1887

Abstract

Efficient, accurate and convenient foreign-gene insertion strategies are crucial for the high-throughput and rapid construction of large DNA viral vectors, but relatively inefficient and labour-intensive methods have limited the application of recombinant viruses. In this study, we applied the nonhomologous insertion (NHI) strategy, which is based on the nonhomologous end joining (NHEJ) repair pathway. Compared to the currently used homologous recombination (HR) strategy, we obtained a higher efficiency of foreign-gene insertion into the herpes simplex virus (HSV) genome that reached 45 % after optimization. By using NHI, we rapidly constructed recombinant reporter viruses using a small amount of clinical viruses, and the recombinant virus was stable for at least ten consecutive passages. The fidelity of NHI ranged from 70–100% and was related to the sequence background of the insertion site according to the sequencing results. Finally, we depict the dynamic process by which the foreign-gene donor plasmid and viral genome are rapidly cleaved by Cas9, as revealed by quantitative pulse analysis. Furthermore, the NHI strategy exerted selection pressure on the wild-type and reverse-integrated viral genomes to efficiently integrate the foreign gene in a predetermined direction. Our results indicate that the use of a rationally designed NHI strategy can allow rapid and efficient foreign gene knock-in into the HSV genome and provide useful guidance for gene insertion into large DNA viral genomes using NHI.

Funding
This study was supported by the:
  • , Talent development project , (Award D2017003)
  • , National Natural Science Foundation of China, http://dx.doi.org/10.13039/501100001809, (Award 81971947)
  • , Major Science and Technology Projects of Yunnan Province , (Award 2019ZF001)
  • , National Science and Technology Major Projects , (Award 2018ZX09738003-007)
  • , CAMS Initiative for Innovative Medicine , (Award 2016-I2M-1-019)
Loading

Article metrics loading...

/content/journal/jgv/10.1099/jgv.0.001451
2020-06-30
2020-07-04
Loading full text...

Full text loading...

/deliver/fulltext/jgv/10.1099/jgv.0.001451/jgv001451.html?itemId=/content/journal/jgv/10.1099/jgv.0.001451&mimeType=html&fmt=ahah

References

  1. Vannucci L, Lai M, Chiuppesi F, Ceccherini-Nelli L, Pistello M. Viral vectors: a look back and ahead on gene transfer technology. New Microbiol 2013; 36:1–22
    [Google Scholar]
  2. Rehman H, Silk AW, Kane MP, Kaufman HL. Into the clinic: Talimogene laherparepvec (T-VEC), a first-in-class intratumoral oncolytic viral therapy. Journal for ImmunoTherapy of Cancer 2016; 4:53 [CrossRef]
    [Google Scholar]
  3. Roizman B, Jenkins F. Genetic engineering of novel genomes of large DNA viruses. Science 1985; 229:1208–1214 [CrossRef]
    [Google Scholar]
  4. Messerle M, Crnkovic I, Hammerschmidt W, Ziegler H, Koszinowski UH. Cloning and mutagenesis of a herpesvirus genome as an infectious bacterial artificial chromosome. Proc Natl Acad Sci U S A 1997; 94:14759–14763 [CrossRef]
    [Google Scholar]
  5. Tanaka M, Kagawa H, Yamanashi Y, Sata T, Kawaguchi Y. Construction of an Excisable bacterial artificial chromosome containing a full-length infectious clone of herpes simplex virus type 1: viruses reconstituted from the clone exhibit wild-type properties in vitro and in vivo. J Virol 2003; 77:1382–1391 [CrossRef]
    [Google Scholar]
  6. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA et al. Genome engineering using the CRISPR-Cas9 system. Nat Protoc 2013; 8:2281–2308 [CrossRef]
    [Google Scholar]
  7. Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell 2014; 157:1262–1278 [CrossRef]
    [Google Scholar]
  8. Bortesi L, Fischer R. The CRISPR/Cas9 system for plant genome editing and beyond. Biotechnol Adv 2015; 33:41–52 [CrossRef]
    [Google Scholar]
  9. Wang T, Wei JJ, Sabatini DM, Lander ES. Genetic screens in human cells using the CRISPR/Cas9 system. Science 2014; 343:80
    [Google Scholar]
  10. Kaminski R, Chen Y, Fischer T, Tedaldi E. Elimination of HIV-1 genomes from human T-lymphoid cells by CRISPR/Cas9 gene editing.; 2016
  11. Li H, Sheng C, Wang S, Yang L, Liang Y et al. Removal of integrated hepatitis B virus DNA using CRISPR-Cas9. Front Cell Infect Microbiol 2017; 7:91 [CrossRef]
    [Google Scholar]
  12. Bi Y, Sun L, Gao D, Ding C, Li Z et al. High-Efficiency targeted editing of large viral genomes by RNA-guided nucleases. PLoS Pathog 2014; 10:e1004090 [CrossRef]
    [Google Scholar]
  13. Wang D, Wang X-W, Peng X-C, Xiang Y, Song S-B et al. Crispr/Cas9 genome editing technology significantly accelerated herpes simplex virus research. Cancer Gene Ther 2018; 25:93–105 [CrossRef]
    [Google Scholar]
  14. Li Z, Bi Y, Xiao H, Sun L, Ren Y et al. Crispr-Cas9 system-driven site-specific selection pressure on herpes simplex virus genomes. Virus Res 2018; 244:286295 [CrossRef]
    [Google Scholar]
  15. Vriend LEM, Prakash R, Chen C-C, Vanoli F, Cavallo F et al. Distinct genetic control of homologous recombination repair of Cas9-induced double-strand breaks, nicks and paired nicks. Nucleic Acids Res 2016; 44:5204–5217 [CrossRef]
    [Google Scholar]
  16. Rodgers K, McVey M. Error-Prone repair of DNA double-strand breaks. J Cell Physiol 2016; 231:15–24 [CrossRef]
    [Google Scholar]
  17. Suzuki K, Tsunekawa Y, Hernandez-Benitez R, Wu J, Zhu J et al. In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration. Nature 2016; 540:144–149 [CrossRef]
    [Google Scholar]
  18. Szpara ML, Tafuri YR, Enquist L. Preparation of viral DNA from nucleocapsids. J Vis Exp 2011; 54:e3151
    [Google Scholar]
  19. Haeussler M, Schönig K, Eckert H, Eschstruth A, Mianné J et al. Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR. Genome Biol 2016; 17:148 [CrossRef]
    [Google Scholar]
  20. Reyon D, Tsai SQ, Khayter C, Foden JA, Sander JD et al. FLASH assembly of TALENs for high-throughput genome editing. Nat Biotechnol 2012; 30:460–465 [CrossRef][PubMed]
    [Google Scholar]
  21. Reed LJ, Muench H. A simple method of estimating fifty per cent endpoints. Am J Epidemiol 1938; 27:493–497 [CrossRef]
    [Google Scholar]
  22. Kay MA, He C-Y, Chen Z-Y. A robust system for production of minicircle DNA vectors. Nat Biotechnol 2010; 28:1287–1289 [CrossRef]
    [Google Scholar]
  23. Degrève B, Johansson M, De Clercq E, Karlsson A, Balzarini J. Differential intracellular compartmentalization of herpetic thymidine kinases (TKs) in TK gene-transfected tumor cells: molecular characterization of the nuclear localization signal of herpes simplex virus type 1 tk. J Virol 1998; 72:9535–9543 [CrossRef]
    [Google Scholar]
  24. Garneau JE, Dupuis Marie-Ève, Villion M, Romero DA, Barrangou R et al. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 2010; 468:6771 [CrossRef]
    [Google Scholar]
  25. Tang Y-D, Guo J-C, Wang T-Y, Zhao K, Liu J-T et al. Crispr/Cas9-Mediated 2-sgRNA cleavage facilitates pseudorabies virus editing. Faseb J 2018; 32:fj.201701129R4293–4301 [CrossRef][PubMed]
    [Google Scholar]
  26. Bétermier M, Bertrand P, Lopez BS. Is non-homologous end-joining really an inherently error-prone process?. PLoS Genet 2014; 10:e1004086 [CrossRef]
    [Google Scholar]
  27. Brinkman EK, Chen T, de Haas M, Holland HA, Akhtar W et al. Kinetics and fidelity of the repair of Cas9-Induced double-strand DNA breaks. Mol Cell 2018; 70:801–813 [CrossRef]
    [Google Scholar]
  28. Shou J, Li J, Liu Y, Wu Q. Precise and predictable CRISPR chromosomal rearrangements reveal principles of Cas9-Mediated nucleotide insertion. Mol Cell 2018; 71:498–509 [CrossRef]
    [Google Scholar]
  29. Allen F, Crepaldi L, Alsinet C, Strong AJ, Kleshchevnikov V et al. Predicting the mutations generated by repair of Cas9-induced double-strand breaks. Nature Biotechnology 2018
    [Google Scholar]
  30. Richardson CD, Ray GJ, DeWitt MA, Curie GL, Corn JE. Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA. Nat Biotechnol 2016; 34:nbt.3481344 [CrossRef]
    [Google Scholar]
  31. Kalousi A, Soutoglou E. Nuclear compartmentalization of DNA repair. Curr Opin Genet Dev 2016; 37:148–157 [CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/jgv.0.001451
Loading
/content/journal/jgv/10.1099/jgv.0.001451
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error