Abstract: The present disclosure provides compositions, methods, and systems for targeted plant genome editing. In some embodiments, the present disclosure provides recombinant self-replicating RNAs derived from a plant virus vector, such as those derived from Tobacco Mosaic Virus (TMV). In some embodiments, the recombinant self-replicating RNAs direct the expression of both a CRISPR endonuclease and a guide RNA capable of directing sequence-specific binding of the CRISPR endonuclease to a target DNA in the genome of plant cell.

Abstract: Modified expression vectors, including Tobacco Mosaic Virus (TMV) expression vectors, methods for modifying such vectors, and uses of the same are disclosed.

Abstract: Modified expression vectors, including Tobacco Mosaic Virus (TMV) expression vectors, methods for modifying such vectors, and uses of the same are disclosed.

Abstract: The present invention relates to codon optimization utilizing DNA shuffling. A method of producing gene sequences optimized for a desired functional property is described involving synthesizing a library of parental codon variant genes encoding some or all codon choices at some or all amino acid positions of a gene, reassorting the variant codons among the parental codon variant genes using DNA shuffling thereby forming progeny codon variant genes, expressing the progeny codon variant genes in a host; and screening or selecting for progeny codon variant genes encoding a desired functional property.

Abstract: Herein-described are various methods for making a vaccine that are made of re-assembled virus like particles (VLP). First, the VLPs are disassembled into encapsidation intermediate populations. Each encapsidation intermediate population undergoes, for instance, chemical conjugation of unique peptide or nucleic moieties to form separate populations. Thereafter, a predetermined amount of each of the several (one or more) different encapsidation intermediates from the different populations is mixed and joined, forming intact VLPs, surrounding a nucleic acid core, that are composed of different encapsidation intermediate such that the reassembled VLP displays more than one peptide or nucleic acid. The nucleic acid can function either as a scaffold alone or can be engineered for the expression of an immunomodulatory protein in a eukaryotic cell.

Abstract: We describe here an in vitro method of increasing complementarity in a heteroduplex polynucleotide sequence. The method uses annealing of opposite strands to form a polynucleotide duplex with mismatches. The heteroduplex polynucleotide is combined with an effective amount of enzymes having strand cleavage activity, 3? to 5? exonuclease activity, and polymerase activity, and allowing sufficient time for the percentage of complementarity to be increased within the heteroduplex. Not all heteroduplex polynucleotides will necessarily have all mismatches resolved to complementarity. The resulting polynucleotide is optionally ligated. Several variant polynucleotides result. At sites where either of the opposite strands has templated recoding in the other strand, the resulting percent complementarity of the heteroduplex polynucleotide sequence is increased. The parent polynucleotides need not be cleaved into fragments prior to annealing heterologous strands. Therefore, no reassembly is required.

Abstract: We describe here an in vitro method of increasing complementarity in a heteroduplex polynucleotide sequence. The method uses annealing of opposite strands to form a polynucleotide duplex with mismatches. The heteroduplex polynucleotide is combined with an effective amount of enzymes having strand cleavage activity, 3? to 5? exonuclease activity, and polymerase activity, and allowing sufficient time for the percentage of complementarity to be increased within the heteroduplex. Not all heteroduplex polynucleotides will necessarily have all mismatches resolved to complementarity. The resulting polynucleotide is optionally ligated. Several variant polynucleotides result. At sites where either of the opposite strands has templated recoding in the other strand, the resulting percent complementarity of the heteroduplex polynucleotide sequence is increased. The parent polynucleotides need not be cleaved into fragments prior to annealing heterologous strands. Therefore, no reassembly is required.

Abstract: Modified expression vectors, including Tobacco Mosaic Virus (TMV) expression vectors, methods for modifying such vectors, and uses of the same are disclosed.

Abstract: We describe here an in vitro method of redistributing sequence variations between non-identical polynucleotide sequences, by making a heteroduplex polynucleotide from two non-identical polynucleotides; introducing a nick in one strand at or near a base pair mismatch site; removing mismatched base(s) from the mismatch site where the nick occurred; and using the opposite strand as template to replace the removed base(s) with bases that complement base(s) in the first strand. By this method, information is transferred from one strand to the other at sites of mismatch.

Abstract: Display of peptides or proteins in an ordered, repetitive array, such as on the surface of a virus-like particle, is known to induce an enhanced immune response relative to vaccination with the “free” protein antigen. The 2100 coat proteins comprising the rod-shaped capsid of Tobacco mosaic virus (TMV) can accommodate short peptide insertions into the primary sequence, but the display of larger protein moieties on the virion surface by genetic fusions to the capsid protein has not been possible. Since TMV lacks surface exposed residues compatible with commonly available linker chemistries, we employed a randomized library approach to introduce a reactive lysine at the externally located at the amino-terminus of the coat protein. We found that we could easily control the extent of virion conjugation and demonstrated stoichiometric biotinylation of the introduced lysine.

Abstract: The present invention relates to foreign peptide sequences fused to recombinant plant viral structural proteins and a method of their production. Fusion proteins are economically synthesized in plants at high levels by biologically contained tobamoviruses. The fusion proteins of the invention have are useful as antigens for inducing the production of antibodies having desired binding properties, e.g., protective antibodies, or for use as vaccine antigens for the induction of protective immunity against the parvovirus. Feline parvovirus epitopes were fused to the N-terminus of the TMV coat protein, expressed in Nicotiana plants, extracted, purified, characterized and administered to animals, resulting in protective immunity.

Abstract: We describe here an in vitro method of increasing complementarity in a heteroduplex polynucleotide sequence. The method uses annealing of opposite strands to form a polynucleotide duplex with mismatches. The heteroduplex polynucleotide is combined with an effective amount of enzymes having strand cleavage activity, 3? to 5? exonuclease activity, and polymerase activity, and allowing sufficient time for the percentage of complementarity to be increased within the heteroduplex. Not all heteroduplex polynucleotides will necessarily have all mismatches resolved to complementarity. The resulting polynucleotide is optionally ligated. Several variant polynucleotides result. At sites where either of the opposite strands has templated recoding in the other strand, the resulting percent complementarity of the heteroduplex polynucleotide sequence is increased. The parent polynucleotides need not be cleaved into fragments prior to annealing heterologous strands. Therefore, no reassembly is required.

Abstract: Disclosed are methods and compositions for creating a DNA, RNA or protein molecule with two or more nucleic acid or polypeptide domains, respectively, joined by a linker region. These methods are used to generate random linker libraries of nucleic acids that encode dual-domain or multi-domain polypeptides. The linker regions are characterized by both length and sequence variability.

Abstract: The present invention relates to foreign peptide sequences fused to recombinant plant viral structural proteins and a method of their production. Fusion proteins are economically synthesized in plants at high levels by biologically contained tobamoviruses. The fusion proteins of the invention have are useful as antigens for inducing the production of antibodies having desired binding properties, e.g., protective antibodies, or for use as vaccine antigens for the induction of protective immunity against the parvovirus. Feline parvovirus epitopes were fused to the N-terminus of the TMV coat protein, expressed in Nicotiana plants, extracted, purified, characterized and administered to animals, resulting in protective immunity.

Abstract: We describe here an in vitro method of increasing complementarity in a heteroduplex polynucleotide sequence. The method uses annealing of opposite strands to form a polynucleotide duplex with mismatches. The heteroduplex polynucleotide is combined with an effective amount of enzymes having strand cleavage activity, 3? to 5? exonuclease activity, and polymerase activity, and allowing sufficient time for the percentage of complementarity to be increased within the heteroduplex. Not all heteroduplex polynucleotides will necessarily have all mismatches resolved to complementarity. The resulting polynucleotide is optionally ligated. Several variant polynucleotides result. At sites where either of the opposite strands has templated recoding in the other strand, the resulting percent complementarity of the heteroduplex polynucleotide sequence is increased. The parent polynucleotides need not be cleaved into fragments prior to annealing heterologous strands. Therefore, no reassembly is required.

Abstract: We describe here an in vitro method of increasing complementarity in a heteroduplex polynucleotide sequence. The method uses annealing of opposite strands to form a polynucleotide duplex with mismatches. The heteroduplex polynucleotide is combined with an effective amount of enzymes having strand cleavage activity, 3? to 5? exonuclease activity, and polymerase activity, and allowing sufficient time for the percentage of complementarity to be increased within the heteroduplex. Not all heteroduplex polynucleotides will necessarily have all mismatches resolved to complementarity. The resulting polynucleotide is optionally ligated. Several variant polynucleotides result. At sites where either of the opposite strands has templated recoding in the other strand, the resulting percent complementarity of the heteroduplex polynucleotide sequence is increased. The parent polynucleotides need not be cleaved into fragments prior to annealing heterologous strands. Therefore, no reassembly is required.

Abstract: The present invention provides nucleic acid sequences having an altered viral movement protein and 126/183 kDa replicase proteins further characterized in its ability tostabilize a transgene contained in a virus that expresses the altered movement protein. The present invention also provides viral vectors expressing the altered movement protein, cells transformed with the vectors, and host plants infected by the viral vectors.

Abstract: Herein-described are various methods for making a vaccine that are made of re-assembled virus like particles (VLP). First, the VLPs are disassembled into encapsidation intermediate populations. Each encapsidation intermediate population undergoes, for instance, chemical conjugation of unique peptide or nucleic moieties to form separate populations. Thereafter, a predetermined amount of each of the several (one or more) different encapsidation intermediates from the different populations is mixed and joined, forming intact VLPs, surrounding a nucleic acid core, that are composed of different encapsidation intermediate such that the reassembled VLP displays more than one peptide or nucleic acid. The nucleic acid can function either as a scaffold alone or can be engineered for the expression of an immunomodulatory protein in a eukaryotic cell.

Abstract: A polypeptide self-antigen useful in a tumor-specific vaccine mimics one or more epitopes of an antigen uniquely expressed by cells of the tumor. The polypeptide is preferably produced in a plant that has been transformed or transfected with nucleic acid encoding the polypeptide and is obtainable from the plant in correctly folded, preferably soluble form without a need for denaturation and renaturation. This plant-produced polypeptide is immunogenic without a need for exogenous adjuvants or other immunostimulatory materials. The polypeptide is preferably an scFv molecule that bears the idiotype of the surface immunoglobulin of a non-Hodgkin's (or B cell) lymphoma. Upon administration to a subject with lymphoma, the plant-produced, tumor-unique scFv polypeptide induces an idiotype-specific antibody or cell-mediated immune response against the lymphoma.

Abstract: A plurality of proteins of interest, or peptides of interest, or other genetically expressed materials, are screened and subsequently produced using any of a variety of expression systems. The plurality of proteins are extracted from a plurality of separate, processed green juices, each green juice containing one of the proteins of interest. A multi-channel apparatus processes the various green juices, one green juice per channel. The apparatus is computer controlled such that the various valves in each channel and pump are controlled in an automated manner to extract each protein of interest and deliver each protein of interest into its own storage vessel.