Herpes simplex viruses (HSVs) are important pathogens and ideal for gene therapy due to its large genome size. Previous researches on HSVs were hampered because the technology to construct recombinant HSVs were based on DNA homology-dependent repair (HDR) and plaque assay, which are inefficient, laborious, and time-consuming. Fortunately, clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9 (CRISPR/Cas9) recently provided the possibility to precisely, efficiently, and rapidly edit genomes and indeed is successfully being used in HSVs. Importantly, CRISPR/Cas9 technology increased HSV HDR efficiency exponentially by a 10,000–1,000,000 times when making recombinant HSVs, and its combination with flow cytometric technology made HSV recombination practically automatic. These may have a significant impact on virus and gene therapy researches. This review will summarize the latest development and molecular mechanisms of CRISPR/Cas9 genome editing technology and its recent application in HSVs.
Subscribe to Journal
Get full journal access for 1 year
only $9.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Naldini L. Gene therapy returns to centre stage. Nature. 2015;526:351–60.
Sander JD, Joung JK. CRISPR-Cas systems for editing, regulating and targeting genomes. Nat Biotechnol. 2014;32:347–55.
Zhang F, Wen Y, Guo X. CRISPR/Cas9 for genome editing: progress, implications and challenges. Hum Mol Genet. 2014;23:R40–6. R1
Peng Z. Current status of gendicine in China: recombinant human Ad-p53 agent for treatment of cancers. Hum Gene Ther. 2005;16:1016–27.
Kohlhapp FJ, Zloza A, Kaufman HL. Talimogene laherparepvec (T-VEC) as cancer immunotherapy. Drugs Today. 2015;51:549–58.
Liu H, Yuan SJ, Chen YT, Xie YB, Cui L, Yang WZ, et al. Preclinical evaluation of herpes simplex virus armed with granulocyte-macrophage colony-stimulating factor in pancreatic carcinoma. World J Gastroenterol. 2013;19:5138–43.
Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337:816–21.
Bolotin A, Quinquis B, Sorokin A, Ehrlich SD. Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology. 2005;151:2551–61. Pt 8
Jansen R, Embden JD, Gaastra W, Schouls LM. Identification of genes that are associated with DNA repeats in prokaryotes. Mol Microbiol. 2002;43:1565–75.
Ishino Y, Shinagawa H, Makino K, Amemura M, Nakata A. Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. J Bacteriol. 1987;169:5429–33.
Doudna JA, Charpentier E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science. 2014;346:1258096.
Bhakta MS, Henry IM, Ousterout DG, Das KT, Lockwood SH, Meckler JF, et al. Highly active zinc-finger nucleases by extended modular assembly. Genome Res. 2013;23:530–8.
Gonzalez B, Schwimmer LJ, Fuller RP, Ye Y, Asawapornmongkol L, Barbas CF 3rd. Modular system for the construction of zinc-finger libraries and proteins. Nat Protoc. 2010;5:791–810.
Townsend JA, Wright DA, Winfrey RJ, Fu F, Maeder ML, Joung JK, et al. High-frequency modification of plant genes using engineered zinc-finger nucleases. Nature. 2009;459:442–5.
Wood AJ, Lo TW, Zeitler B, Pickle CS, Ralston EJ, Lee AH, et al. Targeted genome editing across species using ZFNs and TALENs. Science. 2011;333:307.
Kim YG, Cha J, Chandrasegaran S. Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc Natl Acad Sci USA. 1996;93:1156–60.
Schmid-Burgk JL, Schmidt T, Kaiser V, Honing K, Hornung V. A ligation-independent cloning technique for high-throughput assembly of transcription activator-like effector genes. Nat Biotechnol. 2013;31:76–81.
Briggs AW, Rios X, Chari R, Yang L, Zhang F, Mali P, et al. Iterative capped assembly: rapid and scalable synthesis of repeat-module DNA such as TAL effectors from individual monomers. Nucleic Acids Res. 2012;40:e117.
Deng D, Yan C, Pan X, Mahfouz M, Wang J, Zhu JK, et al. Structural basis for sequence-specific recognition of DNA by TAL effectors. Science. 2012;335:720–3.
Boch J, Scholze H, Schornack S, Landgraf A, Hahn S, Kay S, et al. Breaking the code of DNA binding specificity of TAL-type III effectors. Science. 2009;326:1509–12.
Kim Y, Kweon J, Kim A, Chon JK, Yoo JY, Kim HJ, et al. A library of TAL effector nucleases spanning the human genome. Nat Biotechnol. 2013;31:251–8.
Pennisi E. The CRISPR craze. Science. 2013;341:833–6.
Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339:819–23.
Gasiunas G, Barrangou R, Horvath P, Siksnys V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci USA. 2012;109:E2579–86.
Grissa I, Vergnaud G, Pourcel C. The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats. BMC Bioinformatics. 2007;8:172.
Wei C, Liu J, Yu Z, Zhang B, Gao G, Jiao R. TALEN or Cas9 - rapid, efficient and specific choices for genome modifications. J Genet Genomics. 2013;40:281–9.
Pourcel C, Salvignol G, Vergnaud G. CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies. Microbiology. 2005;151:653–63. Pt 3
Mojica FJ, Diez-Villasenor C, Garcia-Martinez J, Soria E. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J Mol Evol. 2005;60:174–82.
Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, et al. RNA-guided human genome engineering via Cas9. Science. 2013;339:823–6.
Ma H, Tu LC, Naseri A, Huisman M, Zhang S, Grunwald D, et al. CRISPR-Cas9 nuclear dynamics and target recognition in living cells. J Cell Biol. 2016;214:529–37.
Gebler C, Lohoff T, Paszkowski-Rogacz M, Mircetic J, Chakraborty D, Camgoz A, et al. Inactivation of cancer mutations utilizing CRISPR/Cas9. J Natl Cancer Inst. 2017;109:djw183.
Perli SD, Cui CH, Lu TK. Continuous genetic recording with self-targeting CRISPR-Cas in human cells. Science. 2016;353:aag0511.
Abudayyeh OO, Gootenberg JS, Essletzbichler P, Han S, Joung J, Belanto JJ, et al. RNA targeting with CRISPR-Cas13. Nature. 2017;550:280–4.
Liang P, Ding C, Sun H, Xie X, Xu Y, Zhang X, et al. Correction of beta-thalassemia mutant by base editor in human embryos. Protein Cell. 2017;8:811–22.
Chen JS, Dagdas YS, Kleinstiver BP, Welch MM, Sousa AA, Harrington LB, et al. Enhanced proofreading governs CRISPR-Cas9 targeting accuracy. Nature. 2017;550:407–10.
Makarova KS, Haft DH, Barrangou R, Brouns SJ, Charpentier E, Horvath P, et al. Evolution and classification of the CRISPR-Cas systems. Nat Rev Microbiol. 2011;9:467–77.
Mali P, Esvelt KM, Church GM. Cas9 as a versatile tool for engineering biology. Nat Methods. 2013;10:957–63.
Honma M, Sakuraba M, Koizumi T, Takashima Y, Sakamoto H, Hayashi M. Non-homologous end-joining for repairing I-SceI-induced DNA double strand breaks in human cells. DNA Repair (Amst). 2007;6:781–8.
Rouet P, Smih F, Jasin M. Introduction of double-strand breaks into the genome of mouse cells by expression of a rare-cutting endonuclease. Mol Cell Biol. 1994;14:8096–106.
Shen B, Zhang W, Zhang J, Zhou J, Wang J, Chen L, et al. Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects. Nat Methods. 2014;11:399–402.
Ran FA, Hsu PD, Lin CY, Gootenberg JS, Konermann S, Trevino AE, et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell. 2013;154:1380–9.
Nunez JK, Harrington LB, Doudna JA. Chemical and biophysical modulation of Cas9 for tunable genome engineering. ACS Chem Biol. 2016;11:681–8.
Pattanayak V, Lin S, Guilinger JP, Ma E, Doudna JA, Liu DR. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat Biotechnol. 2013;31:839–43.
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;34:184–91.
Schaefer KA, Wu WH, Colgan DF, Tsang SH, Bassuk AG, Mahajan VB. Unexpected mutations after CRISPR-Cas9 editing in vivo. Nat Methods. 2017;14:547–8.
Cho SW, Kim S, Kim Y, Kweon J, Kim HS, Bae S, et al. Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases. Genome Res. 2014;24:132–41.
Mali P, Aach J, Stranges PB, Esvelt KM, Moosburner M, Kosuri S, et al. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat Biotechnol. 2013;31:833–8.
Fu Y, Foden JA, Khayter C, Maeder ML, Reyon D, Joung JK, et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol. 2013;31:822–6.
Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol. 2013;31:827–32.
Kleinstiver BP, Pattanayak V, Prew MS, Tsai SQ, Nguyen NT, Zheng Z, et al. High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature. 2016;529:490–5.
Lin Y, Cradick TJ, Brown MT, Deshmukh H, Ranjan P, Sarode N, et al. CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences. Nucleic Acids Res. 2014;42:7473–85.
Whitley RJ, Roizman B. Herpes simplex virus infections. Lancet. 2001;357:1513–8.
Hill GM, Ku ES, Dwarakanathan S. Herpes simplex keratitis. Dis-a-Mon: DM. 2014;60:239–46.
Rabinstein AA. Herpes virus encephalitis in adults: current knowledge and old myths. Neurol Clin. 2017;35:695–705.
Koyanagi N, Imai T, Shindo K, Sato A, Fujii W, Ichinohe T, et al. Herpes simplex virus-1 evasion of CD8+T cell accumulation contributes to viral encephalitis. J Clin Invest. 2017;127:3784–95.
Thellman NM, Triezenberg SJ. Herpes simplex virus establishment, maintenance, and reactivation: in vitro modeling of latency. Pathogens. 2017;6:E28.
Russell SJ, Peng KW, Bell JC. Oncolytic virotherapy. Nat Biotechnol. 2012;30:658–70.
Russell SJ, Peng KW. Viruses as anticancer drugs. Trends Pharmacol Sci. 2007;28:326–33.
Park BH, Hwang T, Liu TC, Sze DY, Kim JS, Kwon HC, et al. Use of a targeted oncolytic poxvirus, JX-594, in patients with refractory primary or metastatic liver cancer: a phase I trial. Lancet Oncol. 2008;9:533–42.
Au GG, Lindberg AM, Barry RD, Shafren DR. Oncolysis of vascular malignant human melanoma tumors by Coxsackievirus A21. Int J Oncol. 2005;26:1471–6.
Senzer NN, Kaufman HL, Amatruda T, Nemunaitis M, Reid T, Daniels G, et al. Phase II clinical trial of a granulocyte-macrophage colony-stimulating factor-encoding, second-generation oncolytic herpesvirus in patients with unresectable metastatic melanoma. J Clin Oncol. 2009;27:5763–71.
Todo T. Oncolytic virus therapy using genetically engineered herpes simplex viruses. Front Biosci. 2008;13:2060–4.
Markert JM, Medlock MD, Rabkin SD, Gillespie GY, Todo T, Hunter WD, et al. Conditionally replicating herpes simplex virus mutant, G207 for the treatment of malignant glioma: results of a phase I trial. Gene Ther. 2000;7:867–74.
Feng YP, Liu QC, Zhu JF, Xie FK, Li L. Development and applications of a nasopharyngeal carcinoma Tet-Off cell line. Oncol Lett. 2011;2:525–30.
Saha D, Martuza RL, Rabkin SD. Macrophage polarization contributes to glioblastoma eradication by combination immunovirotherapy and immune checkpoint blockade. Cancer Cell. 2017;32:253–67 e5.
Wong HH, Jiang G, Gangeswaran R, Wang P, Wang J, Yuan M, et al. Modification of the early gene enhancer-promoter improves the oncolytic potency of adenovirus 11. Mol Ther. 2012;20:306–16.
Mastrangelo MJ, Maguire HC Jr., Eisenlohr LC, Laughlin CE, Monken CE, McCue PA, et al. Intratumoral recombinant GM-CSF-encoding virus as gene therapy in patients with cutaneous melanoma. Cancer Gene Ther. 1999;6:409–22.
Becker TC, Noel RJ, Coats WS, Gomez-Foix AM, Alam T, Gerard RD, et al. Use of recombinant adenovirus for metabolic engineering of mammalian cells. Methods Cell Biol. 1994;43:161–89. Pt A
Yuan M, Gao X, Chard LS, Ali Z, Ahmed J, Li Y, et al. A marker-free system for highly efficient construction of vaccinia virus vectors using CRISPR Cas9. Mol Ther Methods Clin Dev. 2015;2:15035.
Li Z, Bi Y, Xiao H, Sun L, Ren Y, Li Y, et al. CRISPR-Cas9 system-driven site-specific selection pressure on herpes simplex virus genomes. Virus Res. 2017;244:286–95.
Lin C, Li H, Hao M, Xiong D, Luo Y, Huang C, et al. Increasing the efficiency of CRISPR/Cas9-mediated precise genome editing of HSV-1 virus in human cells. Sci Rep. 2016;6:34531.
van Diemen FR, Kruse EM, Hooykaas MJ, Bruggeling CE, Schurch AC, van Ham PM, et al. CRISPR/Cas9-mediated genome editing of herpesviruses limits productive and latent infections. PLoS Pathog. 2016;12:e1005701.
Xu X, Fan S, Zhou J, Zhang Y, Che Y, Cai H, et al. The mutated tegument protein UL7 attenuates the virulence of herpes simplex virus 1 by reducing the modulation of alpha-4 gene transcription. Virol J. 2016;13:152.
Johnson RD, Jasin M. Double-strand-break-induced homologous recombination in mammalian cells. Biochem Soc Trans. 2001;29:196–201. Pt 2
Yuan M, Webb E, Lemoine NR, Wang Y. CRISPR-Cas9 as a powerful tool for efficient creation of oncolytic viruses. Viruses. 2016;8:72.
Smith KO, Galloway KS, Kennell WL, Ogilvie KK, Radatus BK. A new nucleoside analog, 9-[[2-hydroxy-1-(hydroxymethyl)ethoxyl]methyl]guanine, highly active in vitro against herpes simplex virus types 1 and 2. Antimicrob Agents Chemother. 1982;22:55–61.
Van Rompay AR, Johansson M, Karlsson A. Phosphorylation of nucleosides and nucleoside analogs by mammalian nucleoside monophosphate kinases. Pharmacol Ther. 2000;87:189–98.
Chen ZH, Yu YP, Zuo ZH, Nelson JB, Michalopoulos GK, Monga S, et al. Targeting genomic rearrangements in tumor cells through Cas9-mediated insertion of a suicide gene. Nat Biotechnol. 2017;35:543–50.
Nelles DA, Fang MY, O’Connell MR, Xu JL, Markmiller SJ, Doudna JA, et al. Programmable RNA tracking in live cells with CRISPR/Cas9. Cell. 2016;165:488–96.
Bi Y, Sun L, Gao D, Ding C, Li Z, Li Y, et al. High-efficiency targeted editing of large viral genomes by RNA-guided nucleases. PLoS Pathog. 2014;10:e1004090.
Suenaga T, Kohyama M, Hirayasu K, Arase H. Engineering large viral DNA genomes using the CRISPR-Cas9 system. Microbiol Immunol. 2014;58:513–22.
Komor AC, Kim YB, Packer MS, Zuris JA, Liu DR. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature. 2016;533:420–4.
We thank Hubei province for funding HWX as a Chutian Scholar Distinguished Professor, the National Natural Science Foundation of China (31700736 to XWW), Hubei Province Natural Science Foundation of China (2016CFB180 to XWW), and Yangtze University for fellowship to DW as a graduate student.
This study was funded by 20160527 (Hubei province of China for funding to HWX as a Chutian Scholar Distinguished Professor), the National Natural Science Foundation of China (31700736 to XWW), and Hubei Province Natural Science Foundation of China (2016CFB180 to XWW).
Conflict of interest
The authors declare that they have no conflict of interest.
About this article
Cite this article
Wang, D., Wang, XW., Peng, XC. et al. CRISPR/Cas9 genome editing technology significantly accelerated herpes simplex virus research. Cancer Gene Ther 25, 93–105 (2018). https://doi.org/10.1038/s41417-018-0016-3
Novel transcription regulatory sequences and factors of the immune evasion protein ICP47 (US12) of herpes simplex viruses
Virology Journal (2020)
Journal of Hematology & Oncology (2020)
Cell & Bioscience (2018)