Mesoporous Silica Nanoparticle-Mediated Intracellular Cre Protein Delivery for Maize Genome Editing via loxP Site Excision,

The delivery of proteins instead of DNA into plant cells allows for a transient presence of the protein or enzyme that can be useful for biochemical analysis or genome modifications. This may be of particular interest for genome editing, because it can avoid DNA (transgene) integration into the genome and generate precisely modified "nontransgenic" plants. In this work, we explore direct protein delivery to plant cells using mesoporous silica nanoparticles (MSNs) as carriers to deliver Cre recombinase protein into maize (Zea mays) cells. Cre protein was loaded inside the pores of gold-plated MSNs, and these particles were delivered by the biolistic method to plant cells harboring loxP sites flanking a selection gene and a reporter gene. Cre protein was released inside the cell, leading to recombination of the loxP sites and elimination of both genes. Visual selection was used to select recombination events from which fertile plants were regenerated. Up to 20% of bombarded embryos produced calli with the recombined loxP sites under our experimental conditions. This direct and reproducible technology offers an alternative for DNA-free genome-editing technologies in which MSNs can be tailored to accommodate the desired enzyme and to reach the desired tissue through the biolistic method.