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.

Disclosed are compositions and virus-like particles (VLPs) self-assembled from the expression of human immunodeficiency virus (HIV) Gag protein and a fragment of gp41 protein containing its N-terminus ectodomain. The fragment of gp41 protein is linked to an antigen that is not a peptide or protein from HIV, which is presented by HIV VLP. In some aspects, the presented antigen is trimerized. Also disclosed are methods of inducing an immune response against the antigen.

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.

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.

The disclosure relates to a T-DNA binary vector based on bean yellow dwarf virus (BeYDV) that reduces plant cell death and increases transgene expression. In one aspect, the T-DNA region comprise a replicon cassette comprising a rep gene or a repA gene with a mutated translation initiation region. The disclosure also relates to replicating geminiviral expression system based on BeYDV comprising with an expression cassette a sequence encoding Rep and a sequence encoding the promoter of ubiquitin-3 from potato with ubiquitin fusion; an expression cassette comprising a sequence encoding RepA and a sequence encoding the promoter of ubiquitin-3 from potato with ubiquitin fusion; and an expression cassette comprising a promoter region, a 5′ UTR, a sequence encoding a recombinant protein, and a 3′ UTR. These expression cassettes are on different T-DNA cloning vectors or on one T-DNA cloning vector.

Disclosed herein are compositions comprising a polypeptide with at least two domains, wherein the first domain is capable of binding CD3 and the second domain is capable of binding to a cancer cell. Also disclosed herein are methods of treating cancer in a subject, comprising: providing a composition comprising a polypeptide with at least two domains, wherein the first domain is capable of binding CD3 and the second domain is capable of binding to a cancer cell; and treating the cancer by administering a therapeutically effective dosage of the composition to the subject.

Systems and methods for gene targeting in plants, including systems and methods that include the use of geminiviruses and customizable endonucleases.

Improved plant transient expression systems using optimized geminiviral vectors that efficiently produce heteromultimeric proteins are described herein. Examples of high yields are shown herein, including two, three, or four fluorescent proteins coexpressed simultaneously. Various antibodies were produced using the optimized vectors with special focus given to the creation and production of a chimeric broadly neutralizing anti-flavivirus antibody. The variable regions of this murine antibody, 2A10G6, were codon optimized and fused to a human IgG1. Analysis of the chimeric antibody showed that it was efficiently expressed in plants, can be purified to near homogeneity by a simple one-step purification process, retains its ability to recognize the Zika virus envelope protein, and induce an immune response against Zika virus. Two other monoclonal antibodies were produced at similar levels. This technology is versatile tool for the production of a wide spectrum of pharmaceutical multi-protein complexes in a fast, powerful, and cost-effective way.

Systems and methods for gene targeting in plants, including systems and methods that include the use of geminiviruses and customizable endonucleases.

The present disclosure relates to plant-based recombinant protein production systems and their methods of production and use. The plant-based recombinant protein production system is a vector comprising a 5 UTR and a 3 UTR, wherein the 3 UTR comprises at least one terminator selected from the group consisting of: EU, IEU, NbACT3, NbACT617, NbACT567, Pin2, BDB501, BDB282, NbHSP, NbHSPb, Rep, RbcS, SIR, SIR 5/3, SIR 3, AtHSP, 35S, RepA, NOS, TMV, TNVD, PEMV, and BYDV. In certain implementations, the vector comprises two terminators in the 3 UTR, where the two terminators are fused to form a double terminator. For example, the double terminator comprises two members selected from the group consisting of: EU, IEU, NbACT3, NbACT617, NbACT567, Pin2, BDB501, BDB282, NbHSP, NbHSPb, Rep, RbcS, SIR, SIR 5/3, SIR 3, AtHSP, 35S, RepA, NOS, TMV, TNVD, PEMV, and BYDV. In some aspects, the vector further comprises a chromatin scaffold/matrix attachment region (MAR) that is downstream of the terminators.