阅读说明：本技术 xCas9n-epBE碱基编辑系统在基因编辑中的应用 (application of xCas9n-epBE base editing system in gene editing ) 是由 杨进孝 徐雯 刘亚 王飞鹏 袁爽 于 2019-08-30 设计创作，主要内容包括：本发明公开了xCas9n-epBE碱基编辑系统在基因编辑中的应用。本发明公开的xCas9n-epBE碱基编辑系统包括xCas9n、PmCDA1、UGI和tRNA-esgRNA；所述tRNA-esgRNA靶向靶点序列；所述tRNA-esgRNA如式I所示：tRNA-所述靶点序列转录的RNA-esgRNA骨架(式I)。通过实验证明：本发明的xCas9n-epBE碱基编辑系统在植物基因组中实现了靶点序列的编辑,尤其是在PAM序列为NGT、NGA、NGG、GAA或GAT时实现了靶点序列中由碱基C到碱基T的替换。本发明的xCas9n-epBE碱基编辑系统在植物或动物基因编辑中具有广泛的应用前景。(The invention discloses an application of an xCas9n-epBE base editing system in gene editing. The xCas9n-epBE base editing system disclosed by the invention comprises xCas9n, PmCDA1, UGI and tRNA-esgRNA; the tRNA-esgRNA targets a target sequence; the tRNA-esgRNA is shown as a formula I: tRNA-RNA transcribed from the target sequence-esgRNA backbone (formula I). Experiments prove that: the xCas9n-epBE base editing system realizes the editing of a target point sequence in a plant genome, and particularly realizes the replacement from a base C to a base T in the target point sequence when the PAM sequence is NGT, NGA, NGG, GAA or GAT. The xCas9n-epBE base editing system has wide application prospect in plant or animal gene editing.)

1. the editing method of the genome target sequence is a method (I), a method (II), a method (III) or a method (IV):

the method (one) comprises the following steps: introducing a coding gene of xCas9n, a DNA molecule for transcribing tRNA-esgRNA, a coding gene of PmCDA1 and a coding gene of UGI into an organism or biological cells, so that the xCas9n, the tRNA-esgRNA, the PmCDA1 and the coding gene of the UGI are expressed, and the genome target sequence is edited;

The method (II) comprises the following steps: introducing an encoding gene of xCas9n, a DNA molecule for transcribing tRNA-esgRNA and an encoding gene of PmCDA1 into an organism or biological cell, so that the xCas9n, the tRNA-esgRNA and the encoding gene of PmCDA1 are expressed, and the genome target sequence is edited;

The method (III) comprises the following steps: introducing xCas9n, tRNA-esgRNA, PmCDA1 and UGI into an organism or an organism cell to realize the editing of a genome target sequence;

The method (IV) comprises the following steps: introducing xCas9n, tRNA-esgRNA and PmCDA1 into an organism or an organism cell to realize the editing of a genome target sequence;

The tRNA-esgRNA targets the target sequence;

the tRNA-esgRNA is shown as a formula I: tRNA-the RNA transcribed from the target sequence-the esgRNA backbone (formula I);

the tRNA is m1) or m2) or m 3):

m1) replacing T in the 474-550 th position of the sequence 1 with U to obtain an RNA molecule;

m2) carrying out substitution and/or deletion and/or addition of one or more nucleotides on the RNA molecule shown in m1) and having the same function;

m3) and m1) or m2) and has the same function;

The esgRNA backbone is n1) or n2) or n 3):

n1) replacing T in the 571-656 position of the sequence 1 with U to obtain an RNA molecule;

n2) the RNA molecule shown in n1) is subjected to substitution and/or deletion and/or addition of one or more nucleotides, and the RNA molecule has the same function.

n3) and n1) or n2) and has the same function;

The PAM sequence of the target sequence is any one of the following sequences: NGT, NGA, NGG, GAA, GAT; n is A, T, C or G.

2. The method of claim 1, wherein:

The xCas9n is xCas9n 3.7.7;

The xCas9n 3.7.7 is A1) or A2) or A3):

A1) the amino acid sequence is a protein shown in a sequence 2;

A2) The protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 2 in the sequence table and has the same function;

A3) A fusion protein obtained by connecting a label to the N-terminal or/and the C-terminal of A1) or A2).

3. The method according to claim 1 or 2, characterized in that:

The PmCDA1 is C1) or C2) or C3):

C1) the amino acid sequence is a protein shown in a sequence 3;

C2) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 3 in the sequence table and has the same function;

C3) C1) or C2) at the N-terminus or/and the C-terminus.

4. A method according to any one of claims 1-3, characterized in that:

The UGI is E1) or E2) or E3):

E1) The amino acid sequence is a protein shown in a sequence 4;

E2) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 4 in the sequence table and has the same function;

E3) A fusion protein obtained by connecting a label to the N-terminal or/and the C-terminal of E1) or E2).

5. The method according to any one of claims 2-4, wherein:

the coding gene of the xCas9n 3.7.7 is b1) or b2) or b 3):

b1) A cDNA molecule or DNA molecule shown in 2857-7125 site of a sequence 1 in a sequence table;

b2) A cDNA or DNA molecule having 75% or more identity with the nucleotide sequence defined in b1) and encoding said xCas9n3.7;

b3) Hybridizes under stringent conditions with a nucleotide sequence defined by b1) or b2) and encodes said xCas9n3.7 cDNA molecule or DNA molecule.

6. The method according to any one of claims 1 to 5, wherein:

The coding gene of the PmCDA1 is d1) or d2) or d 3):

d1) A cDNA molecule or DNA molecule shown in the 7417-8040 site of the sequence 1 in the sequence table;

d2) a cDNA molecule or DNA molecule which has 75 percent or more identity with the nucleotide sequence defined by d1) and codes the PmCDA 1;

d3) Hybridizes with the nucleotide sequence limited by d1) or d2) under strict conditions and encodes the cDNA molecule or DNA molecule of the PmCDA 1.

7. The method according to any one of claims 1 to 6, wherein:

the encoding gene of the UGI is f1), f2) or f 3):

f1) A cDNA molecule or DNA molecule shown in the 8062-8358 position of the sequence 1 in the sequence table;

f2) A cDNA molecule or DNA molecule having 75% or more identity to the nucleotide sequence defined in f1) and encoding said UGI;

f3) hybridizing with the nucleotide sequence defined by f1) or f2) under strict conditions, and encoding the cDNA molecule or DNA molecule of UGI.

8. the preparation method of the biological mutant comprises the following steps: editing a genomic target sequence of an organism according to the method of any one of claims 1 to 7 to obtain an organism mutant.

9. The method according to any one of claims 1 to 8, wherein: editing the genome target sequence to mutate C in the target sequence into T.

10. the method according to any one of claims 1 to 9, wherein: the organism is p1) or p2) or p3) or p 4):

p1) plants or animals;

p2) monocotyledonous or dicotyledonous plants;

p3) gramineous plants;

p4) rice;

And/or, the biological cell is q1) or q2) or q3) or q 4):

q1) plant cells or animal cells;

q2) a monocotyledonous or dicotyledonous plant cell;

q3) a graminaceous plant cell;

q4) rice cells.

Technical Field

The invention relates to the field of biotechnology, in particular to application of an xCas9n-epBE base editing system in gene editing.

Background

The CRISPR-Cas9 technology has become a powerful genome editing means and is widely applied to many tissues and cells. The CRISPR/Cas9 protein-RNA complex is localized on the target by a guide RNA (guide RNA), cleaved to generate a DNA double strand break (dsDNA break, DSB), and the organism will then instinctively initiate a DNA repair mechanism to repair the DSB. Repair mechanisms are generally of two types, one being non-homologous end joining (NHEJ) and the other being homologous recombination (HDR). In general, NHEJ dominates, and repair produces random indels (insertions or deletions) much higher than precise repair. For base exact substitution, the application of using HDR to achieve base exact substitution is greatly limited because of the low efficiency of HDR and the need for a DNA template.

in 2016, two laboratories such as David Liu and Akihiko Kondo independently report two different types of Cytosine Base Editors (CBEs), respectively, and use two different types of cytidine deaminases rAPOBEC1(rat APOBEC1) and PmCDA1(activation-induced Cytosine deaminase (AID) orthogonal template), which are based on the principle that the base editing of a single Cytosine (C) base is directly realized by using the cytidine deaminase, but not by generating DSB and initiating HDR repair, so that the base editing efficiency of C to be replaced by Thymine (Thymine, T) is greatly improved. Specifically, dead Cas9(dCas9) or the Cas9 nickase (Cas9n) is positioned to a target point through a guide RNA together with rAPOBEC1 or PmCDA1, rAPOBEC1 or PmCDA1 catalyzes C on unpaired single-stranded DNA to generate cytosine deamination reaction to become Uracil (Uracil, U), U is paired with Adenine (Adenine, A) through DNA repair, and T is finally paired with A through DNA replication, so that C-to-T conversion is realized. In the editor tested, the mean mutation rate of the SpCas9n (D10A) & rAPOBEC1/PmCDA1& UGI base editing system (containing uracil DNA glycosylase inhibitor, UGI) was higher because one was that UGI could inhibit Uracil DNA Glycosylase (UDG) from catalytically clearing U in DNA, and another was that SpCas9n (D10A) made a cut on the non-editing strand, inducing a eukaryotic mismatch repair mechanism or a long-patch BER (base-evolution repair) repair mechanism, promoting more preferential repair of U: G mismatch to U: a.

at present, a SpCas9n (D10A) & rAPOBEC1/PmCDA1& UGI base editing system is widely applied to rice to realize C-to-T conversion, but the editing target point is mainly limited to a sequence of which PAM (Protospace Adjacent Motif) is NGG, and the range of editable C is greatly limited. In human cells, the variant xCas9 of SpCas9 was able to recognize NG, GAA and GAT targets, and was successfully developed as CBE, greatly expanding the range of editable C in the genome. Researchers also try to develop new rice CBE by using xCas9 for many times, but in rice transgenic T0 seedlings, except the fact that PAM can be edited as a target of NGG, other NG PAM (including NGT, NGA and NGC) and GAA and GAT PAM targets are not edited, and application of xCas9 in expanding the range of editable C in rice genomes is limited.

Disclosure of Invention

The technical problem to be solved by the present invention is how to use variant xCas9 of SpCas9 to mutate C to T in the target sequences of NG (including NGT, NGA and NGG) and GAA and GAT from PAM sequence to NG in an organism or a cell of an organism.

In order to solve the above technical problems, the present invention firstly provides a method for editing a genomic target sequence.

the editing method of the genome target sequence provided by the invention is a method (I), a method (II), a method (III) or a method (IV):

The method (one) comprises the following steps: introducing a coding gene of xCas9n, a DNA molecule for transcribing tRNA-esgRNA, a coding gene of PmCDA1 and a coding gene of UGI into an organism or biological cells, so that the xCas9n, the tRNA-esgRNA, the PmCDA1 and the coding gene of the UGI are expressed, and the genome target sequence is edited;

The method (II) comprises the following steps: introducing an encoding gene of xCas9n, a DNA molecule for transcribing tRNA-esgRNA and an encoding gene of PmCDA1 into an organism or biological cell, so that the xCas9n, the tRNA-esgRNA and the encoding gene of PmCDA1 are expressed, and the genome target sequence is edited;

The method (III) comprises the following steps: introducing xCas9n, tRNA-esgRNA, PmCDA1 and UGI into an organism or an organism cell to realize the editing of a genome target sequence;

The method (IV) comprises the following steps: introducing xCas9n, tRNA-esgRNA and PmCDA1 into an organism or an organism cell to realize the editing of a genome target sequence;

The tRNA-esgRNA targets the target sequence;

The tRNA-esgRNA is shown as a formula I: tRNA-the RNA transcribed from the target sequence-the esgRNA backbone (formula I);

The tRNA is m1) or m2) or m 3):

m1) replacing T in the 474-550 th position of the sequence 1 with U to obtain an RNA molecule;

m2) carrying out substitution and/or deletion and/or addition of one or more nucleotides on the RNA molecule shown in m1) and having the same function;

m3) and m1) or m2) and has the same function;

The esgRNA backbone is n1) or n2) or n 3):

n1) replacing T in the 571-656 position of the sequence 1 with U to obtain an RNA molecule;

n2) the RNA molecule shown in n1) is subjected to substitution and/or deletion and/or addition of one or more nucleotides, and the RNA molecule has the same function.

n3) and n1) or n2) and has the same function;

The PAM sequence of the target sequence is any one of the following sequences: NGT, NGA, NGG, GAA, GAT; n is A, T, C or G.

the PAM sequence is a DNA sequence connected with the 3' end of the target sequence. And N in the PAM sequences (NGT, NGA and NGG) or G in the PAM sequences (GAA and GAT) is connected with the 3' end of the target sequence. The size of the target sequence can be 15-25bp, further 18-22bp, and further 20 bp.

in the above method, the xCas9n may be xCas9n 3.6.6 or xCas9n 3.7.7. In a specific embodiment of the present invention, said xCas9n is xCas9n 3.7.7.

The xCas9n 3.7.7 is A1) or A2) or A3):

A1) The amino acid sequence is a protein shown in a sequence 2;

A2) The protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 2 in the sequence table and has the same function;

A3) a fusion protein obtained by connecting a label to the N-terminal or/and the C-terminal of A1) or A2).

The PmCDA1 is C1) or C2) or C3):

C1) The amino acid sequence is a protein shown in a sequence 3;

C2) The protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 3 in the sequence table and has the same function;

C3) c1) or C2) at the N-terminus or/and the C-terminus.

The UGI is E1) or E2) or E3):

E1) the amino acid sequence is a protein shown in a sequence 4;

E2) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 4 in the sequence table and has the same function;

E3) A fusion protein obtained by connecting a label to the N-terminal or/and the C-terminal of E1) or E2).

In order to facilitate the purification of the protein of A1), C1) or E1), the amino terminal or the carboxyl terminal of the protein consisting of the amino acid sequence shown in the sequence 2 or the sequence 3 or the sequence 4 in the sequence listing is labeled as shown in the following table.

sequence of Table, tag

Label (R)	residue of	sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

The protein of A2), C2) or E2) is a protein having 75% or more identity to or having 75% or more identity to the amino acid sequence of the protein represented by SEQ ID NO. 2, SEQ ID NO. 3 or SEQ ID NO. 4 and having the same function. The identity of 75% or more than 75% is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity.

The protein of A2), C2) or E2) may be artificially synthesized, or may be obtained by synthesizing the encoding gene and then performing biological expression.

the gene encoding the protein in A2), C2) or E2) above can be obtained by deleting one or several codons of amino acid residues from the DNA sequence shown in 2857-7125, 7417-8040 or 8062-8358 of the sequence 1, and/or by carrying out missense mutation of one or several base pairs, and/or by connecting the coding sequence of the tag shown in the above table to the 5 'end and/or 3' end thereof. The 2857-7125, 7417-8040 and 8062-8358 of the sequence 1 encode proteins shown in the sequence 2, the sequence 3 and the sequence 4, respectively.

The coding gene of the xCas9n 3.7.7 is b1) or b2) or b 3):

b1) A cDNA molecule or DNA molecule shown in 2857-7125 site of a sequence 1 in a sequence table;

b2) A cDNA or DNA molecule having 75% or more identity to the nucleotide sequence defined in b1) and encoding said xCas9n 3.7.7;

b3) A cDNA molecule or a DNA molecule which hybridizes with the nucleotide sequence defined by b1) or b2) under strict conditions and codes for the xCas9n 3.7.7.

The coding gene of the PmCDA1 is d1) or d2) or d 3):

d1) a cDNA molecule or DNA molecule shown in the 7417-8040 site of the sequence 1 in the sequence table;

d2) a cDNA molecule or DNA molecule which has 75 percent or more identity with the nucleotide sequence defined by d1) and codes the PmCDA 1;

d3) Hybridizes with the nucleotide sequence limited by d1) or d2) under strict conditions and encodes the cDNA molecule or DNA molecule of the PmCDA 1.

The encoding gene of the UGI is f1), f2) or f 3):

f1) A cDNA molecule or DNA molecule shown in the 8062-8358 position of the sequence 1 in the sequence table;

f2) a cDNA molecule or DNA molecule having 75% or more identity to the nucleotide sequence defined in f1) and encoding said UGI;

f3) Hybridizing with the nucleotide sequence defined by f1) or f2) under strict conditions, and encoding the cDNA molecule or DNA molecule of UGI.

The nucleotide sequence encoding said xCas9n, said PmCDA1, or said UGI of the present invention can be easily mutated by a person of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which have been artificially modified to have an identity of 75% or more with the nucleotide sequence of said xCas9n, said PmCDA1 or said UGI of the present invention are derived from the nucleotide sequence of the present invention and are identical to the sequence of the present invention as long as they encode said xCas9n, said PmCDA1 or said UGI and have the same function.

the term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes nucleotide sequences that are 75% or greater, or 85% or greater, or 90% or greater, or 95% or greater, identical to the nucleotide sequence of a protein consisting of the amino acid sequence set forth in coding sequence 2, 3, or 4 of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

the stringent conditions are hybridization and washing of the membrane 2 times, 5min each, at 68 ℃ in a solution of 2 XSSC, 0.1% SDS, and 2 times, 15min each, at 68 ℃ in a solution of 0.5 XSSC, 0.1% SDS; alternatively, hybridization was carried out at 65 ℃ in a solution of 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS, and the membrane was washed.

The above-mentioned identity of 75% or more may be 80%, 85%, 90% or 95% or more.

in the above method, the tRNA-esgRNA obtained by transcribing the DNA molecule of the tRNA-esgRNA is an immature RNA precursor, and the tRNA in the RNA precursor is cleaved by two enzymes (RNase P and RNase Z) to obtain mature RNA. How many targets there are in a recombinant expression vector, how many independent mature RNAs are obtained, and each mature RNA is composed of RNA transcribed by the target sequence and the esgRNA skeleton in turn, or composed of individual bases remaining in the tRNA, RNA transcribed by the target sequence and the esgRNA skeleton in turn.

In the above method (i), the number of UGIs may be one or two or more. In a specific embodiment of the present invention, the number of the UGIs is specifically one.

in the above method, in the method (one), the gene encoding xCas9n, the DNA molecule encoding tRNA-esgRNA that is transcribed, the gene encoding PmCDA1, and the gene encoding UGI are introduced into an organism or a cell of an organism via a recombinant expression vector containing an expression cassette for the gene encoding xCas9n, an expression cassette for the DNA molecule encoding tRNA-esgRNA that is transcribed, an expression cassette for the gene encoding PmCDA1, and an expression cassette for the gene encoding UGI. Each of the above-mentioned expression cassettes may be introduced into an organism or a biological cell by the same recombinant expression vector, or may be introduced into an organism or a biological cell by two or more recombinant expression vectors together.

in a specific embodiment of the invention, each expression cassette is introduced into an organism or a biological cell through the same recombinant expression vector, wherein the recombinant expression vector is specifically an xCas9n-epBE-1 recombinant expression vector, an xCas9n-epBE-2 recombinant expression vector, an xCas9n-epBE-3 recombinant expression vector, an xCas9n-epBE-4 recombinant expression vector, an xCas9n-epBE-5 recombinant expression vector or an xCas9n-epBE-6 recombinant expression vector.

the sequence of the xCas9n-epBE-1 recombinant expression vector is sequence 1 in a sequence table. The xCas9n-epBE-1 recombinant expression vector contains the following two target points T1-1 and T5-1 of OsMPK2 gene, and the sequences are shown in Table 1.

The sequence of the xCas9n-epBE-2 recombinant expression vector is obtained by respectively replacing sequences of two target points T1-1 and T5-1 of an OsMPK2 gene in an xCas9n-epBE-1 recombinant expression vector sequence with sequences of two target points T2-1 and T5-2 of an OsMPK5 gene and keeping other sequences unchanged. The sequences of two targets T2-1 and T5-2 of the OsMPK5 gene are shown in Table 1.

the sequence of the xCas9n-epBE-3 recombinant expression vector is obtained by replacing the DNA molecule shown in the 474 nd-839 th position of the sequence 1 in the sequence of the xCas9n-epBE-1 recombinant expression vector with the DNA molecule shown in the sequence 8 and keeping other sequences unchanged. The xCas9n-epBE-3 recombinant expression vector contains the following four target points T4-1, T6-1, T1-2 and T5-3 of OsMPK5 gene, and the sequence is shown in Table 2.

the sequence of the xCas9n-epBE-4 recombinant expression vector is obtained by respectively replacing sequences of two target points T1-1 and T5-1 of an OsMPK2 gene in the sequence of the xCas9n-epBE-1 recombinant expression vector with sequences of the other two target points T4-2 and T2-2 of an OsMPK2 gene and keeping other sequences unchanged. The sequences of two other targets T4-2 and T2-2 of the OsMPK2 gene are shown in Table 2.

The sequence of the xCas9n-epBE-5 recombinant expression vector is obtained by respectively replacing the sequences of two target points T1-1 and T5-1 of an OsMPK2 gene in the sequence of the xCas9n-epBE-1 recombinant expression vector with the sequence of one target point T3-1 of an OsNRT1.1B gene and the sequence of one target point T3-2 of an OsWaxy gene and keeping other sequences unchanged. The sequence of a target point T3-1 of the OsNRT1.1B gene and the sequence of a target point T3-2 of the OsWaxy gene are shown in a table 2.

The sequence of the xCas9n-epBE-6 recombinant expression vector is obtained by respectively replacing the sequences of two target points T1-1 and T5-1 of an OsMPK2 gene in the sequence of the xCas9n-epBE-1 recombinant expression vector with the sequences of two target points T6-2 and T6-3 of an OsWaxy gene and keeping other sequences unchanged. The sequences of two targets T6-2 and T6-3 of the OsWaxy gene are shown in Table 2.

In order to solve the technical problems, the invention also provides a preparation method of the biological mutant.

The preparation method of the biological mutant provided by the invention comprises the following steps: and editing the genome target sequence of the organism according to the editing method of the genome target sequence to obtain the biological mutant.

In any of the above methods, the target sequence may be one or two or more.

in any of the above methods, the editing of the genomic target sequence may be to mutate C in the target sequence to T. The C may be a base C located anywhere in the target sequence.

In any of the above methods, the organism is p1), p2), p3) or p 4): p1) plants or animals; p2) monocotyledonous or dicotyledonous plants; p3) gramineous plants; p4) rice (e.g., Nipponbare rice);

The biological cell is q1) or q2) or q3) or q 4): q1) plant cells or animal cells; q2) a monocotyledonous or dicotyledonous plant cell; q3) a graminaceous plant cell; q4) rice cells (e.g., Nipponbare rice cells).

the invention provides an application of an xCas9n-epBE base editing system in gene editing. The xCas9n-epBE base editing system comprises xCas9n, PmCDA1, UGI and tRNA-esgRNA; the tRNA-esgRNA targets a target sequence; the tRNA-esgRNA is shown as a formula I: tRNA-RNA transcribed from the target sequence-esgRNA backbone (formula I). Experiments prove that: the xCas9n-epBE base editing system realizes the editing of a target point sequence in a plant genome, and particularly realizes the replacement from a base C to a base T in the target point sequence when the PAM sequence is NGT, NGA, NGG, GAA or GAT. The xCas9n-epBE base editing system has wide application prospect in plant or animal gene editing.

Drawings

FIG. 1 is a schematic diagram showing the structure of each element in a four-base editing system.

FIG. 2 shows the efficiency of C.T base substitution in the four base editing system.

FIG. 3 shows the ratio of the number of target spots at which C.T base substitution occurs at the corresponding position C to the number of all editable target spots at the corresponding position which are exactly C.

FIG. 4 shows the ratio of the number of positive T0 seedlings with C.T base substitution at the corresponding position C to the number of all edited positive T0 seedlings with the corresponding position being exactly C.

Detailed Description

the present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA/RNA.

the primer pair T1-1 consists of a primer T1-1-F: 5'-tgtgacaaaatctagccatttttc-3' and primer T1-1-R: 5'-caacattcccatgaacagatg-3', and is used for amplifying target T1-1.

The primer pair T1-2 consists of a primer T1-2-F: 5'-tcaagtacatccactcggcg-3' and primer T1-2-R: 5'-gccagtcaaagaagcagtgc-3', and is used for amplifying the target T1-2.

The primer pair T2-1 consists of a primer T2-1-F: 5'-tcaagctcctcaggcacctc-3' and primer T2-1-R: 5'-cagcaagcacgagttggg-3', and is used for amplifying target T2-1.

The primer pair T2-2 consists of a primer T2-2-F: 5'-tgtgacaaaatctagccatttttc-3' and primer T2-2-R: 5'-gctgggatcaaacacaagc-3', and is used for amplifying the target T2-2.

the primer pair T3-1 consists of a primer T3-1-F: 5'-aacacggtcaccaacttcatc-3' and primer T3-1-R: 5'-cccacatgaatgatgcatatg-3', and is used for amplifying target T3-1.

the primer pair T3-2 consists of a primer T3-2-F: 5'-taagcacacacaaacttcgatc-3' and primer T3-2-R: 5'-gcagacgaacacaacatcctc-3', and is used for amplifying the target T3-2.

The primer pair T4-1 consists of a primer T4-1-F: 5'-ttccctttttaatagctgccttc-3' and primer T4-1-R: 5'-gtgttgttgtagcagttagtgacag-3', and is used for amplifying target T4-1.

The primer pair T4-2 consists of a primer T4-2-F: 5'-tgtgacaaaatctagccatttttc-3' and primer T4-2-R: 5'-tgccctgtaccatcgagtaac-3', and is used for amplifying the target T4-2.

the primer pair T5-1 consists of a primer T5-1-F: 5'-cataggttgaagctttggattatg-3' and primer T5-1-R: 5'-gttcttcattggaaattcatctagtg-3', and is used for amplifying target T5-1.

the primer pair T5-2 consists of a primer T5-2-F: 5'-tcaagctcctcaggcacctc-3' and primer T5-2-R: 5'-cagcaagcacgagttggg-3', and is used for amplifying the target T5-2.

The primer pair T5-3 consists of a primer T5-3-F: 5'-tcaagtacatccactcggcg-3' and primer T5-3-R: 5'-gccagtcaaagaagcagtgc-3', and is used for amplifying target T5-3.

The primer pair T6-1 consists of a primer T6-1-F: 5'-ttccctttttaatagctgccttc-3' and primer T6-1-R: 5'-gtgttgttgtagcagttagtgacag-3', and is used for amplifying target T6-1.

the primer pair T6-2 consists of a primer T6-2-F: 5'-ttcaggtcatccttcgatttc-3' and primer T6-2-R: 5'-gatacttctcctccatgctcttg-3', and is used for amplifying the target T6-2.

The primer pair T6-3 consists of a primer T6-3-F: 5'-gcgaagaactgggagaatgtg-3' and primer T6-3-R: 5'-acacacataaattcagggtccg-3', and is used for amplifying target T6-3.

In the following examples, C.T base substitutions refer to mutations from C to T at any position in the target sequence.

The efficiency of C · T base substitution is equal to the number of positive resistant calli (or positive T0 seedlings) in which C · T base substitution occurred/the number of total positive resistant calli analyzed (or total positive T0 seedlings) × 100%.

homozygous mutants are defined as T0 seedlings in which all sites where C.T base substitutions occur are homozygous mutations. Otherwise, the mutant is a heterozygous mutant.

Japanese fine rice: reference documents: the effects of sodium nitroprusside and its photolysis products on the growth of Nippon rice seedlings and the expression of 5 hormone marker genes [ J ]. proceedings of university of Master Henan (Nature edition), 2017(2): 48-52.; the public is available from the agroforestry academy of sciences of Beijing.

recovering the culture medium: n6 solid medium containing 200mg/L timentin.

Screening a culture medium: n6 solid medium containing 50mg/L hygromycin.

Differentiation medium: n6 solid culture medium containing 2mg/L KT, 0.2mg/L NAA, 0.5g/L glutamic acid and 0.5g/L proline.

rooting culture medium: n6 solid medium containing 0.2mg/L NAA, 0.5g/L glutamic acid, 0.5g/L proline.