Methods and compositions for targeted genomic insertion
The present invention relates to methods and compositions for chimeric RNA comprising a guide RNA and bait RNA for modifying a target site in the genome of a cell. Such modifications include integration of a transgene and allelic mutations and modifications of native genes. Also provided are plants comprising a modified nucleic acid sequence compared to the native gene integrated into a targeted genomic site in the plant genome.
1. A chimeric RNA for targeted genomic modification of a cell, wherein the chimeric RNA comprises:
a) a guide RNA comprising a polynucleotide targeting guide sequence of at least 8 contiguous nucleotides and at least 80% identical to a target nucleic acid sequence of at least 8 contiguous nucleotides within a target nucleic acid molecule; and
b) a bait RNA comprising at least 8 contiguous nucleic acids, wherein the nucleic acid sequence of the bait RNA is at least 70% complementary to at least 8 contiguous nucleic acids of a donor DNA molecule, wherein the donor DNA molecule is intended for integration into the genome of a cell, wherein a tracrRNA is operably linked to said bait RNA sequence.
2. The chimeric RNA of claim 1 , wherein said bait RNA is operably linked to said guide RNA.
3. The chimeric RNA of claim 2 , wherein the guide RNA further comprises a nucleic acid sequence which is a binding site for a site-directed modifying polypeptide.
4. The chimeric RNA of claim 1 , wherein the guide RNA comprises a crRNA.
5. The chimeric RNA of claim 4 , wherein the crRNA is operably linked to said bait RNA.
6. The chimeric RNA of claim 1 , wherein the guide RNA comprises a crRNA operably linked to said tracrRNA.
7. The chimeric RNA of claim 1 , wherein the guide RNA further comprises a tracrRNA, wherein the tracrRNA interacts with the guide RNA to create an RNA duplex that is a binding site for a site-directed modifying polypeptide.
8. The chimeric RNA of claim 7 , wherein the guide RNA comprises a tracrRNA operably linked to a bait RNA.
9. An expression cassette comprising a nucleic acid sequence encoding the chimeric RNA of claim 1 .