CRISPR Target-based Single-guide RNA (sgRNA) for Diagnostic Testing of Hepatitis B Virus
Abstract
Background: Indonesia is the second-highest country with hepatitis B cases in the South East Asian region. Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 12 (Cas12) could be developed as a diagnostic tool to detect hepatitis B infection. This study was aimed to develop a diagnostic method for hepatitis B virus by designing CRISPR target-based single-guide RNA (sgRNA).
Materials and method: The preCore/Core-gene sequences of hepatitis B virus were collected from the National Center for Biotechnology Information (NCBI) website. The selected sequence was submitted to Cas Designer and CRISPOR tools to design sgRNA. The resulting sgRNA was cloned in silico into an expression vector using Benchling software.
Results: The 23-nucleotide sequence 5'- GTAGTCAGTTATGTCAATGTTAA-3’ had 30% GC content, 68.3 out-of-frame and 76 predicted efficiencies. This sequence had no mismatch based on analysis.
Conclusion: This preliminary study will help design a CRISPR-based diagnostic kit for the detection of hepatitis B virus in Indonesia. However, further in vitro and in vivo studies are required to demonstrate its potential and efficiency.
Keywords: CRISPR-Cas12b, diagnostic, HBV, sgRNA
Full Text:
PDFReferences
Fatmawati, Nasrul E, Zubir N, Sandra F. Programmed cell death protein 1-overexpressed CD8+ T lymphocytes play a role in increasing chronic hepatitis B disease progression. Indones Biomed J. 2021; 13(3): 310–5, CrossRef.
World Health Organization [Internet]. Geneva: World Health Organization; ©2022. Hepatitis B [cited 2022 Jul 28]. Available from: https://www.who.int/.
Mastutik G, Juniastuti J, Rohman A, Amin M, Setiawan PB. Genotipe dan subtipe virus hepatitis B penderita yang terinfeksi kronik aktif. Indones J Clin Pathol Med Lab. 2014; 20(2): 111–5, CrossRef.
Lukito B, Suriapranata I, Pendrianto P, Sulaiman A, Yusuf I, Suhadi FB, et al. Vascular endothelial growth factor level as a predictor of hepatocellular carcinoma in liver cirrhosis patients. Indones Biomed J. 2014; 6(3): 167, CrossRef.
Centers for Diseases Control and Prevention [Internet]. Atlanta: Centers for Diseases Control and Prevention; ©2020. People Born Outside of the United States and Viral Hepatitis [cited 2022 Jul 28]. Available from: https://www.cdc.gov/.
Song JE, Kim DY. Diagnosis of hepatitis B. Ann Transl Med. 2016; 4(18): 338, CrossRef.
World Health Organization. Guidelines on Hepatitis B and C Testing. Geneva: World Health Organization; 2017, article.
Chen X, Tan Y, Wang S, Wu X, Liu R, Yang X, et al. A CRISPR-Cas12b-based platform for ultrasensitive, rapid, and highly specific detection of hepatitis B virus genotypes B and C in clinical application. Front Bioeng Biotechnol. 2021; 9: 743322, CrossRef.
Teng F, Cui T, Feng G, Guo L, Xu K, Gao Q, et al. Repurposing CRISPR-Cas12b for mammalian genome engineering. Cell Discov. 2018; 4: 63, CrossRef.
Liang TJ. Hepatitis B: The virus and disease. Hepatology. 2009; 49(5 Suppl): S13-21, CrossRef.
Yano Y, Utsumi T, Lusida MI, Hayashi Y. Hepatitis B virus infection in Indonesia. World J Gastroenterol. 2015; 21(38): 10714–20, CrossRef.
Lusida MI, Nugrahaputra VE, Soetjipto, Handajani R, Nagano-Fujii M, Sasayama M, et al. Novel subgenotypes of hepatitis B virus genotypes C and D in Papua, Indonesia. J Clin Microbiol. 2008; 46(7): 2160–6, CrossRef.
Hayer J, Jadeau F, Deléage G, Kay A, Zoulim F, Combet C. HBVdb: A knowledge database for Hepatitis B Virus. Nucleic Acids Res. 2013; 41(Database issue): D566-70, CrossRef.
BAE Lab [Internet]. Seoul: BAE Lab; ©2022. Cas-Designer [cited 2022 Jul 12]. Available from: http://www.rgenome.net/.
Tefor Infrastructure [Internet]. Gif-sur-Yvette: CRISPOR; ©2022. CRISPOR [cited 2022 Jul 12]. Available from: http://crispor.tefor.net/.
Benchling [Internet]. San Francisco: Benchling; ©2022. Benchling: Cloud-based platform for biotech R&D [cited 2022 Jul 13]. Available from: https://www.benchling.com.
Siburian MD, Utama A, Dhenni R, Arnelis, Fanany I, Intan MDB, et al. High prevalence of hepatitis B virus genotype C/C1 in the Minangkabau ethnic group in Indonesia. Virol J. 2013; 10: 1-11, CrossRef.
Haeussler M, Concordet J. CRISPOR Manual. Gif-sur-Yvette: CRISPOR; 2018, article.
Park J, Bae S, Kim J. Cas-Designer: A web-based tool for choice of CRISPR-Cas9 target sites. 2015; 31: 4014–6, CrossRef.
Strecker J, Jones S, Koopal B, Schmid-Burgk J, Zetsche B, Gao L, et al. Engineering of CRISPR-Cas12b for human genome editing. Nat Commun. 2019; 10(1): 212, CrossRef.
Meiliana A, Dewi NM, Wijaya A. Genome editing with CRISPR-Cas9 systems: Basic research and clinical applications. Indones Biomed J. 2017; 9(1): 1–16, CrossRef.
Addgene [Internet]. Watertown: Addgene; ©2022. pCAG-AaCas12b-2AeGFP [cited 2022 Jul 13]. Available from: https://www.addgene.org/.
DOI: https://doi.org/10.21705/mcbs.v7i2.301
Copyright (c) 2023 Cell and BioPharmaceutical Institute
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
Indexed by:
Cell and BioPharmaceutical Institute
