Targeted viral-mediated plant genome editing using CRISPR /Cas9
View Patent ↗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.
1. A method for making a heritable genomic modification at a target site in the genome of one or more plant germline cells, the method comprising
delivering a Tobacco Rattle Virus RNA2 (TRV2) T-DNA vector comprising a pea early browning virus (PEBV) promotor operably linked to a nucleic acid sequence encoding a single guide RNA (sgRNA) and a Tobacco Rattle Virus RNA1 (TRV1) T-DNA vector to one or more plant cells of a whole transgenic plant, and expression thereof to form recombinant Tobacco Rattle Virus, and
allowing recombinant Tobacco Rattle Virus infection of, and sgRNA expression in, one or more germline cells of the plant expressing a heterologous Cas endonuclease from a stably integrated genomic construct encoding the Cas endonuclease, wherein the sgRNA and Cas endonuclease form a complex and introduce a single or double strand break at a target site in the genome of the germline cell or cells leading to a heritable genomic modification.
2. The method of claim 1 , wherein the construct comprises a heterologous nucleic acid encoding a Cas9 endonuclease operably linked to a promoter.
3. The method of claim 2 , wherein the heterologous nucleic acid encoding the Cas9 endonuclease is a plant optimized nucleic acid sequence.
4. The method of claim 1 , wherein delivery of the RNA2 (TRV2) T-DNA vector to the plant cell or cells comprises infiltrating the cell or cells with bacteria comprising the RNA1 (TRV2) T-DNA vector.
5. The method of claim 1 , wherein delivery of the RNA1 (TRV1) T-DNA vector to the plant cell or cells comprises infiltrating the cell or cells with bacteria comprising the RNA1 (TRV1) T-DNA vector.
6. The method of claim 1 , further comprising delivering to the plant cell or cells a heterologous donor DNA, wherein the heterologous donor DNA comprises a polynucleotide of interest to be incorporated into the genome of the plant cell.
7. The method of claim 5 , wherein the bacteria is an Agrobacterium.
8. The method of claim 1 , further comprising delivering to the plant cell or cells one or more additional RNA2 (TRV2) T-DNA vectors, wherein each additional RNA2 (TRV2) T-DNA vector encodes a different sgRNA.
9. The method of claim 8 , wherein delivery of the one or more additional RNA2 (TRV2) T-DNA vectors to the plant cell or cells comprises infiltrating the cell or cells with bacteria comprising the additional RNA2 (TRV2) T-DNA vector(s).
10. The method of claim 1 , further comprising selecting seeds comprising the heritable genomic modification.
11. The method of claim 10 , further comprising growing a plant or plants from one or more of the selected seeds.
12. The method of claim 4 , wherein the bacteria is an Agrobacterium.
13. The method of claim 4 , wherein the bacteria is Agrobacterium tumefaciens.
14. The method of claim 5 , wherein the bacteria is Agrobacterium tumefaciens.
15. The method of claim 1 , wherein the modification comprises at least one deletion or substitution of one or more nucleotides in the target site.
16. The method of claim 8 , comprising modifications at two or more target sites.
17. The method of claim 16 , wherein the two or more modifications each comprise at least one deletion or substitution of one or more nucleotides in the target site.
18. The method of claim 1 , wherein a double strand break is introduced.
19. The method of claim 1 , wherein the Cas endonuclease is a nickase.
20. The method of claim 1 , wherein the PEBV promoter comprises the PREV promoter sequence of SEQ ID NO:8.