Multipart DNA Assembly Using Site-Specific Recombinases from the Large Serine Integrase Family

Olorunniji, Femi J.; Merrick, Christine; Rosser, Susan J.; Smith, Margaret C. M.; Stark, W. Marshall; Colloms, Sean D.

doi:10.1007/978-1-4939-7169-5_19

Cited by 15 publications

(17 citation statements)

References 11 publications

(5 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We began with a plasmid (p(BEIZY)) made in previous work ( 8 , 9 ), which contains an array of genes expressing enzymes for a metabolic pathway leading to the carotenoid zeaxanthin. The five carotenoid genes crtB, crtE, crtI, crtZ and crtY were assembled (in this order) from PCR products using ϕC31 integrase-mediated attP × attB recombination, so that each gene cassette in p(BEIZY) is flanked by attL sites (Figure 6A ).…”

Section: Resultsmentioning

confidence: 99%

“…A mixture of p(BEIZY) and Cm R PCR product was treated with the ϕC31.Int-gp3 fusion protein, or with integrase plus gp3 (two different concentrations of gp3 were compared). Aliquots of the reactions were stopped after 1, 2 and 18 h, and the products were used to transform E. coli cells by a standard protocol ( 8 , 9 ), selecting for ampicillin and chloramphenicol resistance. The numbers of colonies were then counted (Figure 6B ).…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Control of serine integrase recombination directionality by fusion with the directionality factor

Olorunniji

McPherson

Rosser

et al. 2017

Nucleic Acids Research

Self Cite

View full text Add to dashboard Cite

Bacteriophage serine integrases are extensively used in biotechnology and synthetic biology for assembly and rearrangement of DNA sequences. Serine integrases promote recombination between two different DNA sites, attP and attB, to form recombinant attL and attR sites. The ‘reverse’ reaction requires another phage-encoded protein called the recombination directionality factor (RDF) in addition to integrase; RDF activates attL × attR recombination and inhibits attP × attB recombination. We show here that serine integrases can be fused to their cognate RDFs to create single proteins that catalyse efficient attL × attR recombination in vivo and in vitro, whereas attP × attB recombination efficiency is reduced. We provide evidence that activation of attL × attR recombination involves intra-subunit contacts between the integrase and RDF moieties of the fusion protein. Minor changes in the length and sequence of the integrase–RDF linker peptide did not affect fusion protein recombination activity. The efficiency and single-protein convenience of integrase–RDF fusion proteins make them potentially very advantageous for biotechnology/synthetic biology applications. Here, we demonstrate efficient gene cassette replacement in a synthetic metabolic pathway gene array as a proof of principle.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Control of serine integrase recombination directionality by fusion with the directionality factor

Olorunniji

McPherson

Rosser

et al. 2017

Nucleic Acids Research

Self Cite

View full text Add to dashboard Cite

show abstract

“…The approach can be used on multiple genetic backgrounds extending ideas of genetic diversity and crucially providing a uniform integration site for expression of transgenes. The attP/B approach also makes feasible ideas of subsequent sequential gene addition or "gene-stacking" by embedding other heterologous att sites in introduced constructs or by multipart assembly in vivo (44,73,74). The advent of advanced sequencing protocol/methods exemplified here in the single molecule reading of ~80kb further advances the reality of precision in the genetic modification of animals.…”

Section: Discussionmentioning

confidence: 99%

Efficient targeted transgenesis of large donor DNA into multiple mouse genetic backgrounds using bacteriophage Bxb1 integrase

Low

Hosur

Lesbirel

et al. 2021

Preprint

View full text Add to dashboard Cite

Efficient, targeted integration of large DNA constructs represent a significant hurdle in genetic engineering for the development of mouse models of human disease and synthetic biology research. To address this, we developed a system for efficient and precise, targeted single-copy integration of large transgenes directly into the zygote using multiple mouse genetic backgrounds. Conventional approaches, such as random transgenesis, CRISPR/Cas9-mediated homology-directed repair (HDR), lentivirus-based insertion, or DNA transposases all have significant limitations. Our strategy uses in vivo Bxb1 mediated recombinase-mediated cassette exchange (RMCE) to efficiently generate precise single-copy integrations of transgenes. This is achieved using a transgene landing pad composed of dual heterologous Bxb1 attachment (att) sites in cis, pre-positioned in the Gt(ROSA)26Sor safe harbor locus. Successful RMCE is achieved in att carrier zygotes using donor DNA carrying cognate attachment sites flanking the desired donor transgene microinjected along with Bxb1-integrase mRNA. This approach routinely achieves perfect vector-free integration of donor constructs at efficiencies as high as 43% and has generated transgenic animals containing inserts up to ~43kb. Furthermore, when coupled with a nanopore-based Cas9-targeted sequencing (nCATS) approach, complete verification of the precise insertion sequence can be achieved. As a proof-of-concept we describe the creation and characterization of C57BL/6J and NSG Krt18-ACE2 transgenic mouse models for SARS-CoV2 research with verified heterozygous N1 animals available for experimental use in ~4 months. In addition, we created a diverse series of mouse backgrounds carrying a single att site version of the landing pad allele in C57BL/6J, NSG, B6(Cg)-Tyrc-2J/J, FVB/NJ, PWK/PhJ, 129S1/SvImJ, A/J, NOD/ShiLtJ, NZO/HILtJ, CAST/EiJ, and DBA/2J for rapid transgene insertion. Combined, this system enables predictable, rapid creation of precisely targeted transgenic animals across multiple genetic backgrounds, simplifying characterization, speeding expansion and use.

show abstract

“…The approach can be used on multiple genetic backgrounds extending ideas of genetic diversity and crucially providing a uniform integration site for expression of transgenes. The attP/B approach also makes feasible ideas of subsequent sequential gene addition or "gene-stacking" by embedding other heterologous att sites in introduced constructs or by multipart assembly in vivo 32,60,61 . The advent of advanced sequencing protocol/methods exempli ed here in the single molecule reading of ~80kb further advances the reality of precision in the genetic modi cation of animals.…”

Section: Conclusion Future Directionsmentioning

confidence: 99%

Efficient Targeted Transgenesis of Large Donor DNA Into Multiple Mouse Genetic Backgrounds Using Bacteriophage Bxb1 Integrase

Low

Hosur

Lesbirel

et al. 2021

Preprint

View full text Add to dashboard Cite

The development of mouse models of human disease and synthetic biology research by targeted transgenesis of large DNA constructs represent a significant genetic engineering hurdle. We developed an efficient, precise, single-copy integration of large transgenes directly into zygotes using multiple mouse genetic backgrounds. We used in vivo Bxb1 mediated recombinase-mediated cassette exchange (RMCE) with a transgene “landing pad” composed of dual heterologous Bxb1 attachment (att) sites in cis, within the Gt(ROSA)26Sor safe harbor locus. RMCE of donor was achieved by microinjection of vector DNA carrying cognate attachment sites flanking the donor transgene with Bxb1-integrase mRNA. This approach achieves perfect vector-free integration of donor constructs at efficiencies >40% with up to ~43kb transgenes. Coupled with a nanopore-based Cas9-targeted sequencing (nCATS), complete verification of precise insertion sequence was achieved. As a proof-of-concept we describe the development of C57BL/6J and NSG Krt18-ACE2 models for SARS-CoV2 research with verified heterozygous N1 animals within ~4 months. Additionally, we created a series of mice with diverse backgrounds carrying a single att site including FVB/NJ, PWK/PhJ, NOD/ShiLtJ, CAST/EiJ and DBA/2J allowing for rapid transgene insertion. Combined, this system enables predictable, rapid development combined with simplified characterization of precisely targeted transgenic animals across multiple genetic backgrounds.

show abstract

Multipart DNA Assembly Using Site-Specific Recombinases from the Large Serine Integrase Family

Cited by 15 publications

References 11 publications

Control of serine integrase recombination directionality by fusion with the directionality factor

Control of serine integrase recombination directionality by fusion with the directionality factor

Efficient targeted transgenesis of large donor DNA into multiple mouse genetic backgrounds using bacteriophage Bxb1 integrase

Efficient Targeted Transgenesis of Large Donor DNA Into Multiple Mouse Genetic Backgrounds Using Bacteriophage Bxb1 Integrase

Contact Info

Product

Resources

About