Anna Cruz

Demonstrating Heritable Modifications through Viral Delivery of Site-Specific Recombinases in Nicotiana benthamiana

The utilization of viral engineering has become increasingly useful for the delivery of reagents in plants. More specifically, RNA viral vectors, such as those based on Tobacco Rattle Virus (TRV), have been used in applications including silencing genes (VIGS) and delivering editing reagents. However, these viral vectors often have a carrying capacity that acts a limiting factor in their use. Site-specific recombinases (SSRs) are enzymes, varying in size, that execute precise rearrangements between DNA target sites. They are used for genetic engineering, to manipulate genome structure and control gene expression, and in plant biotechnology for marker gene excision and transgene integration. In this work, we demonstrate the delivery and stable expression of four recombinases of different sizes (~0.6 kb to ~1.5 kb) on TRV viral vectors in transgenic Nicotiana benthamiana target lines. Through an excision reaction, we attain both marker removal and the activation of a Ruby reporter. Cre, FLP, CinH, and Integrase13, cloned onto TRV vectors, were able to carry out recombination within infected somatic tissues, and also led to heritable modifications. The use of a Ruby reporter, turned on by excision, allowed for the tracking of infection within the plants and the transmission of modifications within the progeny. This experiment allows researchers more power to design genetic​ switches in the future. Additionally, it offers further information regarding the use of viral recombinase delivery, which might be helpful in optimizing future genetic engineering techniques involving similar systems.​