DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins

Editing plant genomes without introducing foreign DNA into cells may alleviate regulatory concerns related to genetically modified plants. We transfected preassembled complexes of purified Cas9 protein and guide RNA into plant protoplasts of Arabidopsis thaliana, tobacco, lettuce and rice and achieved targeted mutagenesis in regenerated plants at frequencies of up to 46%. The targeted sites contained germline-transmissible small insertions or deletions that are indistinguishable from naturally occurring genetic variation

Plastid-Based Expression Strategies

Since the development of methodologies for achieving genetic modification of chloroplast genomes, termed transplastomic technologies, a straightforward use in the field of plant-based vaccines was initiated. Chloroplast transformation is mediated by homologous recombination that allows for site-specific insertion of foreign DNA into the plastome. This focus offers substantial advantages that include high yields, improved biosafety given by maternal inheritance in most plant species, and multigene expression through polycistrons allowing in theory for the straightforward production of multicomponent vaccines. One limitation of the system consists of the lack of glycosylation pathways, which are of relevance in some cases. State of the art in this area reflects a number of well-characterized vaccination models, although no one has passed clinical evaluations, which contrasts with other nuclear transient expression systems. Transplastomic technologies are envisioned as a relevant tool for developing new convenient vaccines