Compositions and methods for site directed genomic modification
The disclosure provides novel corn, tomato, and soybean U6, U3, U2, U5, and 7SL snRNA promoters which are useful for CRISPR/Cas-mediated targeted gene modifications in plants. The disclosure also provides methods for use for U6, U3, U2, U5, and 7SL promoters in driving expression of sgRNA polynucleotides which function in a CRISPR/Cas system of targeted gene modification in plants. The disclosure also provides methods of genome modification by insertion of blunt-end DNA fragments at a site of genomic cleavage.
1. A recombinant DNA construct comprising a U6 snRNA promoter fragment, wherein said U6 snRNA promoter fragment is operably linked to:
(i) a sequence encoding a single-guide RNA (sgRNA); or
(ii) a sequence specifying a non-coding RNA;
and wherein the sequence of said U6 snRNA promoter fragment consists of a sequence selected from the group consisting of SEQ ID NO:7 and SEQ ID NO:8, wherein the U6 snRNA promoter fragment and the sgRNA or the non-coding RNA come from different sources.
2. The recombinant DNA construct of claim 1 , wherein the sequence of said U6 snRNA promoter fragment consists of the sequence of SEQ ID NO:7.
3. The recombinant DNA construct of claim 1 , further comprising a transcription termination sequence.
4. The recombinant DNA construct of claim 1 , further comprising a sequence encoding a promoter operably linked to a sequence encoding a clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated Cas endonuclease gene product.
5. The recombinant DNA construct of claim 4 , wherein the Cas endonuclease gene product is further operably linked to a nuclear localization sequence (NLS).
6. The recombinant DNA construct of claim 4 , wherein the sequence encoding said Cas endonuclease is SEQ ID NO:27, SEQ ID NO:68, SEQ ID NO:97, SEQ ID NO:119, or SEQ ID NO:136.
7. The recombinant DNA construct of claim 1 , wherein the non- coding RNA is a microRNA (miRNA), a miRNA precursor, a small interfering RNA (siRNA), a small RNA (22-26 nt in length) and precursor encoding same, a heterochromatic siRNA (hc-siRNA), a Piwi-interacting RNA (piRNA), a hairpin double strand RNA (hairpin dsRNA), a trans-acting siRNA (ta-siRNA), or a naturally occurring antisense siRNA (nat-siRNA).
8. A cell comprising the recombinant DNA construct of claim 1 .
9. The cell of claim 8 , wherein the cell is a plant cell.
10. A method of introducing a double-strand break in the genome of a cell, comprising introducing in said cell:
a) at least one recombinant DNA construct of claim 1 ; and
b) a second recombinant DNA construct comprising a sequence encoding a promoter operably linked to a sequence encoding a clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated Cas endonuclease gene product operably linked to a nuclear localization sequence (NLS).
11. The method of claim 10 , wherein the U 6 snRNA promoter fragment consists essentially of the sequence of SEQ ID NO:7.
12. The method of claim 10 , wherein the sequence encoding said Cas endonuclease is SEQ ID NO:27, SEQ ID NO:68, SEQ ID NO:97, SEQ ID NO:119, or SEQ ID NO:136.
13. A method of introducing a double-strand break in the genome of a cell, comprising introducing to said cell at least one recombinant DNA construct of claim 4 .
14. The method of claim 13 , wherein the U6 snRNA promoter fragment consists of the sequence of SEQ ID NO:7.
15. The method of claim 13 , wherein the sequence encoding the Cas endonuclease is SEQ ID NO:27, SEQ ID NO:68, SEQ ID NO:97, SEQ ID NO:119, or SEQ ID NO:136.
16. A method of genome modification comprising:
a) introducing a double-strand break in the genome of a plant cell by the method according to claim 10 ; and
b) introducing into said plant cell a recombinant blunt-end double-strand DNA fragment,
wherein said recombinant blunt-end double-strand DNA fragment is incorporated into said double strand break by endogenous DNA repair.
17. A method of genome modification comprising:
a) introducing a double-strand break in the genome of a plant cell by the method according to claim 13 ; and
b) introducing into said plant cell a recombinant blunt-end double-strand DNA fragment,
wherein said recombinant blunt-end double-strand DNA fragment is incorporated into said double strand break by endogenous DNA repair.