The present invention belongs to the field of genetic engineering. Specifically, the present invention relates to a directed evolution method based on geminivirus. More specifically, the present invention relates to a directed evolution method for in vivo screening of a genetic element in a plant cell by using primary and secondary replicons of geminivirus.

A method for increasing the efficiency of homologous recombination-based gene editing in a plant according to an embodiment of the present invention includes optimizing temperature and photoperiod conditions during tissue culture of plant cells, expressing factors required for homology-directed DNA repair (HDR) and factors for increasing the HDR efficiency by using a multiple replicon, or regulating the HDR pathway or non-homologous end joining (NHEJ) pathway.

The present disclosure provides a viral-mediated genome-editing platform that facilitates multiplexing, obviates stable transformation, and is applicable across plant species. The RNA2 genome of the tobacco rattle virus (TRV) was engineered to carry and systemically deliver a guide RNA molecules into plants overexpressing Cas9 endonuclease. High genomic modification frequencies were observed in inoculated as well as systemic leaves including the plant growing points. This system facilitates multiplexing and can lead to germinal transmission of the genomic modifications in the progeny, thereby obviating the requirements of repeated transformations and tissue culture. The editing platform of the disclosure is useful in plant genome engineering and applicable across plant species amenable to viral infections for agricultural biotechnology applications.

The present invention relates to methods and hybrids for the targeted modification of a nucleic acid-target region in a plant target structure using CRISPR/Cas systems. The invention specifically relates to methods and hybrids for directly obtaining a plant or plant material which comprises an editing of a nucleic acid introduced in a targeted manner into a meristematic cell. The hybrids can be introduced in a transient and/or stable manner. The invention also relates to novel plant-optimized introduction strategies. The invention further relates to a method for carrying out an in vitro screening assay in order to first check the suitable gRNA candidates in vitro with respect to their efficiency.

Severe Acute Respiratory Syndrome Coronavirus 2 antigen virus-like particles (VLPs) and recombinant immune complexes (RICs) are described, along with methods of making said VLPs and RICs in plants and using said VLPs and RICs to induce an immune response in a subject.

Methods and materials for increasing somatic and germline genome editing are provided herein. For example, provided herein are methods and materials for using augmented sgRNAs to increase somatic and germline genome editing.

The present disclosure provides DNA molecules and constructs, including their nucleotide sequences, useful for expressing proteins in plants to promote formation of nodule-like structures in the presence of Rhizobia. The present disclosure also provides DNA molecules and constructs, including their nucleotide sequences, useful for expressing proteins in plants to increase drought resistance. The present disclosure also provides plants and plant cells transgenic plants, plant cells, plant parts, seeds, and commodity products comprising the DNA molecules, along with methods of their use.

The present invention provides plant virus vectors developed from the Sugarcane mosaic virus (SCMV). The vectors include a nucleic acid sequence encoding an infectious Sugarcane mosaic virus (SCMV) operably linked to one or more regulatory elements functional in a plant. The plant virus vectors may be used to infect monocot plants, such as maize, for gene expression applications.

A recombinant vector according to an embodiment is for genome editing without inserting a replicon into the plant genome in a T.sub.0 generation plant. The recombinant vector includes a geminivirus-based replicon between the sequence of LB (left border) and sequence of RB (right border) of Ti plasmid. A method of genome editing without inserting a replicon into the plant genome in a T.sub.0 generation plant according to an embodiment includes transforming a plant cell by inserting a foreign gene to the aforementioned recombinant vector.

This invention relates to methods for the transformation of Setaria species such as Setaria viridis and transformed plants produced according to the method. Specifically, this invention relates to direct transformation of callus derived from mature embryos using Agrobacterium-mediated transformation, and plants regenerated from the transformed callus tissue. The methods comprise utilizing Setaria mature embryos as the source of plant material for callus induction; induced calli can be infected by Agrobacterium hosting an appropriate vector. Transgenic plants are regenerated from transgenic calli grown under conditions favoring growth of transformed cells while substantially inhibiting growth of non-transformed cells. These methods provide for significantly increased plant transformation efficiency with minimal ratio of escapes.