US 2026/0275371 A1 · METHOD FOR GENERATING NEW GENE IN ORGANISM AND USE THEREOF

Abstract

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.

Claims20

Dependent claims

A method for creating a new gene in an organism, characterized by comprising the following steps:

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 by a non-homologous end joining (NHEJ) or homologous repair, generating a new combination of the different genetic elements or different protein domains different from the original genomic sequence, thereby creating the new gene;
preferably said gene elements are selected from the group consisting of a promoter, a 5′ untranslated region, a coding region or non-coding RNA region, a 3′ untranslated region, a terminator of the gene, or any combination thereof;
and
wherein said two or more different specific sites locate on the same chromosome or on different chromosomes.

1 more element

An editing method for increasing the gene expression level of a target endogenous gene in an organism, which is independent of an exogenous DNA donor fragment, which comprises 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 optional endogenous highly-expressing gene;
ligating the DNA breaks to each other by means of non-homologous end joining (NHEJ) or homologous repair, thereby generating an in vivo fusion of the coding region of the target endogenous gene and the optional strong endogenous promoter to form a new highly-expressing endogenous gene, and
wherein the target endogenous gene and the optional endogenous highly-expressing gene are located on the same chromosome or on different chromosomes.

A highly-expressing rice endogenous gene, which is selected from the group being consisted of:

(1) a rice endogenous HPPD gene, which has a sequence selected from the group consisting of:
(a) the nucleic acid sequence as shown in SEQ ID NO:
9, SEQ ID NO:
10, SEQ ID NO:
18 or SEQ ID NO:
19 or a portion thereof or a complementary sequence thereof;
(b) a sequence having an identity of at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% to any one of the sequences as defined in (1);
or
(c) a nucleic acid sequence capable of hybridizing to the sequence as shown in (1) or (2) under a stringent condition;
(2) a rice endogenous EPSPS gene, which has a sequence selected from the group consisting of:
(a) the nucleic acid sequence as shown in SEQ ID NO:
11, SEQ ID NO:
12, SEQ ID NO:
13 or SEQ ID NO:
14 or a portion thereof or a complementary sequence thereof;
(b) a sequence having an identity of at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% to any one of the sequences as defined in (1);
or
(c) a nucleic acid sequence capable of hybridizing to the sequence as shown in (1) or (2) under a stringent condition;
and
(3) a rice endogenous PPO gene, which has a sequence selected from the following:
(a) the nucleic acid sequence as shown in SEQ ID NO:
15, SEQ ID NO:
16, SEQ ID NO:
17, SEQ ID NO:
20, SEQ ID NO:
21, SEQ ID NO:
22, SEQ ID NO:
23, SEQ ID NO:
24, SEQ ID NO:
25, or SEQ ID NO:
26 or a portion thereof or a complementary sequence thereof;
(b) a sequence having an identity of at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% to any one of the sequences as defined in (1);
or
(c) a nucleic acid sequence capable of hybridizing to the sequence as shown in (1) or (2) under a stringent condition.

31 more elements

A rice resistant to a herbicide, which comprises one or a combination of two or more of the highly-expressing rice endogenous genes selected from the group being consisted of:

(1) a highly-expressing rice endogenous HPPD gene, which has a sequence selected from the group consisting of:
(a) the nucleic acid sequence as shown in SEQ ID NO:
9, SEQ ID NO:
10, SEQ ID NO:
18 or SEQ ID NO:
19 or a portion thereof or a complementary sequence thereof;
(b) a sequence having an identity of at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% to any one of the sequences as defined in (1);
or
(c) a nucleic acid sequence capable of hybridizing to the sequence as shown in (1) or (2) under a stringent condition;
(2) a highly-expressing rice endogenous EPSPS gene, which has a sequence selected from the group consisting of:
(a) the nucleic acid sequence as shown in SEQ ID NO:
11, SEQ ID NO:
12, SEQ ID NO:
13 or SEQ ID NO:
14 or a portion thereof or a complementary sequence thereof;
(b) a sequence having an identity of at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% to any one of the sequences as defined in (1);
or
(c) a nucleic acid sequence capable of hybridizing to the sequence as shown in (1) or (2) under a stringent condition;
and
(3) a highly-expressing rice endogenous PPO gene, which has a sequence selected from the group consisting of:
(a) the nucleic acid sequence as shown in SEQ ID NO:
15, SEQ ID NO:
16, SEQ ID NO:
17, SEQ ID NO:
20, SEQ ID NO:
21, SEQ ID NO:
22, SEQ ID NO:
23, SEQ ID NO:
24, SEQ ID NO:
25, or SEQ ID NO:
26 or a portion thereof or a complementary sequence thereof;
(b) a sequence having an identity of at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% to any one of the sequences as defined in (1);
or
(c) a nucleic acid sequence capable of hybridizing to the sequence as shown in (1) or (2) under a stringent condition;
preferably the rice is non-transgenic.

32 more elements

Dependencies are inferred from the claim text.

Method for generating new gene in organism and use thereof

Life of this patent

Method for generating new gene in organism and use thereof

Life of this patent

Abstract

Claims20