System for site-specific modification of ALS gene using CRISPR-Cas9 system for production of herbicide-resistant rice and use of same
The present invention discloses a system for site-specific modification of ALS gene by a CRISPR-Cas9 system to produce herbicide-resistant rice, and uses thereof. The system for site-specific modification in a plant genome of the present invention comprises a vector for site-specific modification in a plant genome and a donor DNA; wherein the vector for site-specific modification in a plant genome comprises a Cas9 protein expression cassette, gRNA expression cassettes and a donor DNA; the gRNA expression cassettes encode two gRNAs targeting two target sites of a target DNA of a plant of interest, respectively; the target DNA has a fragment to be site-specifically modified which is positioned between the two target site.
1. A composition for site-specific modification in a plant genome, comprising a vector for site-specific modification in the plant genome and a donor DNA A;
wherein the vector for site-specific modification in the plant genome comprises a Cas9 protein expression cassette, a gRNA expression cassette, and a donor DNA B;
wherein the gRNA expression cassette encodes two gRNAs targeting two target sites in a target DNA of a plant of interest;
wherein the target DNA of the plant of interest comprises a fragment to be site-specifically modified which is positioned between the two target sites in the target DNA of the plant of interest;
wherein of the two target sites, one positioned upstream is an upstream target site,
wherein the other one positioned downstream is a downstream target site;
wherein the donor DNA B comprises the upstream target site, the downstream target site, and a fragment for site-specific modification positioned between the upstream target site and the downstream target site;
wherein the fragment for site-specific modification is a DNA fragment to replace the fragment to be site-specifically modified in the target DNA;
wherein the donor DNA A is other than the vector and has a same nucleotide sequence as the donor DNA B, and
wherein: the upstream target site consists of nucleotides at positions 7590-7609 from 5′-end of SEQ ID NO: 1; the downstream target site consists of nucleotides at positions 8032-8051 from 5′-end of SEQ ID NO: 1; and the fragment for site-specific modification is set forth by the nucleotides at positions 7716-7979 from 5′-end of SEQ ID NO: 1.
2. The composition according to claim 1 , wherein: the plant of interest is a monocotyledonous plant or a dicotyledonous plant.
3. The composition according to claim 2 , wherein: the monocotyledonous plant is a gramineous plant.
4. The composition according to claim 1 , wherein: the target DNA is a gene encoding acetolactate synthase.
5. The composition according to claim 4 , wherein: the acetolactate synthase is a protein with the amino acid sequence as set forth by SEQ ID NO: 2.
6. The composition according to claim 5 , wherein: the target DNA is SEQ ID NO: 3.
7. The composition according to claim 4 , wherein: the gRNA expression cassette includes a gRNA expression cassette 1 encoding gRNA1, and a gRNA expression cassette 2 encoding gRNA2, wherein the gRNA1 targets the upstream target site, and the gRNA2 targets the downstream target site.
8. The composition according to claim 7 , wherein: the gRNA expression cassette 1 is nucleotides at positions 261-747 from 5′-end of SEQ ID NO: 1; and the gRNA expression cassette 2 is nucleotides at positions 8328-8814 from 5′-end of SEQ ID NO: 1.
9. The composition according to claim 8 , wherein:
the vector for the site-specific modification in the plant genome is SEQ ID NO: 1; and
the donor DNA A is nucleotides at positions 7590-8051 from 5′-end of SEQ ID NO: 1.
10. A method for site-specific modification in a plant genome, comprising:
introducing into a plant of interest a vector for site-specific modification in the plant genome and a donor DNA A to obtain a plant with the plant genome site-specifically modified;
wherein the vector for site-specific modification in the plant genome comprises a Cas9 protein expression cassette, gRNA expression cassettes, and a donor DNA B;
wherein the gRNA expression cassettes encode two gRNAs targeting two target sites in a target DNA of a plant of interest, respectively;
wherein the target DNA of the plant of interest comprises a fragment to be site-specifically modified positioned between the two target sites in the target DNA of the plant of interest;
wherein a first of the two target sites is an upstream target site;
wherein a second of the two target sites is a downstream target site;
wherein the donor DNA B comprises the upstream target site, the downstream target site, and a fragment for site-specific modification positioned between the upstream target site and the downstream target site;
wherein the fragment for site-specific modification is a DNA fragment to replace the fragment to be site-specifically modified in the target DNA; and
wherein the donor DNA A has a same nucleotide sequence as the donor DNA B,
wherein: the upstream target site consists of nucleotides at positions 7590-7609 from 5′-end of SEQ ID NO: 1; the downstream target site consists of nucleotides at positions 8032-8051 from 5′-end of SEQ ID NO: 1; and the fragment for site-specific modification is set forth by the nucleotides at positions 7716-7979 from 5′-end of SEQ ID NO: 1.
11. The method according to claim 10 , wherein: the vector for site-specific modification in the plant genome and the donor DNA A is introduced into the plant of interest in a molar ratio of 1:(1-40).
12. A method for producing herbicide-resistance in a plant, comprising:
introducing into a plant of interest a vector for site-specific modification in the plant genome and a donor DNA A to obtain a plant with the plant genome site-specifically modified;
wherein the vector for site-specific modification in the plant genome comprises a Cas9 protein expression cassette, a gRNA expression cassette, and a donor DNA B;
wherein the gRNA expression cassette encodes two gRNAs targeting two target sites in a target DNA of a plant of interest, respectively;
wherein the target DNA of the plant of interest comprises a fragment to be site-specifically modified positioned between the two target sites in the target DNA of the plant of interest;
wherein a first of the two target sites is an upstream target site;
wherein a second of the two target sites is a downstream target site;
wherein the donor DNA B comprises the upstream target site, the downstream target site, and a fragment for site-specific modification positioned between the upstream target site and the downstream target site;
wherein the fragment for site-specific modification is a DNA fragment to replace the fragment to be site-specifically modified in the target DNA; and
wherein the donor DNA A has a same nucleotide sequence as the donor DNA B,
wherein: the upstream target site consists of nucleotides at positions 7590-7609 from 5′-end of SEQ ID NO: 1; the downstream target site consists of nucleotides at positions 8032-8051 from 5′-end of SEQ ID NO: 1; and the fragment for site-specific modification is set forth by the nucleotides at positions 7716-7979 from 5′-end of SEQ ID NO: 1.
13. The method according to claim 12 , wherein the target DNA is a gene encoding acetolactate synthase.
14. The method according to claim 12 , wherein the plant of interest is a monocotyledonous plant or a dicotyledonous plant.