Cloning-free genome engineering in Sinorhizobium meliloti advances applications of Cre/loxP site-specific recombination

Abstract Serine integrases are emerging as core tools in synthetic biology and have applications in biotechnology and genome engineering. We have designed a split-intein serine integrase-based system with potential for regulation of site-specific recombination events at the protein level in vivo. The ϕC31 integrase was split into two extein domains, and intein sequences (Npu DnaEN and Ssp DnaEC) were attached to the two termini to be fused. Expression of these two components followed by post-translational protein trans-splicing in Escherichia coli generated a fully functional ϕC31 integrase. We showed that protein splicing is necessary for recombination activity; deletion of intein domains or mutation of key intein residues inactivated recombination. We used an invertible promoter reporter system to demonstrate a potential application of the split intein-regulated site-specific recombination system in building reversible genetic switches. We used the same split inteins to control the reconstitution of a split Integrase-Recombination Directionality Factor fusion (Integrase-RDF) that efficiently catalysed the reverse attR x attL recombination. This demonstrates the potential for split-intein regulation of the forward and reverse reactions using the integrase and the integrase-RDF fusion, respectively. The split-intein integrase is a potentially versatile, regulatable component for building synthetic genetic circuits and devices.

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Genome editing of different strains of Aureobasidium melanogenum using an efficient Cre/loxp site-specific recombination system

Fungal Biology ◽

10.1016/j.funbio.2019.06.001 ◽

2019 ◽

Vol 123 (10) ◽

pp. 723-731 ◽

Cited By ~ 11

Author(s):

Zhao Zhang ◽

Yi Lu ◽

Zhe Chi ◽

Guang-Lei Liu ◽

Hong Jiang ◽

...

Keyword(s):

Genome Editing ◽

Loxp Site ◽

Site Specific ◽

Site Specific Recombination ◽

Recombination System ◽

Different Strains ◽

Aureobasidium Melanogenum ◽

Site Specific Recombination System

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Controlled Cre/loxP Site-Specific Recombination in the Developing Brain in Medaka Fish, Oryzias latipes

PLoS ONE ◽

10.1371/journal.pone.0066597 ◽

2013 ◽

Vol 8 (6) ◽

pp. e66597 ◽

Cited By ~ 14

Author(s):

Teruhiro Okuyama ◽

Yasuko Isoe ◽

Masahito Hoki ◽

Yuji Suehiro ◽

Genki Yamagishi ◽

...

Keyword(s):

Oryzias Latipes ◽

Loxp Site ◽

Developing Brain ◽

Medaka Fish ◽

Site Specific ◽

Site Specific Recombination

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Transgene Coplacement and High Efficiency Site-Specific Recombination With the Cre/loxP System in Drosophila

Genetics ◽

10.1093/genetics/144.2.715 ◽

1996 ◽

Vol 144 (2) ◽

pp. 715-726 ◽

Cited By ~ 10

Author(s):

Mark L Siegal ◽

Daniel L Hartl

Keyword(s):

Germ Cells ◽

Maternal Effect ◽

High Efficiency ◽

Loxp Site ◽

Position Effects ◽

Site Specific ◽

Site Specific Recombination ◽

Bacteriophage P1 ◽

Eye Color ◽

F2 Generation

Abstract Studies of gene function and regulation in transgenic Drosophila are often compromised by the possibility of genomic position effects on gene expression. We have developed a method, called transgene coplacement, in which any two sequences can be positioned at exactly the same site and orientation in the genome. Transgene coplacement makes use of the bacteriophage P1 system of Cre/loxP site-specific recombination, which we have introduced into Drosophila. In the presence of a cre transgene driven by a dual hsp70-Mosl promoter, a white reporter gene flanked by loxP sites is excised with virtually 100% efficiency both in somatic cells and in germ cells. A strong maternal effect, resulting from Cre recombinase present in the oocyte, is observed as white or mosaic eye color in F1 progeny. Excision in germ cells of the F1 yields a strong grand-maternal effect, observed as a highly skewed ratio of eye-color phenotypes in the F2 generation. The excision reactions of Cre/loxP and the related FLP/FRT system are used to create Drosophila lines in which transgenes are at exactly allelic sites in homologous chromosomes.

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