Development of Int-Plex@ binary memory switch system: plant genome modulation driven by large serine-integrases

Abstract

ABSTRACTThe comprehension of virus-host interactions has allowed numerous advances in developing biotechnological methodologies for plant genome editions, constituting a promising path for plant genetic engineering. Among these advancements, phage- encoded large serine-integrases have emerged as noteworthy tools to modulate plant metabolic pathways by inserting, excising, or inverting DNA stretches in a reversible and specific way. The present work shows the foundation of the Int-Plex@ (INTegrase PLant EXpression) binary memory switch system, which consists of the application of four distinct orthogonal prophage large serine-integrases (Int) (BxB1, phiC31, Int13, and Int9) as an input trigger mechanism for the inversion or excision of genomic DNA. The memory genetic switch is divided into the excision module and the inversion module. The excision module is activated by BxB1 or phiC31 enzymes (input). In this case, the DNA sequence flanked by its attachment sites is excised from the genome (output). The inversion module is activated by Int9 or Int13 (input). The invertedmgfgene sequence is flipped to its functional coding sequence, and the switch output is mGFP. Moreover, prokaryotic-based cell-freein vitrotranscription-translation reactions (TxTl) were used as a fast platform for testing IntsattB/P in tandemsite activity. Furthermore different plasmid delivery strategies for plant cell Int heterologous expression were tested: leaf tissue agroinfiltration ofAgrobacterium tumefacienstransformed with binary plasmids and a biolistic system. After each treatment, the edited genomic DNA sequences were amplified and verified by Sanger and Nanopore sequencing. Despite the challenges of using Ints, the potential benefits are significant and deserve deeper exploration and development. The Int-Plex@ binary genome memory switch system can be applied to produce genetic circuits combined with omics tools and sgRNAs to engineer and modulate plant metabolic pathways temporally and reversibly.