阅读说明：本技术 差异代理技术在a·g碱基替换细胞富集中的应用 (Application of differential proxy technology in enrichment of A.G base substitution cells ) 是由 杨进孝 赵久然 张成伟 徐雯 武莹 吕欣欣 于 2019-09-30 设计创作，主要内容包括：本发明公开了差异代理技术在A·G碱基替换细胞富集中的应用。本发明的差异代理技术载体包括靶向目标基因靶点序列的esgRNA、靶向功能丧失的筛选剂抗性基因靶点序列的sgRNA、A·G碱基替换系统和功能丧失的筛选剂抗性基因；A·G碱基替换系统在靶向功能丧失的筛选剂抗性基因靶点序列的sgRNA的向导下,可通过对所述功能丧失的筛选剂抗性基因靶点序列进行A·G碱基替换使所述功能丧失的筛选剂抗性基因功能恢复。本发明实现了细胞水平上A·G碱基替换细胞富集,大大提高A·G碱基替换效率。(The invention discloses an application of a differential agent technology in enrichment of A.G base-substituted cells. The differential agent technology vector comprises esgRNA of a target gene target sequence, sgRNA of a screening agent resistance gene target sequence with target function loss, an A.G base replacement system and a screening agent resistance gene with function loss; the A.G base substitution system can restore the function of the selection agent resistance gene with the loss of function by carrying out A.G base substitution on the selection agent resistance gene target sequence with the loss of function under the guidance of sgRNA of the selection agent resistance gene target sequence with the loss of function. The invention realizes the enrichment of A.G base substitution cells on the cell level and greatly improves the A.G base substitution efficiency.)

1. A kit comprising a sgRNA or a biological material related to the sgRNA, an a · G base substitution system, and a loss-of-function screener resistance gene or a biological material related to the loss-of-function screener resistance gene;

the sgRNA consists of esgRNA targeting a target gene target sequence and sgRNA targeting the loss-of-function screening agent resistance gene target sequence;

the esgRNA structure of the target gene target sequence is as follows: an RNA-esgRNA backbone transcribed from the target gene sequence;

the sgRNA structure of the target sequence of the screening agent resistance gene targeting the loss of function is as follows: an RNA-sgRNA backbone transcribed from the loss-of-function screener resistance gene target sequence;

the a.g base substitution system comprises Cas9 nuclease or a biological material associated with the Cas9 nuclease and adenine deaminase or a biological material associated with the adenine deaminase;

the A.G base substitution system can restore the function of the screening agent resistance gene with the loss of function by carrying out A.G base substitution on the screening agent resistance gene target sequence with the loss of function under the guidance of sgRNA of the screening agent resistance gene target sequence with the loss of function;

the sgRNA backbone is S1) or S2) or S3):

s1) replacing T in the 2418 th 2493 th site of the sequence 1 with U to obtain an RNA molecule;

s2) carrying out substitution and/or deletion and/or addition of one or more nucleotides on the RNA molecule shown in S1) and having the same function;

s3) and S1) or S2) and has the same function;

the esgRNA backbone is T1) or T2) or T3):

t1) replacing T in the 617-702 th site of the sequence 1 with U to obtain an RNA molecule;

t2) carrying out substitution and/or deletion and/or addition of one or more nucleotides on the RNA molecule shown in T1) and having the same function;

t3) and T1) or T2) and has the same function.

2. The kit of claim 1, wherein: the screening agent resistance gene with the function loss is a sequence obtained by deleting the initiation codon of the screening agent resistance gene and adding an agent target sequence at the 5' end of the screening agent resistance gene; the A/G base substitution system can restore the function of the selection agent resistance gene with the function lost by carrying out A/G base substitution on the surrogate target sequence under the guidance of sgRNA of the selection agent resistance gene target sequence with the function lost.

3. The kit of claim 2, wherein: the surrogate target sequence is sequence 5.

4. The kit of any one of claims 1 to 3, wherein: the screening agent resistance gene is a hygromycin resistance gene.

5. The kit of any one of claims 1 to 4, wherein: the Cas9 nuclease is SpCas9n protein;

and/or said adenine deaminase is an ecTadA protein;

and/or, the SpCas9n protein is a1) or a2) or A3):

A1) the amino acid sequence is a protein shown in a sequence 3;

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 3 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 biological material related to the SpCas9n is any one of B1) to B5):

B1) a nucleic acid molecule encoding the SpCas9 n;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B2);

B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector;

B5) a transgenic cell line comprising B1) the nucleic acid molecule or a transgenic cell line comprising B2) the expression cassette;

the ecTadA protein is E1) or E2) or E3):

E1) the amino acid sequence is a protein shown in a sequence 2;

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 2 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);

the biological material related to said ecTadA protein is any one of F1) to F5):

F1) a nucleic acid molecule encoding said ecTadA protein;

F2) an expression cassette comprising the nucleic acid molecule of F1);

F3) a recombinant vector comprising the nucleic acid molecule of F1) or a recombinant vector comprising the expression cassette of F2);

F4) a recombinant microorganism containing F1) said nucleic acid molecule, or a recombinant microorganism containing F2) said expression cassette, or a recombinant microorganism containing F3) said recombinant vector;

F5) a transgenic cell line comprising the nucleic acid molecule of F1) or a transgenic cell line comprising the expression cassette of F2);

the biological material related to the loss-of-function screener resistance gene is any one of K1) to K4):

K1) an expression cassette containing the loss-of-function selection agent resistance gene;

K2) a recombinant vector containing the selection agent resistance gene having the loss of function, or a recombinant vector containing K1) the expression cassette;

K3) a recombinant microorganism containing the loss-of-function screener resistance gene, or a recombinant microorganism containing K1) the expression cassette, or a recombinant microorganism containing K2) the recombinant vector;

K4) a transgenic cell line containing the loss-of-function screener resistance gene, or a transgenic cell line containing the expression cassette of K1).

6. The sgRNA or a biological material associated with the sgRNA of any one of claims 1 to 5.

7. Use of the kit of any one of claims 1-5 or the sgRNA or the biomaterial associated with the sgRNA of claim 6 in any one of M1) -M6):

m1) enriching the cells with A.G base substitution of the genome target sequence of the organism or organism cells;

m2) preparing a product for enriching cells with A.G base substitution of a target sequence of a genome of an organism or an organism cell;

m3) improving the A.G base replacement efficiency of the genome target sequence of the organism or the organism cell;

m4) preparing a product for improving the A.G base replacement efficiency of the genome target sequence of the organism or the organism cell;

m5) an A.G base substitution in a genomic target sequence of an organism or cell of an organism;

m6) preparing the product of the A.G base substitution in the target sequence of the organism or organism cell.

8, N1) or N2) or N3):

n1) A method for enriching cells with A.G base substitutions of target sequences in genomes of organisms or cells of organisms or a method for improving the A.G base substitution efficiency of the target sequences in genomes of organisms or cells of organisms, comprising the following steps: introducing into an organism or cell of an organism a gene encoding a Cas9 nuclease, a DNA molecule transcribing an esgRNA targeting a target gene target sequence, a DNA molecule transcribing an sgRNA targeting the loss-of-function screener resistance gene target sequence, a gene encoding an adenine deaminase, and a loss-of-function screener resistance gene of any one of claims 1-5, such that the Cas9 nuclease, the sgRNA, the adenine deaminase are all expressed; under the guidance of sgRNA of the target sequence of the screening agent resistance gene with the targeted loss of function, the Cas9 nuclease and the adenine deaminase can restore the function of the screening agent resistance gene with the loss of function by carrying out A.G base substitution on the target sequence of the screening agent resistance gene with the targeted loss of function, thereby enriching cells with the A.G base substitution of the screening agent resistance gene, and further realizing the enrichment of cells with the A.G base substitution of the target sequence of the target gene of the genome of an organism or an organism cell or improving the A.G base substitution efficiency of the target sequence of the target gene of the genome of the organism or the organism cell;

n2) A method for enriching cells with A.G base substitutions of target sequences in genomes of organisms or cells of organisms or a method for improving the A.G base substitution efficiency of the target sequences in genomes of organisms or cells of organisms, comprising the following steps: introducing into an organism or cell of an organism a Cas9 nuclease of any one of claims 1-5, an esgRNA targeting a target gene sequence, an sgRNA targeting the loss-of-function screener resistance gene target sequence, an adenine deaminase, and a loss-of-function screener resistance gene; under the guidance of sgRNA of the target sequence of the screening agent resistance gene with the targeted loss of function, the Cas9 nuclease and the adenine deaminase can restore the function of the screening agent resistance gene with the loss of function by carrying out A.G base substitution on the target sequence of the screening agent resistance gene with the targeted loss of function, thereby enriching cells with the A.G base substitution of the screening agent resistance gene, and further realizing the enrichment of cells with the A.G base substitution of the target sequence of the target gene of the genome of an organism or an organism cell or improving the A.G base substitution efficiency of the target sequence of the target gene of the genome of the organism or the organism cell;

n3) biological mutant, comprising the following steps: editing the genome of the organism according to the method of N1) or N2) to obtain an organism mutant; the biological mutant is an organism in which A.G base substitution occurs.

9. The kit of any one of claims 1 to 5 or the use of claim 7 or the method of claim 8, wherein: the A.G base is replaced by a base A and mutated into a base G.

10. The kit of any one of claims 1 to 5 or the use of claim 7 or the method of claim 8, wherein: the organism is P1) or P2) or P3) or P4):

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 Q4):

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 differential agent technology in enrichment of A.G base-substituted cells.

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 2017, a novel Adenine Base Editor (ABE) was reported by David Liu laboratories. Through seven rounds of evolution, researchers fuse tRNA Adenine deaminase (tRNAadensonsine deaminase, ecTadA) derived from Escherichia coli at the 5' end of Cas9 nickase (Cas9n), can directly realize the replacement of a single base A (Adenine, A) to G (Guanine, G) in cells, and do not generate DSB and start HDR repair, thereby greatly improving the base editing efficiency of A to G. The specific process is as follows: when sgRNA containing a genome targeting sequence binds to ecTadA & Cas9n, the complex targets, ecTadA catalyzes adenine deamination of a on unpaired single stranded DNA to Inosine (Inosine, I), I is considered to be G during DNA repair, Cas9n introduces a Cytosine C (Cytosine) that pairs with I upon cleavage of the phosphodiester bond of the paired DNA strands. Finally, C-G pairing is generated in the next repair process, so that A-G conversion is realized.

At present, the research of enriching A.G base substituted cells in plants by reporter gene mediated cell enrichment technology is very limited, and at present, there is no report of utilizing a selection marker in the transformation process to realize the enrichment of A.G base substituted cells on the cellular level and further improve the A.G base substitution efficiency.

Disclosure of Invention

The invention aims to provide application of a differential proxy technology in cell enrichment of A/G base substitution, and the differential proxy technology can realize enrichment of A/G base substitution cells on a cellular level, so that the A/G base substitution efficiency of a target spot is improved.

In order to achieve the above object, the present invention first provides a kit comprising a sgRNA or a biological material related to the sgRNA, an a · G base substitution system, and a selection agent resistance gene for loss of function or a biological material related to the selection agent resistance gene for loss of function;

the sgRNA consists of esgRNA targeting a target gene target sequence and sgRNA targeting the loss-of-function screening agent resistance gene target sequence;

the esgRNA structure of the target gene target sequence is as follows: an RNA-esgRNA backbone transcribed from the target gene sequence;

the sgRNA structure of the target sequence of the screening agent resistance gene targeting the loss of function is as follows: an RNA-sgRNA backbone transcribed from the loss-of-function screener resistance gene target sequence;

the a.g base substitution system comprises Cas9 nuclease or a biological material associated with the Cas9 nuclease and adenine deaminase or a biological material associated with the adenine deaminase;

the A.G base substitution system can restore the function of the screening agent resistance gene with the loss of function by carrying out A.G base substitution on the screening agent resistance gene target sequence with the loss of function under the guidance of sgRNA of the screening agent resistance gene target sequence with the loss of function;

the sgRNA backbone is S1) or S2) or S3):

s1) replacing T in the 2418 th 2493 th site of the sequence 1 with U to obtain an RNA molecule;

s2) carrying out substitution and/or deletion and/or addition of one or more nucleotides on the RNA molecule shown in S1) and having the same function;

s3) and S1) or S2) and has the same function;

the esgRNA backbone is T1) or T2) or T3):

t1) replacing T in the 617-702 th site of the sequence 1 with U to obtain an RNA molecule;

t2) carrying out substitution and/or deletion and/or addition of one or more nucleotides on the RNA molecule shown in T1) and having the same function;

t3) and T1) or T2) and has the same function.

In the kit, the number of target sequences of the target gene to be targeted can be one or two or more; the number of target sequences of the screening agent resistance gene targeting the loss of function may be one or two or more. The size of the target sequence can be 15-25bp, further 18-22bp, and further 20 bp.

The screening agent resistance gene with the loss of function meets the following conditions: the function or activity of the screening agent resistance gene with the function loss is lost, and the function of the screening agent resistance gene with the function loss can be recovered after the A.G base substitution is carried out on the target sequence of the screening agent resistance gene with the function loss. The target sequence of the screening agent resistance gene with the loss of function can be a target sequence on the screening agent resistance gene with the loss of function (positioned in the screening agent resistance gene with the loss of function), and can also be a target sequence additionally added in the screening agent resistance gene with the loss of function or at the 5 'end or the 3' end. When a target sequence (referred to as a surrogate target sequence) is additionally added to the sequence of the non-functional selection agent resistance gene in order that the non-functional selection agent resistance gene can recover the function after the A.G base substitution, the non-functional selection agent resistance gene sequence includes not only the non-functional selection agent resistance gene itself but also the surrogate target sequence and, if necessary, one or two or more bases additionally added in order to ensure that the selection agent resistance gene can be translated in a normal reading frame after the addition of the surrogate target sequence.

Further, the selection agent resistance gene with loss of function may be a sequence obtained by deleting the initiation codon (e.g., ATG) of the selection agent resistance gene and adding a surrogate target sequence to the 5' end of the selection agent resistance gene. The surrogate target sequence satisfies the following conditions: the A.G base substitution of the surrogate target sequence by the A.G base substitution system can restore the function of the selection agent resistance gene with lost function. The agent target sequence consists of a screening agent resistance gene target sequence with function loss and a PAM sequence in sequence. It should be noted that, in order to ensure that the screener resistance gene with the start codon removed can be translated in normal reading frame after the surrogate target sequence is added, one or two or more bases may be added between the surrogate target sequence and the screener resistance gene with the start codon removed.

In one embodiment of the invention, the surrogate target sequence is sequence 5. The sequence of the target point of the screening agent resistance gene with the loss of function is the 1 st to 20 th sites of the sequence 5. The A.G base substitution system can perform A.G base substitution on the proxy target sequence under the guidance of sgRNA targeting the proxy target sequence, so that the 6 th base A of the proxy target sequence is mutated into the base G to form ATG, and further the function of the screening marker gene is recovered. It should be noted that, in order to ensure that the screener resistance gene with the start codon removed can be translated in normal reading frame after the surrogate target sequence is added, a base C is added between the surrogate target sequence and the screener resistance gene with the start codon removed.

Further, the screening agent resistance gene may be a screening agent resistance gene commonly used in the art, such as Bar/PAT glufosinate-N-acetyltransferase gene, PMI 6-phosphomannose isomerase gene, EPSPS 5-enolpyruvylshikimate-3-phosphate synthase gene, and the like. In one embodiment of the invention, the screener resistance gene is a hygromycin resistance gene.

In the above kit, the Cas9 nuclease includes Cas9 nuclease or its variant, dead inactivating enzyme (dead Cas9, dCas9) or its variant, nickase (Cas9 nickase, Cas9n) or its variant from different sources. The Cas9 nucleases or variants thereof of different origins include Cas9 (such as SaCas9, SaCas9-KKH and the like) derived from bacteria, Cas9-PAM variants (such as xCas9, NG Cas9, Cas9-VQR, Cas9-VRER and the like), Cas9 high fidelity enzyme variants (such as HypaCas9, eSpCas9(1.1), Cas9-HF1 and the like) and the like. In a specific embodiment of the invention, the Cas9 nuclease is Cas9n, specifically SpCas9n protein. In another embodiment of the invention, the Cas9 nuclease is Cas9n, in particular HypaCas9n protein.

The adenine deaminase can be adenine deaminase of different sources, such as an ecTadA protein derived from Escherichia coli, or adenine deaminase derived from a plant endogenous source (such as a rice endogenous OsTadA, an Arabidopsis thaliana derived AtTadA, and the like). In a particular embodiment of the invention, the adenine deaminase is an ecTadA protein derived from escherichia coli.

Further, the SpCas9n protein is a1) or a2) or A3):

A1) the amino acid sequence is a protein shown in a sequence 3;

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 3 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 biological material related to the SpCas9n is any one of B1) to B5):

B1) a nucleic acid molecule encoding the SpCas9 n;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B2);

B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector;

B5) a transgenic cell line comprising B1) the nucleic acid molecule or a transgenic cell line comprising B2) the expression cassette;

the ecTadA protein is E1) or E2) or E3):

E1) the amino acid sequence is a protein shown in a sequence 2;

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 2 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);

the biological material related to said ecTadA protein is any one of F1) to F5):

F1) a nucleic acid molecule encoding said ecTadA protein;

F2) an expression cassette comprising the nucleic acid molecule of F1);

F3) a recombinant vector comprising the nucleic acid molecule of F1) or a recombinant vector comprising the expression cassette of F2);

F4) a recombinant microorganism containing F1) said nucleic acid molecule, or a recombinant microorganism containing F2) said expression cassette, or a recombinant microorganism containing F3) said recombinant vector;

F5) a transgenic cell line comprising the nucleic acid molecule of F1) or a transgenic cell line comprising the expression cassette of F2);

the biological material related to the loss-of-function screener resistance gene is any one of K1) to K4):

K1) an expression cassette containing the loss-of-function selection agent resistance gene;

K2) a recombinant vector containing the selection agent resistance gene having the loss of function, or a recombinant vector containing K1) the expression cassette;

K3) a recombinant microorganism containing the loss-of-function screener resistance gene, or a recombinant microorganism containing K1) the expression cassette, or a recombinant microorganism containing K2) the recombinant vector;

K4) a transgenic cell line containing the loss-of-function screener resistance gene, or a transgenic cell line containing the expression cassette of K1).

In order to facilitate the purification of the proteins A1) and 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 in the sequence table is linked with the tags 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 in A2) or E2) is a protein having 75% or more identity to the amino acid sequence of the protein shown in SEQ ID NO. 2 or SEQ ID NO. 3 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 in A2) and E2) can be artificially synthesized, or can be obtained by synthesizing the coding gene and then performing biological expression.

A2) and E2) as described above, can be obtained by deleting one or more amino acid residues from the DNA sequence shown at positions 4205-4705 (protein shown in coding sequence 2) of sequence 1 and 5396-9496 (protein shown in coding sequence 3) of sequence 1, and/or by performing missense mutation of one or more base pairs, and/or by linking the coding sequences of the tags shown in the above table to the 5 'end and/or 3' end thereof.

Further, B1) the nucleic acid molecule is B1) or B2) or B3):

b1) a cDNA molecule or DNA molecule shown in No. 5396-9496 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 SpCas9 n;

b3) a cDNA or DNA molecule hybridizing under stringent conditions to the nucleotide sequence defined in b1) or b2) and encoding the SpCas9 n;

F1) the nucleic acid molecule is f1) or f2) or f 3):

f1) a cDNA molecule or a DNA molecule shown in the 4205-4705 site of the sequence 1 in the sequence table;

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

f3) a cDNA molecule or DNA molecule which hybridizes under stringent conditions with a nucleotide sequence defined in f1) or f2) and encodes said ecTadA;

K1) the loss-of-function selection agent resistance gene is a DNA molecule shown in the 12278-13324 position of the sequence 1.

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

The nucleotide sequence encoding the SpCas9n or the ecadada of the present invention can be easily mutated by one of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which are artificially modified and have 75% or more identity to the nucleotide sequence of the SpCas9n or the ecTadA of the invention are derived from the nucleotide sequence of the invention and are identical to the sequence of the invention as long as they encode the SpCas9n or the ecTadA 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 more, or 85% or more, or 90% or more, or 95% or more identical to the nucleotide sequence of a protein consisting of the amino acid sequence shown in coding sequence 2 or 3 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.

B2) The expression cassette containing the nucleic acid molecule encoding the SpCas9n protein (SpCas9n gene expression cassette) refers to DNA capable of expressing the SpCas9n protein in host cells, and the DNA may include not only a promoter for starting the transcription of the SpCas9n gene, but also a terminator for terminating the transcription of the SpCas9n gene. Further, the expression cassette may also include an enhancer sequence. The existing expression vector can be used for constructing a recombinant vector containing the SpCas9n gene expression cassette.

F2) The expression cassette containing a nucleic acid molecule encoding an ecTadA protein (ecTadA gene expression cassette) is a DNA capable of expressing an ecTadA protein in a host cell, and the DNA may include not only a promoter which initiates transcription of the ecTadA gene but also a terminator which terminates transcription of the ecTadA gene. Further, the expression cassette may also include an enhancer sequence. Still further, the expression cassette may contain one or two nucleic acid molecules encoding an ecTadA protein. The recombinant vector containing the ecTadA gene expression cassette can be constructed using an existing expression vector.

The vector may be a plasmid, cosmid, phage or viral vector. In a specific embodiment of the invention, the recombinant vector is specifically a DisSUGs-1 recombinant expression vector, a DisSUGs-2 recombinant expression vector or a DisSUGs-3 recombinant expression vector.

The sequence of the DisSUGs-1 recombinant expression vector is sequence 1. The DisSUGs-1 recombinant expression vector contains four target sequences, and the sequences are shown in a table 1.

The sequence of the DisSUGs-2 recombinant expression vector is that the first three target sequences in the sequence 1 are sequentially and respectively replaced by the following three target sequences: DEP1-T2, ACC, NRT1.1B-T4, and the sequences obtained by keeping other sequences unchanged. The corresponding target sequence information is shown in Table 1.

The sequence of the DisSUGs-3 recombinant expression vector is that the first three target sequences in the sequence 1 are sequentially and respectively replaced by the following three target sequences: SPL14, WRKY45, DELLA, and the other sequences were kept unchanged. The corresponding target sequence information is shown in Table 1.

The microorganism may be a yeast, bacterium, algae or fungus. Wherein the bacterium can be an Agrobacterium, such as Agrobacterium EHA 105. In a specific embodiment of the present invention, the recombinant microorganism is specifically agrobacterium EHA105 containing the Dissugs-1 recombinant expression vector or the Dissugs-2 recombinant expression vector or the Dissugs-3 recombinant expression vector.

The transgenic cell line does not include propagation material.

The kit has the following uses:

m1) enriching the cells with A.G base substitution of the genome target sequence of the organism or organism cells;

m2) preparing a product for enriching cells with A.G base substitution of a target sequence of a genome of an organism or an organism cell;

m3) improving the A.G base replacement efficiency of the genome target sequence of the organism or the organism cell;

m4) preparing a product for improving the A.G base replacement efficiency of the genome target sequence of the organism or the organism cell;

m5) an A.G base substitution in a genomic target sequence of an organism or cell of an organism;

m6) preparing the product of the A.G base substitution in the target sequence of the organism or organism cell.

The sgrnas or biological materials related to the sgrnas also belong to the scope of the present invention.

In order to achieve the above object, the present invention also provides a novel use of the above kit or the above sgRNA or a biological material related to the sgRNA.

The invention provides the use of the above described kit or the above described sgRNA or a biological material related to said sgRNA in any of M1) -M6):

m1) enriching the cells with A.G base substitution of the genome target sequence of the organism or organism cells;

m2) preparing a product for enriching cells with A.G base substitution of a target sequence of a genome of an organism or an organism cell;

m3) improving the A.G base replacement efficiency of the genome target sequence of the organism or the organism cell;

m4) preparing a product for improving the A.G base replacement efficiency of the genome target sequence of the organism or the organism cell;

m5) an A.G base substitution in a genomic target sequence of an organism or cell of an organism;

m6) preparing the product of the A.G base substitution in the target sequence of the organism or organism cell.

In order to achieve the above object, the present invention also provides the method described in N1) or N2) or N3):

n1) A method for enriching cells with A.G base substitutions of a target sequence in the genome of an organism or an organism cell or a method for improving the A.G base substitution efficiency of the target sequence in the genome of an organism or an organism cell, comprising the following steps: introducing the coding gene of the Cas9 nuclease, the DNA molecule of the esgRNA transcribed and targeted to the target gene target sequence, the DNA molecule of the sgRNA transcribed and targeted to the target gene target sequence of the screening agent resistance gene with lost function, the coding gene of the adenine deaminase and the screening agent resistance gene with lost function into an organism or an organism cell so as to express the Cas9 nuclease, the sgRNA and the adenine deaminase; under the guidance of sgRNA of the target sequence of the screening agent resistance gene with the targeted loss of function, the Cas9 nuclease and the adenine deaminase can restore the function of the screening agent resistance gene with the lost function by carrying out A.G base substitution on the target sequence of the screening agent resistance gene with the targeted loss of function, thereby enriching cells with the A.G base substitution of the screening agent resistance gene, and further realizing the enrichment of cells with the A.G base substitution of the target sequence of the target gene of the genome of an organism or an organism cell or improving the A.G base substitution efficiency of the target sequence of the target gene of the genome of the organism or the organism cell;

n2) A method for enriching cells with A.G base substitutions of a target sequence in the genome of an organism or an organism cell or a method for improving the A.G base substitution efficiency of the target sequence in the genome of an organism or an organism cell, comprising the following steps: introducing the Cas9 nuclease, esgRNA targeting a target gene target sequence, sgRNA targeting the loss-of-function screening agent resistance gene target sequence, adenine deaminase and a loss-of-function screening agent resistance gene into an organism or an organism cell; under the guidance of sgRNA of the target sequence of the screening agent resistance gene with the targeted loss of function, the Cas9 nuclease and the adenine deaminase can restore the function of the screening agent resistance gene with the lost function by carrying out A.G base substitution on the target sequence of the screening agent resistance gene with the targeted loss of function, thereby enriching cells with the A.G base substitution of the screening agent resistance gene, and further realizing the enrichment of cells with the A.G base substitution of the target sequence of the target gene of the genome of an organism or an organism cell or improving the A.G base substitution efficiency of the target sequence of the target gene of the genome of the organism or the organism cell;

n3) biological mutant, comprising the following steps: editing the genome of the organism according to the method of N1) or N2) to obtain a biological mutant; the biological mutant is an organism in which A.G base substitution occurs.

In the above method, the gene encoding Cas9 nuclease, the DNA molecule of esgRNA that is transcription-targeted to the target gene sequence, the DNA molecule of sgRNA that is transcription-targeted to the loss-of-function screening agent-resistant gene target sequence, and the gene encoding adenine deaminase in N1) are introduced into an organism or a biological cell via a recombinant vector containing the expression cassette of the gene encoding Cas9 nuclease, the expression cassette of the DNA molecule of esgRNA that is transcription-targeted to the target gene target sequence, the expression cassette of the DNA molecule of sgRNA that is transcription-targeted to the loss-of-function screening agent-resistant gene target sequence, and the expression cassette of the gene encoding adenine deaminase. Each of the above-mentioned expression cassettes may be introduced into an organism or a cell of an organism using the same recombinant expression vector, or may be introduced into an organism or a cell of an organism using two or more recombinant expression vectors.

In a specific embodiment of the present invention, each of the above-described expression cassettes is introduced into an organism or a cell of an organism via the same recombinant expression vector. The expression cassette of the adenine deaminase coding gene in the recombinant expression vector contains two coding genes of adenine deaminase. The recombinant expression vector is specifically the DisSUGs-1 recombinant expression vector, the DisSUGs-2 recombinant expression vector or the DisSUGs-3 recombinant expression vector.

In the kit of parts or the use or the method, the base substitution A.G is mutated to the base G. The base A can be any position in the target sequence.

In the above kit of parts or use or method, the organism is P1) or P2) or P3) or P4):

p1) plants or animals;

p2) monocotyledonous or dicotyledonous plants;

p3) gramineous plants;

p4) rice (e.g., japanese fine rice);

the biological cell is Q1) or Q2) or Q3) or Q4):

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 technical principle of the difference agent of the invention is as follows: the optimized esgRNA is applied to the cell enrichment technology of A.G base replacement, the optimized esgRNA is used for editing a target sequence of an endogenous target gene of a genome, the sgRNA is used for editing a surrogate target sequence of a reporter gene, and the A.G base replacement efficiency of the target sequence of the endogenous target gene is further improved.

The cell enrichment technology principle of the A.G base substitution is as follows: a cell enrichment technique using the inactivated screening agent resistance gene as a reporter gene for A.G base substitution is established, so that cells in which A.G base substitution has occurred on the reporter gene can grow in a medium containing the screening agent, and cells in which A.G base substitution has not occurred cannot grow in a medium containing the screening agent. On the basis of the reporter gene, if A.G base replacement editing is carried out on the endogenous target gene target spot, cells growing in a culture medium containing a screening agent have higher probability of A.G base replacement of the endogenous target gene target spot, so that enrichment of the cells with A.G base replacement of the endogenous target gene target spot is realized, and the A.G base replacement efficiency of the endogenous target gene target spot is improved.

The invention has the following advantages:

1. there are many different types of genes that can be used as reporter genes for cell enrichment in plants by A.G base substitution. Because genetic transformation methods (such as an agrobacterium transformation method and a gene gun transformation method) of various crops have relatively mature and stable screening systems, the genetic transformation methods have more broad spectrum and universality compared with other genetic transformation methods such as a fluorescent reporter gene and an endogenous herbicide resistance gene and the like by using a resistance gene corresponding to a screening agent for transformation as a reporter gene to enrich endogenous mutant cells of a genome.

2. The technical design is simple and convenient, and the agent target and the design form can be more widely applied to resistance genes corresponding to more screening agents so as to meet the requirements of different transformation screening systems of different crops.

3. The differential agent technology is suitable for cell enrichment technologies based on different deaminase-mediated base editors or different Cas9 enzyme-mediated base editors, can realize enrichment of A.G base-substituted cells on the cell level, and greatly improves A.G base substitution efficiency.

Drawings

Fig. 1 is a schematic structural diagram of dispugs as a differential proxy technology bearer.

FIG. 2 is a schematic diagram showing the operation principle of the cell for enriching A.G base substitutions by the differential proxy technology.

FIG. 3 is a comparison of the efficiency of A.G base substitution on target in rice resistance-curing by differential agent technique and conventional technique.

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.

Primer pair T1 was composed of primer T1-F: 5'-ctgtcttcggctggtctggg-3' and primer T1-R: 5'-tgccaagcacatcaaacaagtaaa-3', and is used for amplifying target ALS-T4.

Primer pair T2 was composed of primer T2-F: 5'-tctagactgtagtggtgataac-3' and primer T2-R: 5'-tttcttctttctgattaatggcc-3', and is used for amplifying target CDC 48-T3.

Primer pair T3 was composed of primer T3-F: 5'-aatccaccaccaatccaatcc-3' and primer T3-R: 5'-caccatggcgtcgtcgtccg-3', for amplifying the target AAT.

Primer pair T4 was composed of primer T4-F: 5'-tcagcctgcagtactgaattatc-3' and primer T4-R: 5'-gggcctaagtgtgacatacaag-3', and is used for amplifying the target DEP 1-T2.

Primer pair T5 was composed of primer T5-F: 5'-gcattgctggacttcaacc-3' and primer T5-R: 5'-caaaccgtatcgcaatctgag-3', for amplifying the target ACC.

Primer pair T6 was composed of primer T6-F: 5'-agcatatatagcaagccaggttg-3' and primer T6-R: 5'-aataagccactgtgttatgtacgc-3', and is used for amplifying target NRT1.1B-T4.

Primer pair T7 was composed of primer T7-F: 5'-gatgtgttgtttgttgcgattc-3' and primer T7-R: 5'-agtgggcatgatggctagg-3', and is used for amplifying the target SPL 14.

Primer pair T8 was composed of primer T8-F: 5'-ctacagggtcacctacatcgg-3' and primer T8-R: 5'-tgagacgacacatcaacaagg-3', and is used for amplifying target WRKY 45.

Primer pair T9 was composed of primer T9-F: 5'-gaagcgcgagtaccaagaag-3' and primer T9-R: 5'-atccgcttggtgtccctc-3', for amplifying target DELLA.

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

The efficiency of base substitution between A and G was 100% of the number of positive resistant calli in which base substitution between A and G occurred/the total number of positive resistant calli analyzed.

Japanese fine rice: reference documents: the effects of sodium nitroprusside and its photolysis products on the growth of Nippon clear rice seedlings and the expression of 5 hormone marker genes [ J ]. proceedings of university of south 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.