The CRISPR‒Cas9 system for genome editing of the ASS1 gene in human cells to predict its effect on HSV-1 replication

Nastaran Khodadad; Mona  Fani; Mahboobeh  Khodadad; Shokufeh  Akbarinia; Zahra  Goodarzi; Ali  Teimoori

doi:10.61882/jcbior.6.3.326

Authors

Nastaran Khodadad HIV/AIDS Research Center, Institute of Health, Shiraz University of Medical Sciences, Shiraz, Iran
Mona Fani Department of Pathobiology and Laboratory Sciences, School of Medicine, North Khorasan University of Medical Sciences, Bojnurd, Iran.
Mahboobeh Khodadad HIV/AIDS Research Center, Institute of Health, Shiraz University of Medical Sciences, Shiraz, Iran
Shokufeh Akbarinia HIV/AIDS Research Center, Institute of Health, Shiraz University of Medical Sciences, Shiraz, Iran
Zahra Goodarzi HIV/AIDS Research Center, Institute of Health, Shiraz University of Medical Sciences, Shiraz, Iran
Ali Teimoori Department of Virology, School of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran https://orcid.org/0000-0003-0766-8591

DOI:

https://doi.org/10.61882/jcbior.6.3.326

Keywords:

Genome editing, HSV-1, CRISPR-Cas9, ASS1 gene

Abstract

Herpes simplex virus type 1 (HSV-1) is a highly contagious pathogen that establishes lifelong latent infections. The replication of HSV-1 is potentially influenced by the arginine succinate synthase (ASS1) gene, a key regulator of cellular metabolism. This study utilized the CRISPR–Cas9 genome editing platform to specifically target and disrupt the ASS1 gene to examine its effect on viral propagation. A guide RNA (gRNA) was designed to complement a sequence within the ASS1 gene. A donor plasmid and the pCas-guide plasmid were cloned and cotransfected into the human embryonic kidney (HEK) cells with sheared adenovirus (Ad)5 DNA (HEK293-AD) cells. Potential ASS1-knockout clones were identified and validated via polymerase chain reaction (PCR) and DNA sequencing analysis. The impact on HSV-1 replication was quantified via a plaque assay to determine the viral titer. Sequencing data from ASS1-gRNA/Cas9-treated cells did not confirm successful gene knockout, as the intended ASS1 disruption was not achieved. The viral titer did not significantly differ between the HSV-1 infection group (MOI=0.01) and the control group. These findings indicate that the single gRNA designed for this study lacked sufficient specificity to elicit a CRISPR-Cas9-mediated gene knockout. Therefore, employing a set of more specific gRNAs is recommended to increase targeting efficiency. Further investigations are needed to elucidate the desired genetic modification and observe its subsequent effects on HSV-1.

References

1. Dutton AJ, Hayes CK, Leib DA, Akhtar LN. Herpes Simplex Virus Neurovirulence Across the Human Lifespan. J Infect Dis. 2025;232(1):14-24.

DOI: 10.1093/infdis/jiaf275 PMID: 40418737

2. Birkmann A, Saunders R. Overview on the management of herpes simplex virus infections: Current therapies and future directions. Antiviral Res. 2025;237:106152.

DOI: 10.1016/j.antiviral.2025.106152 PMID: 40154924

3. Szpara ML, Parsons L, Enquist LW. Sequence variability in clinical and laboratory isolates of herpes simplex virus 1 reveals new mutations. J Virol. 2010;84(10):5303-13.

DOI: 10.1128/JVI.00312-10 PMID: 20219902

4. Sawtell NM, Thompson RL. Alphaherpesvirus Latency and Reactivation with a Focus on Herpes Simplex Virus. Curr Issues Mol Biol. 2021;41:267-356. DOI: 10.21775/cimb.041.267 PMID: 32883886

5. Dochnal S, Merchant HY, Schinlever AR, Babnis A, Depledge DP, Wilson AC, et al. DLK-Dependent Biphasic Reactivation of Herpes Simplex Virus Latency Established in the Absence of Antivirals. J Virol. 2022;96(12):e0050822.

DOI: 10.1128/jvi.00508-22 PMID: 35608347

6. Grady SL, Purdy JG, Rabinowitz JD, Shenk T. Argininosuccinate synthetase 1 depletion produces a metabolic state conducive to herpes simplex virus 1 infection. Proc Natl Acad Sci U S A. 2013;110(51):E5006-15. DOI: 10.1073/pnas.1321305110

PMID: 24297925

7. Guo YF, Liao MQ, Cai WL, Yu XX, Li SN, Ke XY, et al. Physical activity, screen exposure and sleep among students during the pandemic of COVID-19. Sci Rep. 2021;11(1):8529.

DOI: 10.1038/s41598-021-88071-4 PMID: 33879822

8. Ohshima K, Nojima S, Tahara S, Kurashige M, Hori Y, Hagiwara K, et al. Argininosuccinate Synthase 1-Deficiency Enhances the Cell Sensitivity to Arginine through Decreased DEPTOR Expression in Endometrial Cancer. Sci Rep. 2017;7:45504.

DOI: 10.1038/srep45504 PMID: 28358054

9. Jamehdor S, Sangtarash MH, Farivar S, Amini R, Teimoori A. Correlation of arginine metabolism with neurodegenerative diseases in infected cells with herpes simplex virus-1. Reviews and Research in Medical Microbiology. 2022;33(1):45-50.

DOI: 10.1097/MRM.0000000000000264

10. Li T. An Essential Role of Argininosuccinate Synthase 1 in Kaposi's Sarcoma-Associated Herpesvirus-Induced Cellular Transformation: University of Southern California; 2016. URL: https://search.proquest.com/openview/761c21ac2e9910afc28e810f92f47a0c/1?pq-origsite=gscholar&cbl=18750

11. Dogrammatzis C, Waisner H, Kalamvoki M. "Non-Essential" Proteins of HSV-1 with Essential Roles In Vivo: A Comprehensive Review. Viruses. 2020;13(1):17.

DOI: 10.3390/v13010017 PMID: 33374862

12. Khatun R, Sengupta S, Bhattacharya M. Bacterial Microbial Defense Systems: From CRISPR-Cas Mechanisms to Emerging Anti-Phage Strategies. IJoI. 2025;13(3):52-8.

DOI: 10.11648/j.iji.20251303.12

13. Ganger S, Harale G, Majumdar P. Clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) systems: Discovery, structure, classification, and general mechanism. CRISPR/Cas-Mediated Genome Editing in Plants: Apple Academic Press; 2023. p. 65-97.

DOI: 10.1201/9781003331759

14. Ebina H, Misawa N, Kanemura Y, Koyanagi Y. Harnessing the CRISPR/Cas9 system to disrupt latent HIV-1 provirus. Sci Rep. 2013;3:2510. DOI: 10.1038/srep02510 PMID: 23974631

15. Song J, Kim S, Kim HY, Hur KH, Kim Y, Park HG. A novel method to detect mutation in DNA by utilizing exponential amplification reaction triggered by the CRISPR-Cas9 system. Nanoscale. 2021;13(15):7193-7201. DOI: 10.1039/d1nr00438g PMID: 33720266

16. Kozovska Z, Rajcaniova S, Munteanu P, Dzacovska S, Demkova L. CRISPR: History and perspectives to the future. Biomed Pharmacother. 2021;141:111917.

DOI: 10.1016/j.biopha.2021.111917 PMID: 34328110

17. Vasques Raposo J, Rodrigues Carvalho Barros L, Ribas Torres L, Barbosa da Silva Pinto R, De Oliveira Lopes A, Mello de Souza E, et al. CRISPR/Cas-9 vector system: targets UL-39 and inhibits Simplexvirus humanalpha1 (HSV-1) replication in vitro. Cell Mol Biol (Noisy-le-grand). 2023;69(7):19-23.

DOI: 10.14715/cmb/2023.69.7.3 PMID: 37715442

18. Sachkova M, Modepalli V, Kittelmann M. The Deep Evolutionary Roots of the Nervous System. Annu Rev Neurosci. 2025;48(1):311-329. DOI: 10.1146/annurev-neuro-112723-040945 PMID: 40198851

19. Graham FL, Smiley J, Russell WC, Nairn R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol. 1977;36(1):59-74. DOI: 10.1099/0022-1317-36-1-59 PMID: 886304

20. Khodadad N, Fani M, Jamehdor S, Nahidsamiei R, Makvandi M, Kaboli S, et al. A knockdown of the herpes simplex virus type-1 gene in all-in-one CRISPR vectors. Folia Histochem Cytobiol. 2020;58(3):174-181. DOI: 10.5603/FHC.a2020.0020

PMID: 32937678

21. Kohli M, Rago C, Lengauer C, Kinzler KW, Vogelstein B. Facile methods for generating human somatic cell gene knockouts using recombinant adeno-associated viruses. Nucleic Acids Res. 2004;32(1):e3. DOI: 10.1093/nar/gnh009 PMID: 14704360

22. Bollen Y, Post J, Koo BK, Snippert HJG. How to create state-of-the-art genetic model systems: strategies for optimal CRISPR-mediated genome editing. Nucleic Acids Res. 2018;46(13):6435-6454. DOI: 10.1093/nar/gky571 PMID: 29955892

23. Liu YC, Cai ZM, Zhang XJ. Reprogrammed CRISPR-Cas9 targeting the conserved regions of HPV6/11 E7 genes inhibits proliferation and induces apoptosis in E7-transformed keratinocytes. Asian J Androl. 2016;18(3):475-9.

DOI: 10.4103/1008-682X.157399 PMID: 26228041

24. Ge X, Xi H, Yang F, Zhi X, Fu Y, Chen D, et al. CRISPR/Cas9-AAV Mediated Knock-in at NRL Locus in Human Embryonic Stem Cells. Mol Ther Nucleic Acids. 2016;5(11):e393.

DOI: 10.1038/mtna.2016.100 PMID: 27898094

25. Wang T, Wei JJ, Sabatini DM, Lander ES. Genetic screens in human cells using the CRISPR-Cas9 system. Science. 2014;343(6166):80-4. DOI: 10.1126/science.1246981

PMID: 24336569

26. GSL Biotech LLC. SnapGene Viewer (Version 8.0.4). 2023. URL: https://www.snapgene.com/snapgene-viewer

27. Sambrook J, Russell DW. Molecular Cloning: Ch. 8. In Vitro amplification of DNA by the polymerase chain reaction: Cold Spring Harbor Laboratory Press; 2001. URL: https://www.gob.mx/cms/uploads/attachment/file/685877/5098_-Sambrook_J.-_Molecular_Clonning_compressed.pdf

28. Okasha H, Samir S. Synthesis and molecular cloning of antimicrobial peptide chromogranin A N-46 gene using conventional PCR. Gene Reports. 2020;18:100571.

DOI: 10.1016/j.genrep.2019.100571

29. Soltani M, Sadrebazzaz A, Nassiri M, Tahmoorespoor M. Cloning, Nucleotide Sequencing and Bioinformatics Study of NcSRS2 Gene, an Immunogen from Iranian Isolate of Neospora caninum. Iran J Parasitol. 2013;8(1):114-27. PMID: 23682269

30. Roehm PC, Shekarabi M, Wollebo HS, Bellizzi A, He L, Salkind J, et al. Inhibition of HSV-1 Replication by Gene Editing Strategy. Sci Rep. 2016;6:23146. DOI: 10.1038/srep23146 PMID: 27064617

31. Fani M, Khodadad N, Ebrahimi S, Ghafari S, Makvandi M, Teimoori A, et al. Zinc Sulfate in Narrow Range as an In Vitro Anti-HSV-1 Assay. Biol Trace Elem Res. 2020;193(2):410-413. DOI: 10.1007/s12011-019-01728-0 PMID: 31028520

32. Nahid Samiei R, Ebrahimi S, Fani M, Ghafari S, Makvandi M, Khodadad N, et al. In vitro effect of some nucleoside reverse transcriptase inhibitors against HSV-1 replication. Eur Rev Med Pharmacol Sci. 2020;24(3):1454-1459.

DOI: 10.26355/eurrev_202002_20204 PMID: 32096195

33. Yuan F, Guo D, Liu Y, Xu Y, Gao G, Wu Y, et al. Generation of an ASS1 heterozygous knockout human embryonic stem cell line, WAe001-A-13, using CRISPR/Cas9. Stem Cell Res. 2018;26:67-71. DOI: 10.1016/j.scr.2017.11.007 PMID: 29247816

34. Ariav Y, Ch'ng JH, Christofk HR, Ron-Harel N, Erez A. Targeting nucleotide metabolism as the nexus of viral infections, cancer, and the immune response. Sci Adv. 2021;7(21):eabg6165. DOI: 10.1126/sciadv.abg6165 PMID: 34138729

35. Becker Y, Olshevsky U, Levitt J. The role of arginine in the replication of herpes simplex virus. J Gen Virol. 1967;1(4):471-8. DOI: 10.1099/0022-1317-1-4-471 PMID: 4295376

36. Inglis VB. Requirement of arginine for the replication of herpes virus. J Gen Virol. 1968;3(1):9-17. DOI: 10.1099/0022-1317-3-1-9 PMID: 4300568

37. Meingast C, Heldt CL. Arginine-enveloped virus inactivation and potential mechanisms. Biotechnol Prog. 2020;36(2):e2931.

DOI: 10.1002/btpr.2931 PMID: 31622532

38. Grimes JM, Khan S, Badeaux M, Rao RM, Rowlinson SW, Carvajal RD. Arginine depletion as a therapeutic approach for patients with COVID-19. Int J Infect Dis. 2021;102:566-570. DOI: 10.1016/j.ijid.2020.10.100 PMID: 33160064

39. Wildy P, Gell PG, Rhodes J, Newton A. Inhibition of herpes simplex virus multiplication by activated macrophages: a role for arginase? Infect Immun. 1982;37(1):40-5.

DOI: 10.1128/iai.37.1.40-45.1982 PMID: 6286497

40. Sanchez MD, Ochoa AC, Foster TP. Development and evaluation of a host-targeted antiviral that abrogates herpes simplex virus replication through modulation of arginine-associated metabolic pathways. Antiviral Res. 2016;132:13-25.

DOI: 10.1016/j.antiviral.2016.05.009 PMID: 27192555

41. Jin YY, Zhang P, Liu DP. Optimizing homology-directed repair for gene editing: the potential of single-stranded DNA donors. Trends Genet. 2025;41(9):788-803.

DOI: 10.1016/j.tig.2025.04.014 PMID: 40436684

42. Dickinson DJ, Ward JD, Reiner DJ, Goldstein B. Engineering the Caenorhabditis elegans genome using Cas9-triggered homologous recombination. Nat Methods. 2013;10(10):1028-34. DOI: 10.1038/nmeth.2641 PMID: 23995389

43. Doench JG, Fusi N, Sullender M, Hegde M, Vaimberg EW, Donovan KF, et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol. 2016;34(2):184-191. DOI: 10.1038/nbt.3437

PMID: 26780180