Artificial Nondirectional Site-specific Recombination Systems

Overview

Journal iScience

Publisher Cell Press

Date 2022 Jan 24

PMID 35072008

Authors

Jun-Yi Wang

Yue-Yang Cao

Ya-Nan Chen

Xiao-Le Wu

Bo-Tao He

Si-Yu Zhu

Xiao Zhou

Yi Wu

Bing-Zhi Li

Ying-Jin Yuan

Affiliations

Soon will be listed here.

Abstract

Site-specific recombination systems (SRSs) are widely used in studies on synthetic biology and related disciplines. Nondirectional SRSs can randomly trigger excision, integration, reversal, and translocation, which are effective tools to achieve large-scale genome recombination. In this study, we designed 6 new nondirectional SRSs named Vika/voxsym1-4 and Dre/roxsym1-2. All 6 artificial nondirectional SRSs were able to generate random deletion and inversion in . Moreover, all six SRSs were orthogonal to Cre/loxPsym. The pairwise orthogonal nondirected SRSs can simultaneously initiate large-scale and independent gene recombination in two different regions of the genome, which could not be accomplished using previous orthogonal systems. These SRSs were found to be robust while working in the cells at different growth stages, as well as in the different spatial structure of the chromosome. These artificial pairwise orthogonal nondirected SRSs offer newfound potential for site-specific recombination in synthetic biology.

Citing Articles

Orthogonal LoxPsym sites allow multiplexed site-specific recombination in prokaryotic and eukaryotic hosts.

Cautereels C, Smets J, De Saeger J, Cool L, Zhu Y, Zimmermann A Nat Commun. 2024; 15(1):1113.

PMID: 38326330 PMC: 10850332. DOI: 10.1038/s41467-024-44996-8.

References

Richardson S, Mitchell L, Stracquadanio G, Yang K, Dymond J, DiCarlo J . Design of a synthetic yeast genome. Science. 2017; 355(6329):1040-1044. DOI: 10.1126/science.aaf4557. View

Xie Z, Li B, Mitchell L, Wu Y, Qi X, Jin Z . "Perfect" designer chromosome V and behavior of a ring derivative. Science. 2017; 355(6329). DOI: 10.1126/science.aaf4704. View

Ausubel F . Radiochemical purification of bacteriophage lambda integrase. Nature. 1974; 247(5437):152-4. DOI: 10.1038/247152a0. View

Shen Y, Stracquadanio G, Wang Y, Yang K, Mitchell L, Xue Y . SCRaMbLE generates designed combinatorial stochastic diversity in synthetic chromosomes. Genome Res. 2015; 26(1):36-49. PMC: 4691749. DOI: 10.1101/gr.193433.115. View

Mitchell L, Wang A, Stracquadanio G, Kuang Z, Wang X, Yang K . Synthesis, debugging, and effects of synthetic chromosome consolidation: synVI and beyond. Science. 2017; 355(6329). DOI: 10.1126/science.aaf4831. View

Luo L, Ambrozkiewicz M, Benseler F, Chen C, Dumontier E, Falkner S . Optimizing Nervous System-Specific Gene Targeting with Cre Driver Lines: Prevalence of Germline Recombination and Influencing Factors. Neuron. 2020; 106(1):37-65.e5. PMC: 7377387. DOI: 10.1016/j.neuron.2020.01.008. View

Brown W, Lee N, Xu Z, Smith M . Serine recombinases as tools for genome engineering. Methods. 2011; 53(4):372-9. DOI: 10.1016/j.ymeth.2010.12.031. View

Sauer B, McDermott J . DNA recombination with a heterospecific Cre homolog identified from comparison of the pac-c1 regions of P1-related phages. Nucleic Acids Res. 2004; 32(20):6086-95. PMC: 534624. DOI: 10.1093/nar/gkh941. View

Meinke G, Bohm A, Hauber J, Pisabarro M, Buchholz F . Cre Recombinase and Other Tyrosine Recombinases. Chem Rev. 2016; 116(20):12785-12820. DOI: 10.1021/acs.chemrev.6b00077. View

10.

Hutchison 3rd C, Chuang R, Noskov V, Assad-Garcia N, Deerinck T, Ellisman M . Design and synthesis of a minimal bacterial genome. Science. 2016; 351(6280):aad6253. DOI: 10.1126/science.aad6253. View

11.

Volkert F, Broach J . Site-specific recombination promotes plasmid amplification in yeast. Cell. 1986; 46(4):541-50. DOI: 10.1016/0092-8674(86)90879-2. View

12.

NASH H, Robertson C . Purification and properties of the Escherichia coli protein factor required for lambda integrative recombination. J Biol Chem. 1981; 256(17):9246-53. View

13.

Lin Q, Qi H, Wu Y, Yuan Y . Robust orthogonal recombination system for versatile genomic elements rearrangement in yeast Saccharomyces cerevisiae. Sci Rep. 2015; 5:15249. PMC: 4609996. DOI: 10.1038/srep15249. View

14.

Zhao J, Pokhilko A, Ebenhoh O, Rosser S, Colloms S . A single-input binary counting module based on serine integrase site-specific recombination. Nucleic Acids Res. 2019; 47(9):4896-4909. PMC: 6511857. DOI: 10.1093/nar/gkz245. View

15.

Zhang W, Zhao G, Luo Z, Lin Y, Wang L, Guo Y . Engineering the ribosomal DNA in a megabase synthetic chromosome. Science. 2017; 355(6329). DOI: 10.1126/science.aaf3981. View

16.

Wu Y, Li B, Zhao M, Mitchell L, Xie Z, Lin Q . Bug mapping and fitness testing of chemically synthesized chromosome X. Science. 2017; 355(6329). PMC: 5679077. DOI: 10.1126/science.aaf4706. View

17.

Ma L, Li Y, Chen X, Ding M, Wu Y, Yuan Y . SCRaMbLE generates evolved yeasts with increased alkali tolerance. Microb Cell Fact. 2019; 18(1):52. PMC: 6410612. DOI: 10.1186/s12934-019-1102-4. View

18.

NASH H . Purification of bacteriophage lambda Int protein. Nature. 1974; 247(5442):543-5. DOI: 10.1038/247543a0. View

19.

Wu Y, Zhu R, Mitchell L, Ma L, Liu R, Zhao M . In vitro DNA SCRaMbLE. Nat Commun. 2018; 9(1):1935. PMC: 5964173. DOI: 10.1038/s41467-018-03743-6. View

20.

Gilbertson L . Cre-lox recombination: Cre-ative tools for plant biotechnology. Trends Biotechnol. 2003; 21(12):550-5. DOI: 10.1016/j.tibtech.2003.09.011. View