An open-source system for in planta gene stacking by Bxb1 and Cre recombinases

Area of Science:

• Plant Biotechnology
• Molecular Genetics
• Crop Science

Background:

• Classical breeding for multiple transgenic traits in crops is inefficient due to low probability of trait assortment.
• Developing stacked cultivars necessitates precise integration of new transgenes without disrupting existing traits.
• Site-specific integration offers a solution for adding transgenes to existing transgenic lines.

Purpose of the Study:

• To test a recombinase-mediated gene-stacking system for creating multi-trait transgenic crops.
• To evaluate the efficiency and reproducibility of sequential site-specific DNA integration in tobacco.
• To assess the potential for developing commercial cultivars using a patent-free gene-stacking technology.

Main Methods:

• Utilized mycobacteriophage Bxb1 integrase for site-specific recombination between attP and attB sites.
• Employed Cre recombinase for excision of unwanted DNA via lox site recombination.
• Performed sequential integration of transgenes in tobacco to create stacked lines.

Main Results:

• Site-specific integration was achieved in approximately 10% of integration events.
• Half of the site-specific integration events were suitable for subsequent rounds of gene stacking.
• Gene silencing was observed in one-third of the site-specific integrants.
• A 3% overall success rate was achieved for precise structure and reproducible expression of sequentially added triple traits.

Conclusions:

• Recombinase-mediated gene stacking provides a workable frequency for developing commercial multi-trait cultivars.
• The tested system allows for precise genetic modification and reproducible trait expression.
• The use of Bxb1-att and Cre-lox systems offers freedom to operate, facilitating broader application in crop improvement.