Serine recombinases mediating stable integration into plant genomes
Inventors
Tiwari, Shiv B. • TREMBLAY, Arianne • Carson, Jonathan • Boddupalli, Sekhar • STAMLER, Rio • EDWARDS, Amanda • FRECH, Kornelie
Assignees
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Publication Number
US-12241070-B2
Publication Date
2025-03-04
Expiration Date
Abstract
Described is a serine recombinase that integrates large sections of foreign DNA into plant chromosomes through non-homologous recombination through attP or attB sites on the foreign DNA but without the engineering of corresponding attB or attP site on the plant chromosome. The serine recombinase-based method has several advantages over other systems, such as Agrobacterium. Also described are related methods, composition, and plants containing exogenous DNA.
Core Innovation
The disclosure provides a plant genome engineering approach for incorporating exogenous DNA into a plant genome by co-transfecting a plant cell with exogenous DNA comprising an attP or attB site and a polynucleotide encoding an SPβc2 serine recombinase operably linked to a promoter active in the plant. The method includes selecting for integration at a plant att pseudosite comprising a core sequence defined by specified SEQ ID NOs or consensus sequences, and growing the plant cell to obtain the engineered plant genome outcome.
A key feature is the use of SPβc2 to integrate foreign DNA via plant att pseudosite sequences without prior engineering of canonical att sites. The disclosure describes SPβc2 as capable of attP-directed stable integration and includes experimental confirmation of complete plasmid integration with enrichment of GFP/kanamycin-positive integrants and PCR/sequencing confirmation.
The system is presented as supporting conditional, inducible excision using a cognate RDF, enabling scarless attL/attR excision, and as allowing additional genetic modification and stacking, including engineered inserts containing loxP/FRT or non-cognate att sites. Cross-species applicability is described for lettuce and other plant species, including Nicotiana, rice, corn, and soybean.
Claims Coverage
The document includes two independent claims. Each independent claim defines an SPβc2-based integration method into plant att pseudosites, with one claim addressing general plant cells and one specifying lettuce plant cells.
Co-transfecting plant cell with attP/attB exogenous DNA and SPβc2 serine recombinase under plant-active promoter
A method comprising co-transfecting a plant cell with exogenous DNA comprising an attP or attB site and a polynucleotide encoding an SPβc2 serine recombinase comprising SEQ ID NO:36, or at least 95% identity, operably linked to a promoter active in the plant.
Selecting integration at plant att pseudosite having defined core sequence
Selecting for integration of the exogenous DNA into the plant cell genome at a plant att pseudosite comprising a core sequence of SEQ ID NO:99 or SEQ ID NO:101 or a consensus sequence of SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:130, or SEQ ID NO:131.
Obtaining site-specific recombination in a lettuce plant cell using SPβc2
A method for obtaining site-specific recombination in a lettuce plant cell comprising contacting a lettuce plant cell with exogenous DNA comprising an attP or attB site and a serine recombinase polypeptide active in a lettuce plant, wherein the serine recombinase is SPβc2.
Selecting integration at a lettuce plant att pseudosite and growing the lettuce plant cell
Selecting for integration of the exogenous DNA into the lettuce plant cell genome at a lettuce plant att pseudosite and growing the lettuce plant cell.
Overall, claim coverage focuses on SPβc2-driven site-specific integration of exogenous DNA containing an attP or attB site into plant att pseudosites, including a lettuce-specific embodiment, with integration selection and growth as defined in the independent claims.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Plant genome engineering for incorporating exogenous DNA into a plant genome via attP/attB and SPβc2 integration into plant att pseudosites.
Obtaining site-specific recombination in a lettuce plant cell using SPβc2.
Conditional, inducible excision of the introduced sequence using a cognate RDF, including scarless attL/attR excision.
System expansion for stacking using engineered inserts containing loxP/FRT or non-cognate att sites.
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