The Application of CRISPR-Cas9 on Hepatitis B Virus

Abstract

The key to creating an effective treatment for Hepatitis B Virus (HBV) is to eliminate the covalently closed circular DNA (cccDNA) in the HBV genome. CccDNA in the HBV genome is responsible for allowing the virus to replicate. Scientists aimed to use CRISPR-Cas9 as a gene-editing software to help remove the cccDNA from the HBV genome. The aim of this review paper is to analyze the current state of gene-editing in regards to the creation of a cure for HBV. After conducting the tests, the results confirmed that gene editing could eliminate cccDNA from the HBV genome, with one study eradicating all of the cccDNA present^[2]. A full-length HBV DNA fragment was used, and the cccDNA present in this sample was disrupted entirely through the gene-editing process. In conclusion, CRISPR-Cas9 can help cure HBV, but more testing needs to be conducted to figure out the long-term effects of gene editing in its relation to curing HBV.

Introduction

Over 350 million people worldwide are infected with hepatitis B virus (HBV) and a majority of them are unaware of their infection. Asians, Africans, and Pacific Islanders are some of the main groups highly susceptible to being infected with HBV. Symptoms of HBV include abdominal pain, fevers, joint pain, and jaundice. A patient who is diagnosed with HBV could also have further complications such as liver cirrhosis and liver cancer. There is no permanent cure for HBV, but some medications could reduce symptoms for a short amount of time.
The advent of gene editing strategies has revolutionized treatments for pathogenic viruses, including HBV. Gene editing is a fairly new process, which in recent years, has guided advancements in HBV treatments within the laboratory. The main program which scientists have been using is called CRISPR-Cas9. CRISPR-Cas9 is a gene-editing program which helps insert specific DNA sequences in certain parts of a genome, allowing it to change the properties expressed by that specific genome^[3]. CRISPR is being used in research to attain a cure for HBV with a focus on eliminating cccDNA, a specific part of the genome that allows for transcription of DNA into RNA. The introduction of cccDNA into the HBV genome using CRISPR-Cas9 disrupts genomic expression. Researchers hope that this will prove to be a viable method to stop protein production and reduce symptoms and complications associated with HBV.
Current advancements in the field for finding a cure for HBV show significant short-term effect, but enough testing has not been done to see if the cure lasts in the long term. Limitations in their research include not having enough time to test any long term treatment. This lack of long term research and analysis is the main limitation in the ongoing research, preventing scientists and labs from creating and introducing a full-fledged cure into the market. Some recent advancements, however, confirm that the cccDNA in the HBV genome can be completely eradicated, a major breakthrough in the process^[1].

Method of Analysis

This research review paper was written as part of the UCI GATI comprehensive program. PubMed was used where keywords such as “CRISPR-Cas9”, “HBV Genome”, and “cccDNA” yielded numerous studies about the application of gene editing on HBV. Ten peer-reviewed journal articles published from 2014 to 2019 were chosen and analyzed in this review paper.

Analysis

Gene editing has been shown to be effective in reducing the symptoms of Hepatitis B by successfully removing the cccDNA from the HBV genome. The CRISPR-Cas9 system was used to conduct tests on both in vivo and in vitro cultures to test the efficacy and application of gene editing. Yu et al. aimed to show that CRISPR-Cas9 could be used to cleave cccDNA present in the genome. To do so, cells were incubated and transfected, introduced with the HBV genome for over 24 hours. They were then removed and plated in order to prevent any dilution from occurring. To increase the accuracy of the results, PCR was conducted around the regions where the HBV genome was targeted in order to amplify the results. After the genome was gathered, the new genome was sequenced to figure out the effects of the CRISPR-Cas9 system on the HBV genome. In 2017, Yu et al. successfully demonstrated that CRISPR-Cas9 disrupted the cccDNA, rendering it unable to produce new generations of the virus, successfully in the HBV genome in both in vivo and in vitro cultures^[2]. This preliminary research should help pave the way for more research to be conducted in order to perfect this process of cleaving cccDNA.
Genotyping HBV turned out to be very useful as well since polymorphic versions, HBV genomes with different coding strands but very similar purposes, of the virus exist. The protospacer adjacent motif (PAM) is critical to identify in every genome since it is the exact mark where the CRISPR-Cas9 system will cleave the DNA. This identification of the PAM helps researchers specifically code the gRNA strands to attach to these sequences. A different test, gRNA XCp, was very useful for specific genotypes of the HBV genome. The genotyping also proved that the genotypes B and C had polymorphic differences within their genomes, which affected the PAM and rendered the cure used in genotype A relatively ineffective ^[1]. Different gRNA strands were used since the effectiveness was dependent on the specific genotype and its PAM or binding site^[1]. The different versions of HBV caused a change in the target sequence for the specific genotype resulting in certain gRNA strains being ineffective. This experiment supported that the PAM in the genome is very important to identify since it determines the type of system that needs to be introduced inside the cell and the genotype of the strand of HBV ^[2].

Figure 1. gRNA Insertion . Lin, 2014

Focusing on explicitly eradicating the cccDNA with CRISPR, Lin et al. utilized another gene-editing approach. The researchers used guideRNA (gRNA), RNA that leads to insertion or deletion in the mRNAs present in the cell^[9]. To make the gRNAs, the entire HBV expression genome was sequenced and analyzed to determine points where the gRNAs would be inserted (Figure 3). HBV-specific gRNAs were also created and introduced into the plasmid. With the eight used gRNAs, four were successful in inserting into the genome and cleaving the DNA^[1]. An assay was used to figure out the exact percentage of insertions and deletions resulted from the CRISPR-Cas9 system (Figure 1). It was also shown that a multiplex of gRNA inclusions helped to suppress the expression of HBV proteins present in a cell, with only 60% relatively remaining. The gRNA was most effective in suppressing the production of HBsAg, an important antigen ^[1]. It was shown that the gRNA insertions were a lot more useful in affecting the HBV proteins which were produced, with only 30% remaining ^[1]. The study shows that the inserted gRNAs were effective in limiting the expression of HBV by disrupting the HBV expression vector present in the genome.

Figure 2. HbsAg Repression. Source: Lin, 2014

The CRISPR-Cas9 system was also very successful in cleaving plasmids in an in vivo setting as well. Yu et al. aimed to determine if the HBV genome could be cleaved in an animal and a plasmid. The CRISPR-Cas9 system and the HBV expression vector were introduced into the mice by injection in the tail. Concentrations of the HBsAG and HBV antigen were gathered to measure the efficacy of this treatment. The mice\’s HBsAg levels were lower when they were receiving the specific HBV gRNA templates ^[2]. The indicators as well for the HBV genome were also found in marginally fewer quantities, for the mice who were treated with the gRNA strains. There were also cloning tests that were conducted after which further proved that the HBV genome was corrupted. Out of the 18 clones, only 5 of them were still able to produce the proteins needed to express the HBV genome, and the others were accurately rendered useless.
Overall, after the multitude of tests that occurred, it is safe to assume that the CRISPR-Cas9 system can effectively help prevent HBV from being active in the short term.
Figure 3. Transfection Graph. Li, 2017

Discussion

Although extensive research has been conducted using CRISPR-Cas9, other systems like Transcription activator-like effector nuclease (TALENs) were used before in order to disrupt the HBV genome. In years prior, TALENs was used to help in mutagenesis of the HBV genome, but it was shown to be a very time-consuming process , which was why labs transferred over to CRISPR. Even though there was a decrease in the effectiveness, the less labor-intensive process made CRISPR the most effective tool in disrupting the genome. ZFN, Zinc Finger Nuclease, was another system used, but it was very similar to TALENs in its time-consuming manner.
Most of the tests resulted in significant disruption in the HBV genome; however, the cccDNA was not completely removed in any of the trials. For example, Yu et al. conducted over
10 trials but none of them proved to fully eliminate the cccDNA, with the most successful trial leaving over 25% of cccDNA in the cell . The presence of unremoved cccDNA will be the most challenging obstacle in advancing this treatment clinically since any cccDNA present after the therapy could eventually cause a resurgence of HBV. Additionally, the formation of new cccDNA strands could further complicate the creation of a cure for HBV. Inhibitor sequences and other tools should be utilized to stop the persistent cccDNA from reactivating the symptoms of HBV.
There were also many limitations to these studies^[4]. Some limitations include that the specific sites for insertion were not yet confirmed, so the CRISPR-Cas9 systems were inserted in locations in the genome, which were inferred by analyzing other human sequences^[4]. This can possibly skew results since no exact locations were made, so the gRNAs could have been inserted into wrong nonbinding locations. Another challenge was that the effect of episomal cccDNA was not taken into consideration. However, more research has to be conducted focusing on the long term effects of these treatments and ways to further cleave remaining cccDNA. Finally, for the treatment to truly work in a clinical setting, an effective delivery technique to transport the gRNA strands needs to be developed. Without an effective delivery method, the CRISPR-Cas9 will be ineffective since the gRNA strands won’t reach the targeted sequence in the cell that is meant to be removed.

Conclusion

In this review paper, it was shown that CRISPR-Cas9 is effective in removing cccDNA from the HBV genome. This removal will help suppress the effects of the virus, including its symptoms. Future research must focus on gene-editing applications in Hepatitis B to provide relief for the 880,000 people who die from this disease every year.
Along with more testing, other steps to be taken in the field are to develop an effective way to introduce the CRISPR-Cas9 system clinically and reduce the persistent cccDNA present. In conclusion, CRISPR-Cas9 is a very promising tool to find a permanent cure for HBV.
References:

1. Chen J, Zhang W, Lin J, Wang F, Wu M, Chen C, et al. “An efficient antiviral strategy for targeting hepatitis B virus genome using transcription activator-like effector nucleases.” 2013, Accessed at: https://pubmed.ncbi.nlm.nih.gov/24025750/
2. Pan Y, Xiao L, Li ASS, Zhang X, Sirois P, Zhang J, et al. “Biological and biomedical applications of engineered nucleases.” Molecular Biotechnology.

U.S. National Library of Medicine, 2017 Accessed through: https://pubmed.ncbi.nlm.nih.gov/23089945/

1. Jiang F, Doudna JA . “CRISPR-Cas9 Structures and Mechanisms,” Annual Review of Biophysics. 2017. Accessed Through:

https://www.annualreviews.org/doi/abs/10.1146/annurev-biophys-062215-010822

1. Lin SR, Hai HC, Kuo YT, et al. “The CRISPR/Cas9 System Facilitates Clearance of the Intrahepatic HBV Templates In Vivo.” Molecular Therapy.Nucleic Acids. U.S. National Library of Medicine: 2014,

Accessed: https://pubmed.ncbi.nlm.nih.gov/25137139/

1. Wiktor SZ. “Viral Hepatitis.” In: Holmes KK, Bertozzi S, Bloom BR, Jha P, eds. Major Infectious Diseases. 3rd ed. Washington (DC): The International Bank for Reconstruction and Development / The World Bank, November 3, 2017.

NCBI Bookshelf: https://www.ncbi.nlm.nih.gov/books/NBK525186/

1. Rojo FP, Nyman RKM, Johnson A A T, et al. “CRISPR-Cas systems: ushering in the new genome editing era” Bioengineered. U.S. National Library of Medicine ,2018: Accessed through: https://pubmed.ncbi.nlm.nih.gov/29968520/
2. Naert T, Nieuwenhuysen T, Vleminckx K. “TALENs and CRISPR/Cas9 fuel genetically engineered clinically relevant Xenopus tropicalis tumor models.” Genesis(New York, N.Y.:2000). U.S. National Library of Medicine: 2017, Accessed Through:https://pubmed.ncbi.nlm.nih.gov/28095622/
3. Mohd-Ismail NK, Lim Z, Gunaratne J, Tan YJ. “Mapping the Interactions of HBV cccDNA with Host Factors.” International Journal of Molecular sciences. U.S. National Library of Medicine: 2019:

Accessed through:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6747236/

1. Dong J, Ying J, Qiu X, Lu Y, Zhang M. “Advanced Strategies for Eliminating the cccDNA of HBV.” Digestive Diseases and sciences. U.S. National Library of Medicine:2018, Accessed Through: https://pubmed.ncbi.nlm.nih.gov/29159681/
2. Bloom K, Maepa MB, Ely A, Arbuthnot P. “Gene Therapy for Chronic HBV-Can We Eliminate cccDNA?.” GENES (Basel). U.S. National Library of Medicine:

2018, Accessed through: https://pubmed.ncbi.nlm.nih.gov/29649127/

About the Author

Anirudh Margasahayam is a current junior at Basis Independent Silicon Valley. He is passionate about biology and bringing change into the world. Aside from school, Anirudh is interested in sports and in playing the guitar.