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 us 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 promoter, operably linked to a sequence encoding a single-guide RNA (sgRNA) or a sequence specifying a non-coding RNA, wherein the sequence of said U6 promoter comprises SEQ ID NO:164; or a fragment thereof, wherein the fragment is at least 140 bp in length, and wherein the U6 promoter or fragment thereof and the sgRNA or non-coding RNA come from different sources.
2 . The recombinant DNA construct of claim 1 , further comprising a transcription termination sequence.
3 . 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.
4 . The recombinant DNA construct of claim 3 , wherein the Cas endonuclease gene product is further operably linked to a nuclear localization sequence (NLS).
5 . The recombinant DNA construct of claim 3 , wherein the sequence encoding said Cas endonuclease is selected from the group consisting of SEQ ID NO: 27, SEQ ID NO: 68, SEQ ID NO:97, SEQ ID NO: 119, and SEQ ID NO:136.
6 . The recombinant DNA construct of claim 1 , wherein the non-coding RNA is selected from the group consisting of: 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), and a naturally occurring antisense siRNA (nat-siRNA).
7 . A cell comprising the recombinant DNA construct of claim 1 .
8 . The cell of claim 7 , wherein the cell is a plant cell.
9 . 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).
10 . The method of claim 9 , wherein the sequence encoding said Cas endonuclease is selected from the group consisting of SEQ ID NO:27, SEQ ID NO:68, SEQ ID NO:97, SEQ ID NO:119, and SEQ ID NO:136.
11 . 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 3 .
12 . The method of claim 11 , wherein the sequence encoding the Cas endonuclease is selected from the group consisting of SEQ ID NO:27, SEQ ID NO:68, SEQ ID NO:97, SEQ ID NO:119, and SEQ ID NO:136.
13 . 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 9 ; 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.
14 . 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 11 ; 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.