Methods and compositions for genome modification

A synthetic composition comprising two distinct Rhizobiales bacteria,

• a. wherein the first of the two bacteria comprises a first vector, wherein the first vector comprises a heterologous polynucleotide, wherein the heterologous polynucleotide is flanked by polynucleotides comprising homology to a nucleotide sequence at the target site,
• b. wherein the second of the two bacteria comprises a second vector, wherein the second vector comprises one or more polynucleotides encoding a Cas endonuclease, a guide RNA, and at least one morphogenic factor;
• wherein the ratio of the amounts of the first vector and the second vector is approximately 1:1 to about 10:1.

A synthetic composition comprising two different Rhizobiales bacteria,

• a. wherein the first of the two bacteria comprises a first vector, wherein the first vector comprises a gene encoding a guide RNA and a heterologous polynucleotide, wherein the heterologous polynucleotide is flanked by polynucleotides comprising homology to a nucleotide sequence at the target site,
• b. wherein the second of the two bacteria comprises a second vector, wherein the second vector comprises one or more polynucleotides encoding a Cas endonuclease and at least one morphogenic factor;
• wherein the ratio of the amounts of the first vector and the second vector is approximately 1:1 to about 10:1.

A synthetic composition comprising one Rhizobiales strain comprising first and second plasmids, wherein each of the two plasmids comprise one or more distinct T-DNA sequences,

• a. wherein the first plasmid comprises a low-copy ORI and comprises a T-DNA encoding a Cas endonuclease and a morphogenic factor,
• b. wherein the second plasmid comprises a higher copy ORI and comprises a T-DNA encoding a heterologous polynucleotide,
• wherein the ratio of the amounts of the first plasmid and the second plasmid is approximately 1:1 to about 1:10.

A method of modifying a target site in the genome of a plant cell through endogenous activation of one or more genes, the method comprising providing to the cell (a) a first polynucleotide encoding a deactivated CRISPR-Cas polypeptide (dCas) that is capable of site-specifically binding to an endogenous target site comprising a polynucleotide involved in cellular regeneration, but incapable of introducing a double-strand break at the endogenous target site, wherein the dCas is operably linked to a transcriptional activator, the transcriptional activator capable of increasing the expression of the polynucleotide involved in cellular regeneration;

• (b) providing a second polynucleotide comprising a heterologous polynucleotide flanked by polynucleotides comprising homology to a nucleotide sequence at the target site to be modified;
• (c) providing a third polynucleotide encoding a Cas endonuclease, a guide RNA, and an anti-regeneration factor;
• wherein the ratio of the amounts of the polynucleotide comprising the Cas endonuclease and the polynucleotide comprising the heterologous polynucleotide is approximately 1:1 to about 1:10, and wherein the Cas endonuclease creates a double-strand break at or near the target site of the plant cell but the polynucleotide encoding the Cas endonuclease does not integrate into the genome of the same plant cell.

A synthetic composition comprising (a) a first polynucleotide encoding a deactivated CRISPR-Cas polypeptide (dCas) that is capable of site-specifically binding to an endogenous target site comprising a polynucleotide involved in cellular regeneration, but incapable of introducing a double-strand break at the endogenous target site, wherein the dCas is operably linked to a transcriptional activator, the transcriptional activator capable of increasing the expression of the polynucleotide involved in cellular regeneration;

• (b) providing a second polynucleotide comprising a heterologous polynucleotide flanked by polynucleotides comprising homology to a nucleotide sequence at the target site to be modified;
• (c) providing a third polynucleotide encoding a Cas endonuclease, a guide RNA, and an anti-regeneration factor.

A method of regeneration of a genome modified plant, the method comprising providing one or more polynucleotides that encode a site-specific DNA modifying agent and optionally, a morphogenic factor to a plant cell, wherein the DNA modifying agent and optionally, the morphogenic factor diffuses or is transported into one or more adjacent plant cells;

• modifying the genome of the one or more adjacent plant cells;
• regenerating the one or more adjacent plant cells into one or more genome modified plants, wherein the one or more genome modified plants do not comprise the polynucleotide sequence encoding the DNA modifying agent and optionally the morphogenic factor in the absence of a separate segregation step by crossing with another plant.