Efficient assembly of a large fragment of monkeypox virus genome as a qPCR template using dual-selection based transformation-associated recombination

Lei Yang; Lingqian Tian; Leshan Li; Qiuhong Liu; Xiang Guo; Yuan Zhou; Rongjuan Pei; Xinwen Chen; Yun Wang

doi:10.1016/j.virs.2022.02.009

June 2022

Lei Yang, Lingqian Tian, Leshan Li, Qiuhong Liu, Xiang Guo, Yuan Zhou, Rongjuan Pei, Xinwen Chen and Yun Wang. Efficient assembly of a large fragment of monkeypox virus genome as a qPCR template using dual-selection based transformation-associated recombination[J]. Virologica Sinica, 2022, 37(3): 341-347. doi: 10.1016/j.virs.2022.02.009

Citation: Lei Yang, Lingqian Tian, Leshan Li, Qiuhong Liu, Xiang Guo, Yuan Zhou, Rongjuan Pei, Xinwen Chen, Yun Wang. Efficient assembly of a large fragment of monkeypox virus genome as a qPCR template using dual-selection based transformation-associated recombination .VIROLOGICA SINICA, 2022, 37(3) : 341-347. http://dx.doi.org/10.1016/j.virs.2022.02.009

双向筛选TAR系统可以有效组装用于qPCR检测的MPXV基因组片段模板

杨磊 ^a,b ,
田玲仟 ^a,b ,
李乐山 ^a,b ,
刘秋红 ^a,b ,
郭祥 ^a,b ,
周缘 ^a ,
裴荣娟 ^a ,
陈新文 ^a,, ,
王云 ^a,,

中国科学院武汉病毒研究所中国科学院大学中国科学院广州生物医药与健康研究院

通讯作者： 陈新文, chenxw@wh.iov.cn; 王云, wangyun@wh.iov.cn
收稿日期： 2021-08-10
录用日期： 2022-02-23

摘要

转化相关重组（TAR）已被广泛用于组装较大DNA产物，而影响组装效率的障碍之一是酵母中存在错误修复DNA途径，这导致载体骨架自连或非正确重组产物的产生。为了提高TAR组装效率，我们制备了一种双向筛选载体PGFCS，通过将P_ADH1-URA3添加到细菌酵母人工染色体PGF，含有P_HIS3-HIS3盒作为阳性选择标记，以添加的P_ADH1-URA3用作负选择标记，以确保携带线性化载体自连的酵母不能在5-氟乳清酸（5-FOA）存在下生长。为了防止PGFCS中引入的ura3与酵母基因组中的内源ura3-52重组，利用Blasticidin的抗生素抗性基因替代酵母染色体中的ura3-52，制备高度可转化的酿酒酵母VL6-48B。在VL6-48B中利用PGFCS，通过TAR组装了包含定量PCR的主要检测靶标-猴痘病毒的55kb基因组片段。PGFCS介导的TAR重组显示载体循环化率为零，正确组装产率均值为79％，表明双选策略可优化TAR重组。
- 猴痘病毒
- , 转化相关重组
- , TAR组装

Efficient assembly of a large fragment of monkeypox virus genome as a qPCR template using dual-selection based transformation-associated recombination

Lei Yang ^a,b ,
Lingqian Tian ^a,b ,
Leshan Li ^a,b ,
Qiuhong Liu ^a,b ,
Xiang Guo ^a,b ,
Yuan Zhou ^a ,
Rongjuan Pei ^a ,
Xinwen Chen ^a,, ,
Yun Wang ^a,,

a State Key Laboratory of Virology, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan, 430071, China;

Corresponding author: Xinwen Chen, chenxw@wh.iov.cn Yun Wang, wangyun@wh.iov.cn
Received Date: 10 August 2021
Accepted Date: 23 February 2022

Abstract

Transformation-associated recombination (TAR) has been widely used to assemble large DNA constructs. One of the significant obstacles hindering assembly efficiency is the presence of error-prone DNA repair pathways in yeast, which results in vector backbone recircularization or illegitimate recombination products. To increase TAR assembly efficiency, we prepared a dual-selective TAR vector, pGFCS, by adding a P_ADH1-URA3 cassette to a previously described yeast-bacteria shuttle vector, pGF, harboring a P_HIS3-HIS3 cassette as a positive selection marker. This new cassette works as a negative selection marker to ensure that yeast harboring a recircularized vector cannot propagate in the presence of 5-fluoroorotic acid. To prevent pGFCS bearing ura3 from recombining with endogenous ura3-52 in the yeast genome, a highly transformable Saccharomyces cerevisiae strain, VL6-48B, was prepared by chromosomal substitution of ura3-52 with a transgene conferring resistance to blasticidin. A 55-kb genomic fragment of monkeypox virus encompassing primary detection targets for quantitative PCR was assembled by TAR using pGFCS in VL6-48B. The pGFCS-mediated TAR assembly showed a zero rate of vector recircularization and an average correct assembly yield of 79% indicating that the dual-selection strategy provides an efficient approach to optimizing TAR assembly.
- Monkeypox virus
- , Transformation-associated recombination(TAR)
- , TAR assembly

References
1. Bauman, K.D., Li, J., Murata, K., Mantovani, S.M., Dahesh, S., Nizet, V., Luhavaya, H., Moore, B.S., 2019. Refactoring the cryptic streptophenazine biosynthetic gene cluster unites phenazine, polyketide, and nonribosomal peptide biochemistry. Cell Chem.Biol. 26, 724-736 e727.
2. Brown, K., Leggat, P.A., 2016. Human monkeypox:current state of knowledge and implications for the future. Trav. Med. Infect. Dis. 1, 8.
3. Critchlow, S.E., Jackson, S.P., 1998. DNA end-joining:from yeast to man. Trends Biochem. Sci. 23, 394-398.
4. DiCarlo, J.E., Norville, J.E., Mali, P., Rios, X., Aach, J., Church, G.M., 2013. Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res. 41, 4336-4343.
5. DiEuliis, D., Berger, K., Gronvall, G., 2017. Biosecurity implications for the synthesis of horsepox, an orthopoxvirus. Health Secur. 15, 629-637.
6. DiEuliis, D., Gronvall, G.K., 2018. A holistic assessment of the risks and benefits of the synthesis of horsepox virus. mSphere 3, e00074-18.
7. Furter-Graves, E.M., Hall, B.D., 1990. DNA sequence elements required for transcription initiation of the Schizosaccharomyces pombe ADH gene in Saccharomyces cerevisiae.Mol. Gen. Genet. 223, 407-416.
8. Gibson, D.G., Benders, G.A., Axelrod, K.C., Zaveri, J., Algire, M.A., Moodie, M., Montague, M.G., Venter, J.C., Smith, H.O., Hutchison 3rd, C.A., 2008. One-step assembly in yeast of 25 overlapping DNA fragments to form a complete synthetic Mycoplasma genitalium genome. Proc. Natl. Acad. Sci. U. S. A. 105, 20404-20409.
9. Hou, Z., Zhou, Z., Wang, Z., Xiao, G., 2016. Assembly of long DNA sequences using a new synthetic Escherichia coli-yeast shuttle vector. Virol. Sin. 31, 160-167.
10. Koblentz, G.D., 2017. The de novo synthesis of horsepox virus:implications for biosecurity and recommendations for preventing the reemergence of smallpox.Health Secur. 15, 620-628.
11. Koblentz, G.D., 2018. A critical analysis of the scientific and commercial rationales for the de novo synthesis of horsepox virus. mSphere 3, e00040-18.
12. Kouprina, N., Larionov, V., 2016. Transformation-associated recombination (TAR) cloning for genomics studies and synthetic biology. Chromosoma 125, 621-632.
13. Kuijpers, N.G., Solis-Escalante, D., Bosman, L., van den Broek, M., Pronk, J.T., Daran, J.M., Daran-Lapujade, P., 2013. A versatile, efficient strategy for assembly of multi-fragment expression vectors in Saccharomyces cerevisiae using 60 bp synthetic recombination sequences. Microb. Cell Factories 12, 47.
14. Kulesh, D.A., Baker, R.O., Loveless, B.M., Norwood, D., Zwiers, S.H., Mucker, E., Hartmann, C., Herrera, R., Miller, D., Christensen, D., Wasieloski Jr., L.P., Huggins, J., Jahrling, P.B., 2004. Smallpox and pan-orthopox virus detection by real-time 3'-minor groove binder TaqMan assays on the roche LightCycler and the Cepheid smart Cycler platforms. J. Clin. Microbiol. 42, 601-609.
15. Larionov, V., Kouprina, N., Nikolaishvili, N., Resnick, M.A., 1994. Recombination during transformation as a source of chimeric mammalian artificial chromosomes in yeast(YACs). Nucleic Acids Res. 22, 4154-4162.
16. Lewis, L.K., Resnick, M.A., 2000. Tying up loose ends:nonhomologous end-joining in Saccharomyces cerevisiae. Mutat. Res. 451, 71-89.
17. Li, Y., Zhao, H., Wilkins, K., Hughes, C., Damon, I.K., 2010. Real-time PCR assays for the specific detection of monkeypox virus West African and Congo Basin strain DNA.J. Virol. Methods 169, 223-227.
18. Ma, J.L., Kim, E.M., Haber, J.E., Lee, S.E., 2003. Yeast Mre11 and Rad1 proteins define a Ku-independent mechanism to repair double-strand breaks lacking overlapping end sequences. Mol. Cell Biol. 23, 8820-8828.
19. Miret, J.J., Pessoa-Brandao, L., Lahue, R.S., 1998. Orientation-dependent and sequencespecific expansions of CTG/CAG trinucleotide repeats in Saccharomyces cerevisiae.Proc. Natl. Acad. Sci. U. S. A. 95, 12438-12443.
20. Noskov, V.N., Koriabine, M., Solomon, G., Randolph, M., Barrett, J.C., Leem, S.H., Stubbs, L., Kouprina, N., Larionov, V., 2001. Defining the minimal length of sequence homology required for selective gene isolation by TAR cloning. Nucleic Acids Res. 29, E32.
21. Noskov, V.N., Kouprina, N., Leem, S.H., Ouspenski, I., Barrett, J.C., Larionov, V., 2003.A general cloning system to selectively isolate any eukaryotic or prokaryotic genomic region in yeast. BMC Genom. 4, 16.
22. Noyce, R.S., Lederman, S., Evans, D.H., 2018. Construction of an infectious horsepox virus vaccine from chemically synthesized DNA fragments. PLoS One 13, e0188453.
23. Paques, F., Haber, J.E., 1999. Multiple pathways of recombination induced by doublestrand breaks in Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 63, 349-404.
24. Shang, Y., Wang, M., Xiao, G., Wang, X., Hou, D., Pan, K., Liu, S., Li, J., Wang, J., Arif, B.M., Vlak, J.M., Chen, X., Wang, H., Deng, F., Hu, Z., 2017. Construction and rescue of a functional synthetic baculovirus. ACS Synth. Biol. 6, 1393-1402.
25. Thi Nhu Thao, T., Labroussaa, F., Ebert, N., V'Kovski, P., Stalder, H., Portmann, J., Kelly, J., Steiner, S., Holwerda, M., Kratzel, A., Gultom, M., Schmied, K., Laloli, L., Husser, L., Wider, M., Pfaender, S., Hirt, D., Cippa, V., Crespo-Pomar, S., Schroder, S., Muth, D., Niemeyer, D., Corman, V.M., Muller, M.A., Drosten, C., Dijkman, R., Jores, J., Thiel, V., 2020. Rapid reconstruction of SARS-CoV-2 using a synthetic genomics platform. Nature 582, 561-565.
26. Vrančić, M., Gregorić, S., Paravić Radičević, A., Gjuračić, K., 2008. Mammalian genome recombineering:yeast, still a helper microorganism of choice? Food Technol.Biotechnol. 46, 237-251.
27. Wild, J., Szybalski, W., 2004. Copy-control tightly regulated expression vectors based on pBAC/oriV. Methods Mol. Biol. 267, 155-167.
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Electronic Supplementary Material

10.1016j.virs.2022.02.009-ESM.doc