hope that different lethality rates of sacB can better meet different selection needs.
In order to construct a mutant library of MAD7, we plan to use error-prone PCR technology to introduce random mutations into MAD7, thereby obtaining a large number of MAD7 mutants. Additionally, to acquire more information on mutation sites, we intend to perform medium and high mutation rate mutations on MAD7. After consulting literature and conducting analysis calculations, we aim to achieve medium and high mutation rate MAD7 mutants with DNA template additions of 200 ng and 10 ng in the error-prone PCR (Table 1). Based on the determined reaction template quantity, we will carry out preliminary experiments to establish the specific conditions for the primers and annealing temperatures used in the error-prone PCR.
After obtaining the MAD7 mutant gene, we plan to ligate it into the backbone of the EP7 plasmid. We will use PCR to amplify the EP7-backbone, followed by DNA gel electrophoresis to separate and recover the PCR product, as well as to purify the PCR product. Finally, we will use gibson assembly to ligate the MAD7 mutant gene with the EP7-backbone, forming the EP7-mutant plasmid containing the MAD7 mutant library. We will then transform three types of plasmids into E. coli K-12 competent cells and perform recovery.
In order to efficiently screen for MAD7 mutants with high precision and high activity, we plan to use the spotting method to inoculate E. coli onto Chl + Amp + Sucrose medium (Figure 2). If the growth of E. coli containing the MAD7 mutant is superior to that of E. coli containing MAD7(WT), it can be inferred that the mutant has higher precision than MAD7(WT).
Therapeutic Tools
Finally, we need to individually test and calculate the editing efficiency and off-target rate of each mutant selected, and compare them with MAD7(WT).
Analysis and Calculation of Editing Efficiency:
Transform the EP1 and EP7-mutant plasmids into E. coli K12. Recover the transformed E. coli K12 cells. Spot the recovered cells onto Chl + Sucrose medium and Chl medium. Perform parallel experiments to ensure accuracy. Calculate the average number of colonies on both media.
Analysis and Calculation of Off-Target Rate:
Transform the P15A-off and EP7-mutant plasmids into E. coli K12 competent cells. Recover the transformed E. coli K12 cells. Spot the recovered cells onto Chl + Amp medium and Chl medium. Perform parallel experiments to ensure accuracy. Calculate the average number of colonies on both media.
After obtaining the editing efficiency and off-target rate for each MAD7 mutant, we will compare them with MAD7(WT) and rank them. We will select the MAD7 mutant with the best overall performance in terms of high precision and low off-target effects for using in gene editing therapy for hepatitis B virus.