Viral hepatitis B is a common infectious disease affecting the world, the cause of which is hepatitis B virus (HBV) infection and induced autoimmune reaction. HBV cannot be eradicated with traditional chemical treatments, but it is possible with the safety-enhancing CRISPR-Cas gene editing tool.
Aiming at curing hepatitis B with CRISPR-Cas gene editing technology, our project mainly carried out the following work: (1) Taking HBsAg gene of hepatitis B virus as the gene editing target, we use the screening model of E. coli to screen out 1 on-target sequence from 33 candidates designed based on the HBsAg gene. According to the on-target sequence, we designed the gRNA of MAD7 and the off-target for subsequent off-target detection. (2) A library containing 660 MAD7 mutants was constructed by zero-shot mutational effect prediction and error-prone PCR. A high-precision and high-activity MAD7 nuclease mutant (HF-MAD7) was screened out by high-throughput screening. The off-target rate decreased from 99.02% to 3.17%, and the editing efficiency was 59.51% of that of WT. The project provides a reliable gene editing therapy for viral hepatitis B.
Viral hepatitis type B is a common infectious disease caused by HBV infection, which mainly causes liver lesions. According to WHO statistics, 254 million people are infected globally, with 1.2 million new hepatitis B infections each year (2022)[1].The number of new hepatitis B patients each year in China accounts for one-third of all infectious diseases all over the world.
The pathogenesis of hepatitis B is that after the relaxed DNA (rcDNA) of HBV invades the nucleus, it is repaired into covalent closed circular DNA (cccDNA), and accompanied by hepatitis B surface antigen (HBsAg) and other genes are integrated into the genome of the host cell. cccDNA and HBV DNA integrated into the host genome can persist in host cells for a long time and direct HBV reproduction and antigen expression. HBsAg is expressed on the surface of liver cells, which can be attacked by the patient's own immune system. Long-term damage to liver cells due to immune response may lead to liver cirrhosis, liver cancer (HCC) and other fatal diseases. Therefore, getting effective treatment as early as possible is very critical for hepatitis B patients.
At present, hepatitis B is treated with interferons and nucleotide analogues drugs. They could reduce clinical symptoms of patients by inhibiting HBV DNA replication. However, these two types of drugs can not completely eliminate HBV. Long-term medication greatly aggravates the patient's suffering and the virus may also appear drug resistance. Therefore, it is necessary to develop a treatment that can cure hepatitis B in one go.
Based on the physiological characteristics of HBV infected cells, HBV genes can be knocked out by gene editing technology, which can achieve the radical elimination of HBV.
The CRISPR-Cas9 system can effectively cut the cccDNA of HBV to inhibit the replication of HBV[2]. However, the previous studies have mostly relied on sustained expression of CRISPR-Cas9 to inhibit HBV, and persistent overexpression of the gene-editing complex increases off-target rates and toxicity. Latest studies[3-7] show that with RNPs delivery, combined with isiRNA and interferon, the existing CRISPR-Cas9 technology can eradicate more than 98% of HBV cccDNA, which reflects the technical feasibility of using gene editing to treat hepatitis B. However, it is also necessary to remove residual rcDNA and DNA integrated into the host genome to reduce HBV rebound and continuous antigen expression, and protect liver cells from immune system attack. This requires that our gene editing tools must have a particularly high level of precision and safety. Besides, the target of gene editing needs to be very sophisticated.
The CRISPR-Cas system has a certain chance of mismatching and binding to non-target DNA sequences during gene editing, causing unexpected gene mutations known as off-target effects, which affect the accuracy and safety of gene editing[8]. Therefore, how to improve targeting specificity and reduce off-target rates by modifying the Cas protein structure has always been the focus of current research[9-11]. Researchers have successfully developed many mutant Cas nucleases with lower off-target rates through rational or irrational means, and have detected many amino acid sites that affect the specificity of Cas nucleases[12-13]. What’s more, the Antonio Casini team utilized a Saccharomyces cerevisiae reporter strain to design a dual-plasmid screening system for efficient and precise screening of mutant Cas9 nucleases obtained through random mutagenesis. Through combined mutations, the researchers successfully obtained evoCas9, which has a 9-fold higher precision than wild-type Cas9[14]. This type of modification resembling an evolutionary process is known as directed evolution and has become one of the main approaches to modifying Cas proteins. However, the Cas proteins modified through directed evolution still cannot reach the gene editing accuracy required for clinical trials.
Our modified object, MAD7 nuclease, is an open source Cas12a. Compared to Cas9, the Cas12a protein does not require tracrRNA and HNH domains, and the crRNA of Cas12a is significantly shorter than Cas9, so Cas12a has a smaller molecular weight. The gene editing accuracy of Cas12a is higher than that of Cas9, which can further ensure the safety of in vivo gene editing[15]. Cas12a's ability to cut single-strand DNA allows it to effectively clear hepatitis B virus rcDNA. At present, the modification of MAD7 is far from enough, so there is still a great potential for structural modification and performance improvement. The MAD7 gene we are using is licensed and supplied by Inscripta, Inc.
Elimination of HBsAg is an important indicator of the cure of hepatitis B, and editing HBsAg gene also contributes to the elimination of HBV DNA. Therefore, it is appropriate to take HBsAg gene fragments as gene editing on-target.
On one hand, we are committed to screening gene editing targets and their corresponding gRNA therapeutic molecules, both with high safety. Among the 33 candidate on-targets designed based on HBsAg gene, one on-target, the most conducive to the safety and accuracy of treatment, was selected as the target site for MAD7 editing. According to the spacer sequence corresponding to the on target, we designed the gRNA of MAD7. Based on the on-target sequence characteristics, we designed off-target sequences that predispose to off-target cleavage of MAD7 to examine the off-target rates of MAD7 mutants.
In order to improve the efficiency of the pattern screening system of E. coli, we performed molecular dynamics simulation and mutation scoring of sacB gene to obtain sacB with different lethal efficiency. A high-throughput and high-precision MAD7 nuclease mutation screening system containing the multiple plasmids was constructed by combining on-target and off-target with sacB gene and other resistance genes.
On the other hand, we are devoted to obtaining high-precision gene editing tools. Combining zero-shot mutational effect prediction and error-prone PCR, we constructed a 660-size irrational /rational MAD7 mutation library. The screening system was transformed into E. coli, where the MAD7 mutant library was to be screened. The 38 high precision and high activity nuclease mutants screened were sequenced to determine the location of the mutation. After verification of the off-target rate and editing efficiency, we finally found an ideal MAD7 mutant, which was named HF-MAD7. In the presence of both on-target and off-target, the off-target rate of HF-MAD7 was only 3.17%, compared with 99.02% of that of wild type. And the editing efficiency of MAD7 was 59.51% of that of wild type. Therefore, we obtained high-precision HF-MAD7 to achieve hepatitis B gene editing therapy.
We hope that this project can bring hope to cure hepatitis B and lay a certain technical foundation for gene editing in the treatment of diseases.
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