Methods for generating new genes in organism and use thereof
The present invention relates to the technical fields of genetic engineering and bioinformatics, in particular, to a method for creating a new gene in an organism in the absence of an artificial DNA template, and a use thereof. The method comprises simultaneously generating DNA breaks at two or more different specific sites in the organism's genome, wherein the specific sites are genomic sites capable of separating different genetic elements or different protein domains, and the DNA breaks are ligated to each other through non-homologous end joining (NHEJ) or homologous repair to generate a new combination of the different gene elements or different protein domains that is different from the original genome sequence, thereby creating a new gene. The new gene of the invention can change the growth, development, resistance, yield and other traits of the organism, and has great value in application.
1 . A method for creating a new gene in a plant, comprising the following steps:
simultaneously generating DNA breaks at two or more different specific sites in the plant's genome by delivering a CRISPR-based gene editing system into a cell of the plant which targets the two or more different specific sites of the genomic DNA,
wherein the CRISPR-based gene editing system comprises a nuclease or a polynucleotide encoding thereof, and two or more guide RNAs which target the two or more different specific sites respectively or one or more polynucleotide encoding the guide RNAs, wherein the nuclease and the guide RNAs contact the two or more different specific sites and creates cleavage at the two or more different specific sites, and
wherein the specific sites are genomic sites separating different genetic elements, wherein the genetic element is selected from a group consisting of a promoter, a 5′ untranslated region, a coding region (CDS), a region encoding a non-coding RNA, a 3′ untranslated region, and a terminator of the gene,
providing a condition allowing the DNA breaks to be ligated to each other by a non-homologous end joining (NHEJ),
detecting a new combination of the different genetic elements different from the original genomic sequence, wherein the combination of different genetic elements is a combination of a promoter of a first gene and a coding region of a second gene, wherein the two genes have different expression patterns, wherein the transcription of the second gene is driven by the promoter of the first gene, wherein the promoter of the first gene and the coding region of the second gene have nucleotide sequences selected from:
(1) the promoter of the first gene is a promoter of CP12 gene, wherein the CP12 gene comprises a nucleotide sequence of SEQ ID NO: 8, and the coding region of the second gene is a coding region of PPO gene, wherein the PPO gene comprises a nucleotide sequence of SEQ ID NO: 7;
(2) the promoter of the first gene is a promoter of Ubiquitin gene, wherein the Ubiquitin gene comprises a nucleotide sequence of SEQ ID NO: 2, and the coding region of the second gene is a coding region of PPO gene, wherein the PPO gene comprises a nucleotide sequence of SEQ ID NO: 1;
(3) the promoter of the first gene is a promoter of UBI2 gene, wherein the UBI2 gene comprises a nucleotide sequence of SEQ ID NO: 5, and the coding region of the second gene is a coding region of HPPD gene, wherein the HPPD gene comprises a nucleotide sequence of SEQ ID NO: 6; and
(4) the promoter of the first gene is a promoter of TKT gene, wherein the TKT gene comprises a nucleotide sequence of SEQ ID NO: 3, and the coding region of the second gene is a coding region of EPSPS gene, wherein the EPSPS gene comprises a nucleotide sequence of SEQ ID NO: 4,
the new gene is created if the new combination of the different genetic elements is detected, wherein the new gene improves the resistance or tolerance to an herbicide,
wherein the method does not involve the provision of a foreign DNA template.
2 . The method according to claim 1 , characterized in that said DNA breaks are achieved by delivering a nuclease with targeting property into a cell of the plant to contact with the specific sites of the genomic DNA.
3 . The method according to claim 2 , characterized in that said nuclease with targeting property is selected from the group consisting of Meganuclease, Zinc finger nuclease, TALEN, and CRISPR/Cas system.
4 . The method according to claim 2 , characterized in that the nucleases with targeting property are delivered into the cell by: 1) a PEG-mediated cell transfection method; 2) a liposome-mediated cell transfection method; 3) an electric shock transformation method; 4) a microinjection; 5) a gene gun bombardment; or 6) an Agrobacterium -mediated transformation method.
5 . An editing method for increasing the gene expression level of a target endogenous gene in a plant, which is independent of an exogenous DNA donor fragment, by creating a new highly-expressing endogenous gene in the plant according to the method in claim 1 , comprising the following steps:
simultaneously generating DNA breaks separately at selected sites between the promoter and the coding region of each of the target endogenous gene and an endogenous highly-expressing gene; ligating the DNA breaks to each other by means of non-homologous end joining (NHEJ), thereby generating an in vivo fusion of the coding region of the target endogenous gene and the strong endogenous promoter to form the new highly-expressing endogenous gene, wherein the promoter of the endogenous highly-expressing gene and the coding region of the target endogenous gene have nucleotide sequences selected from:
(1) the promoter of the endogenous highly-expressing gene is a promoter of CP12 gene, wherein the CP12 gene comprises a nucleotide sequence of SEQ ID NO: 8, and the coding region of the target endogenous gene is a coding region of PPO gene, wherein the PPO gene comprises a nucleotide sequence of SEQ ID NO: 7;
(2) the promoter of the endogenous highly-expressing gene is a promoter of Ubiquitin gene, wherein the Ubiquitin gene comprises a nucleotide sequence of SEQ ID NO: 2, and the coding region of the target endogenous gene is a coding region of PPO gene, wherein the PPO gene comprises a nucleotide sequence of SEQ ID NO: 1;
(3) the promoter of the endogenous highly-expressing gene is a promoter of UBI2 gene, wherein the UBI2 gene comprises a nucleotide sequence of SEQ ID NO: 5, and the coding region of the target endogenous gene is a coding region of HPPD gene, wherein the HPPD gene comprises a nucleotide sequence of SEQ ID NO: 6; and
(4) the promoter of the endogenous highly-expressing gene is a promoter of TKT gene, wherein the TKT gene comprises a nucleotide sequence of SEQ ID NO: 3, and the coding region of the target endogenous gene is a coding region of EPSPS gene, wherein the EPSPS gene comprises a nucleotide sequence of SEQ ID NO: 4,
wherein the target endogenous gene and the endogenous highly-expressing gene are located on the same chromosome.
6 . An editing method for knocking up the expression of an endogenous HPPD, EPSPS or PPO gene in a plant by creating a new highly-expressing plant endogenous HPPD, EPSPS or PPO gene in the plant according to the method in claim 1 , characterized in that it comprises fusing the coding region of the HPPD, EPSPS or PPO gene with a strong endogenous promoter of a plant in vivo to form the new highly-expressing plant endogenous HPPD, EPSPS or PPO gene, respectively; wherein the method comprises the following steps: simultaneously generating DNA breaks respectively in selected specific sites between the promoter and the coding region of each of the HPPD, EPSPS or PPO gene and an optional endogenous highly-expressing gene, ligating the DNA breaks to each other through an intracellular repair pathway, generating in vivo a fusion of the coding region of the HPPD, EPSPS or PPO gene and the optional strong endogenous promoter to form the new highly-expressing HPPD, EPSPS or PPO gene, respectively.
7 . The method according to claim 1 , characterized in that said two or more different specific sites locate on the same chromosome, wherein said at least two different genes may have the same or different transcription directions.
8 . The method according to claim 2 , characterized in that the nuclease with targeting property” is in the form of DNA, or exists in the form of mRNA or protein, but not DNA.
9 . The method of claim 1 , characterized in that, in the new combination of the different genetic elements, the promoter of the first gene is a promoter of CP12 gene, wherein the CP12 gene comprises a nucleotide sequence of SEQ ID NO: 8, and the coding region of the second gene is a coding region of PPO gene, wherein the PPO gene comprises a nucleotide sequence of SEQ ID NO: 7; or
the promoter of the first gene is a promoter of Ubiquitin gene, wherein the Ubiquitin gene comprises a nucleotide sequence of SEQ ID NO: 2, and the coding region of the second gene is a coding region of PPO gene, wherein the PPO gene comprises a nucleotide sequence of SEQ ID NO: 1; wherein an expression level of the PPO gene in a genome comprising the new combination is higher than an expression level of the PPO gene in a wild-type genome.
10 . A new gene obtainable by the method according to claim 1 , characterized in that compared with the original gene, the new gene either has a different promoter and therefore is expressed with a different spatial-temporal characteristics or a different intensity characteristics or a different developmental stage characteristics.
11 . The editing method according to claim 6 , characterized in that it comprises fusing the coding region of the HPPD gene with a strong endogenous promoter of a rice, wherein the strong promoter is a promoter of ubiquitin2 gene,
it comprises fusing the coding region of the EPSPS gene with a strong endogenous promoter of a rice, wherein the strong promoter is a promoter of TKT gene, or
it comprises fusing the coding region of the PPO gene with a strong endogenous promoter of a rice or an Arabidopsis , wherein in rice, the strong promoter is a promoter of CP12 gene, and in Arabidopsis , the strong promoter is a promoter of ubiquitin10 gene.
12 . A highly-expressing plant endogenous HPPD, EPSPS or PPO gene obtainable by the editing method according to claim 6 .
13 . A method for producing a plant with an increased resistance or tolerance to an herbicide, which comprises regenerating the plant host cell into a plant and a progeny derived therefrom, wherein the plant host cell comprises the expression cassette comprising the gene according to claim 12 .
14 . A method for controlling a weed in a cultivation site of a plant, wherein the plant is selected from the group consisting of a plant prepared by the method according to claim 13 , wherein the method comprises applying to the cultivation site one or more of HPPD, EPSPS or PPO inhibitory herbicides in an amount for effectively controlling the weed.