CN110669775B

CN110669775B - Application of differential proxy technology in enrichment of A.G base substitution cells

Info

Publication number: CN110669775B
Application number: CN201910938672.XA
Authority: CN
Inventors: 杨进孝; 赵久然; 张成伟; 徐雯; 武莹; 吕欣欣
Original assignee: Beijing Academy of Agriculture and Forestry Sciences
Current assignee: Beijing Academy of Agriculture and Forestry Sciences
Priority date: 2019-09-30
Filing date: 2019-09-30
Publication date: 2021-07-16
Anticipated expiration: 2039-09-30
Also published as: CN110669775A

Abstract

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.

Description

Application of differential proxy technology in enrichment of A.G base substitution 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 (tRNA Adenine 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, so that the base editing efficiency of replacing A with G is greatly improved. 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 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):

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.

Example 1 establishment of differential agent technique for EcTadA & ecTadA & Cas9 n-mediated A.G base substitutions

Firstly, establishing a differential agent technology vector for A.G base substitution mediated by EcTadA & ecTadA & Cas9n

The common technical vector for EcTadA & ecTadA & Cas9n (ABE) -mediated A.G base substitutions was named sgRNA-GT.

The differential proxy technology vector for EcTadA & ecTadA & Cas9n (ABE) -mediated A.G base substitutions was named DisSUGs.

schematic diagrams of sgRNA-GT and DisSUGs vectors are shown in fig. 1.

The difference agent technology carrier is different from the common technology carrier in that:

1) the differential agent technology vector modifies the resistance gene of the screening agent in the common technology vector to lose the function, and adds a corresponding agent target sequence in the sgRNA part. Taking the screening agent resistance gene as Hygromycin resistance gene Hygromycin as an example: the screening agent resistance gene in the vector of the common technology is the complete Hygromycin resistance gene Hygromycin. The screening agent resistance gene in the differential agent technology carrier is a Hygromycin resistance gene Hygromycin (Hygromycin) with loss of function^-ATG) The Hygromycin resistance gene Hygromycin with lost function is a sequence obtained by removing ATG from the complete Hygromycin resistance gene Hygromycin and adding a surrogate target sequence at the 5' end. Wherein, the surrogate target sequences are as follows: ctcatagcactcaatgcggtTGG (capital letters are PAM sequences).

2) The differential surrogate technology uses optimized esgRNA to edit the endogenous target sequence of the genome and sgRNA to edit the surrogate target sequence of the resistance gene of the screening agent.

Second, the working principle of the EcTadA & ecTadA & Cas9n mediated A.G base substitution differential agent technology

The operation principle of the differential agent technique of A.G base substitution is shown in FIG. 2. Taking the screening agent resistance gene as the Hygromycin resistance gene as an example: in the differential agent technology, after ATG is removed from Hygromycin resistance gene Hygromycin, resistance function is lost, and a plant cannot grow resistance callus in a Hygromycin screening culture medium, when an A.G base replacement system (EcTadA & ecTadA & Cas9n) in the differential agent technology mutates A6 in an agent target sequence to G6 (A at the 6 th base is mutated to base G) under guidance of sgRNA, and ATG is formed, the Hygromycin resistance gene Hygromycin can be normally expressed, resistance function is recovered, and the plant can grow resistance callus in the Hygromycin screening culture medium. Because the cells growing the resistant callus have been subjected to A.G base substitution, the efficiency of the A.G base substitution of the endogenous gene corresponding to the cells is relatively higher, so that the purpose of enriching the A.G base substitution cells is achieved, and the A.G base substitution efficiency of the endogenous target of the plant is improved.

Example 2 construction of EcTadA & ecTadA & Cas9 n-mediated differential agent technology vector and application thereof in rice genome editing

Construction of recombinant expression vector

The recombinant expression vectors in this example are classified into the following two types: DissuGs recombinant expression vectors and sgRNA-GT recombinant expression vectors. The structural schematic diagram of each element of the two recombinant expression vectors is shown in FIG. 1. Each vector is a circular plasmid.

Each recombinant expression vector is divided into three types according to different target sequences, and the following six recombinant expression vectors are in total: DisSUGs-1 recombinant expression vector, DisSUGs-2 recombinant expression vector, DisSUGs-3 recombinant expression vector, sgRNA-GT-1 recombinant expression vector, sgRNA-GT-2 recombinant expression vector and sgRNA-GT-3 recombinant expression vector.

The six recombinant expression vectors are artificially synthesized, and the specific structural descriptions of the six recombinant expression vectors are respectively as follows:

the sequence of the DisSUGs-1 recombinant expression vector is sequence 1 in a sequence table. The nucleotide sequence of OsU6a promoter at the 131-position 596 of the sequence 1, the nucleotide sequence of OsU6b promoter at the 712-position 1044, the nucleotide sequence of OsU6c promoter at the 1160-position 1901 and the nucleotide sequence of OsU3 promoter at the 2017-position 2397; the 597-position 616, the 1045-position 1064 and the 1902-position 1921 are three target sequences of ALS-T4, CDC48-T3 and AAT respectively, and the 2398-position 2417 is a reporter gene surrogate target sequence; the 617-702, 1065-1150 and 1922-2007 positions are esgRNA nucleotide sequences, and the 2418-2493 position is an sgRNA nucleotide sequence. The 2511-4224 th site of the sequence 1 is a nucleotide sequence of an OsUbq3 promoter, the 4234 th site-4734 th site and the 4831 th site-5328 th site are ecTadA coding sequences, and the ecTadA proteins are shown as a coding sequence 2; the 5425-9525 th site of the sequence 1 is a coding sequence of SpCas9n protein, and the coding sequence 3 is SpCas9n protein; the 9682-10014 position of the sequence 1 is a 3' UTR sequence of OsUbq 3; the nucleotide sequence of Nos terminator at positions 10015-10267 of the sequence 1; the 10308-12300 th site of the sequence 1 is the nucleotide sequence of the ZmUbi1 promoter, the 12307-12329 th site is the surrogate target sequence, the 12331-13353 th site is the nucleotide sequence of hygromycin phosphotransferase with the initiation codon removed, and the 13380-13595 th site is the nucleotide sequence of the CaMV35S terminator. Four target sequences in the DisSUGs-1 recombinant expression vector are shown in Table 1, and the targets are ALS-T4, CDC48-T3, AAT and ST1152 surrogate targets respectively.

The sequence of the sgRNA-GT-1 recombinant expression vector is obtained by replacing the 12307-13353 position of the sequence 1 with the complete hygromycin phosphotransferase nucleotide sequence shown in the sequence 4 and keeping other sequences unchanged.

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

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

The target nucleotide sequences of the esgrnas or sgrnas of each vector and the corresponding PAM sequences are shown in table 1.

TABLE 1

II, obtaining the rice positive resistance callus

Carrying out operation on the DisSuGs-1 vector, DisSuGs-2 vector, DisSuGs-3 vector, sgRNA-GT-1 vector, sgRNA-GT-2 vector and sgRNA-GT-3 vector obtained in the step one according to the following steps 1-8 respectively:

1. the vector was introduced into Agrobacterium EHA105 (product of Shanghai Diego Biotechnology Ltd., CAT #: AC1010) to obtain recombinant Agrobacterium.

2. Culturing the recombinant Agrobacterium with a medium (YEP medium containing 50. mu.g/ml kanamycin and 25. mu.g/ml rifampicin), shaking at 28 ℃ and 150rpm to OD₆₀₀At room temperature, centrifuging at 10000rpm for 1min, resuspending the thallus with an infection solution (glucose and sucrose are replaced by N6 liquid culture medium, and the concentrations of glucose and sucrose in the infection solution are 10g/L and 20g/L respectively) and diluting to OD₆₀₀And the concentration is 0.2, and an agrobacterium tumefaciens infection solution is obtained.

3. The mature seeds of the rice variety Nipponbare are shelled and threshed, placed in a 100mL triangular flask, added with 70% (v/v) ethanol water solution for soaking for 30sec, then placed in 25% (v/v) sodium hypochlorite water solution, sterilized by shaking at 120rpm for 30min, washed with sterile water for 3 times, sucked with filter paper to remove water, and then placed on an N6 solid culture medium with the embryo downwards, and cultured in dark at 28 ℃ for 4-6 weeks, thus obtaining the rice callus.

4. After the step 3 is completed, the rice callus is soaked in an agrobacterium infection solution A (the agrobacterium infection solution A is a liquid obtained by adding acetosyringone into the agrobacterium infection solution, the addition amount of the acetosyringone meets the volume ratio of the acetosyringone to the agrobacterium infection solution of 25 mul: 50ml) and soaked for 10min, and then the rice callus is placed on a culture dish (containing about 200ml of the agrobacterium-free infection solution) paved with two layers of sterilized filter paper and cultured in the dark at 21 ℃ for 1 day.

5. And (4) putting the rice callus obtained in the step (4) on a recovery culture medium, and performing dark culture at 25-28 ℃ for 3 days.

6. And (4) placing the rice callus obtained in the step (5) on a screening culture medium, and performing dark culture at 28 ℃ for 2 weeks.

7. And (4) putting the rice callus obtained in the step (6) on a screening culture medium again, and performing dark culture at 28 ℃ for 2 weeks to obtain the rice resistance callus.

8. Respectively extracting 20-24 genome DNAs of rice resistant calli and taking the genome DNAs as templates, and performing PCR amplification by using a primer pair consisting of a primer F (5'-attatgtagcttgtgcgtttcg-3') and a primer R (5'-gatgaagagcttatcgacgt-3') to obtain PCR amplification products; the PCR amplification product was subjected to agarose gel electrophoresis, followed by judgment as follows: if the PCR amplification product contains about 1150bp DNA fragment, the corresponding rice resistant callus is rice positive resistant callus; if the PCR amplification product does not contain the DNA fragment of about 1150bp, the corresponding rice resistant callus is not a rice positive resistant callus.

Third, result analysis

1. Taking 20-24 rice positive resistant callus genome DNAs obtained in the second step as templates (independently infecting twice to obtain an average value and a variance) for each vector, and carrying out PCR amplification on the ALS-T4 target by adopting a primer pair T1 to obtain a PCR amplification product; for CDC48-T3 target, carrying out PCR amplification on T2 by using a primer to obtain a PCR amplification product; for the AAT target, adopting a primer pair T3 to carry out PCR amplification to obtain a PCR amplification product; for the DEP1-T2 target, carrying out PCR amplification on T4 by adopting a primer pair to obtain a PCR amplification product; for the ACC target, carrying out PCR amplification on T5 by using a primer pair to obtain a PCR amplification product; for NRT1.1B-T4 target, carrying out PCR amplification on T6 by adopting a primer pair to obtain a PCR amplification product; for the SPL14 target, carrying out PCR amplification on T7 by adopting a primer pair to obtain a PCR amplification product; for WRKY45 target, carrying out PCR amplification on T8 by using a primer to obtain a PCR amplification product; for DELLA target, PCR amplification is carried out by using a primer pair T9 to obtain a PCR amplification product.

2. And (3) carrying out Sanger sequencing and analysis on the PCR amplification product obtained in the step (1). The sequencing results were analyzed only for each target region. The number of the rice positive resistant calluses with A.G base substitution at each target point of each carrier is respectively counted, the A.G base substitution efficiency is calculated, and the result is shown in figure 3.

The results show that: by using a differential proxy technique, the A.G base substitution efficiency of the 5 th base in the ALS-T4 target point is increased from 34% to 93% in the rice resistance healing wound; the A.G base replacement efficiency of the 5 th base of CDC48-T3 target point is increased from 36% to 80%, and the A.G base replacement efficiency of the 9 th base is increased from 0% to 25%; the A.G base replacement efficiency of the 6 th base of the AAT target point is increased from 22 percent to 53 percent; the A.G base substitution efficiency of the 4 th base of DEP1-T2 increased from 21% to 63%; the A.G base replacement efficiency of the 8 th base of the NRT1.1B-T4 target point is increased from 0% to 9%; the substitution efficiency of the A.G base of the SPL14 target site 5 th base is increased from 20% to 90%, and the substitution efficiency of the A.G base of the 7 th base is increased from 18% to 88%; the efficiency of A.G base substitution at base 6 of DELLA target increased from 31% to 95%. In conclusion, the A.G base substitution efficiency of most targets is improved to 2.5-3 times that of the common technology by using the differential agent technology.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Sequence listing

<110> agriculture and forestry academy of sciences of Beijing City

<120> application of differential agent technology in enrichment of cells by A.G base substitution

<160>5

<170>PatentIn version 3.5

<210>1

<211>20001

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>1

ggtggcagga tatattgtgg tgtaaacatg gcactagcct caccgtcttc gcagacgagg 60

ccgctaagtc gcagctacgc tctcaacggc actgactagg tagtttaaac gtgcacttaa 120

ttaaggtacc tggaatcggc agcaaaggat tttttcctgt agttttccca caaccatttt 180

ttaccatccg aatgatagga taggaaaaat atccaagtga acagtattcc tataaaattc 240

ccgtaaaaag cctgcaatcc gaatgagccc tgaagtctga actagccggt cacctgtaca 300

ggctatcgag atgccataca agagacggta gtaggaacta ggaagacgat ggttgattcg 360

tcaggcgaaa tcgtcgtcct gcagtcgcat ctatgggcct ggacggaata ggggaaaaag 420

ttggccggat aggagggaaa ggcccaggtg cttacgtgcg aggtaggcct gggctctcag 480

cacttcgatt cgttggcacc ggggtaggat gcaatagaga gcaacgttta gtaccacctc 540

gcttagctag agcaaactgg actgccttat atgcgcgggt gctggcttgg ctgccgcctc 600

atgaacattc aggagcgttt cagagctatg ctggaaacag catagcaagt tgaaataagg 660

ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttttt ttgcaagaac 720

gaactaagcc ggacaaaaaa aaaaggagca catatacaaa ccggttttat tcatgaatgg 780

tcacgatgga tgatggggct cagacttgag ctacgaggcc gcaggcgaga gaagcctagt 840

gtgctctctg cttgtttggg ccgtaacgga ggatacggcc gacgagcgtg tactaccgcg 900

cgggatgccg ctgggcgctg cgggggccgt tggatgggga tcggtgggtc gcgggagcgt 960

tgaggggaga caggtttagt accacctcgc ctaccgaaca atgaagaacc caccttataa 1020

ccccgcgcgc tgccgcttgt gttgtagcac ccatgacaat gacagtttca gagctatgct 1080

ggaaacagca tagcaagttg aaataaggct agtccgttat caacttgaaa aagtggcacc 1140

gagtcggtgc tttttttttc tcattagcgg tatgcatgtt ggtagaagtc ggagatgtaa 1200

ataattttca ttatataaaa aaggtacttc gagaaaaata aatgcatacg aattaattct 1260

ttttatgttt tttaaaccaa gtatatagaa tttattgatg gttaaaattt caaaaatatg 1320

acgagagaaa ggttaaacgt acggcatata cttctgaaca gagagggaat atggggtttt 1380

tgttgctccc aacaattctt aagcacgtaa aggaaaaaag cacattatcc acattgtact 1440

tccagagata tgtacagcat tacgtaggta cgttttcttt ttcttcccgg agagatgata 1500

caataatcat gtaaacccag aatttaaaaa atattcttta ctataaaaat tttaattagg 1560

gaacgtatta ttttttacat gacacctttt gagaaagagg gacttgtaat atgggacaaa 1620

tgaacaattt ctaagaaatg ggcatatgac tctcagtaca atggaccaaa ttccctccag 1680

tcggcccagc aatacaaagg gaaagaaatg agggggccca caggccacgg cccacttttc 1740

tccgtggtgg ggagatccag ctagaggtcc ggcccacaag tggcccttgc cccgtgggac 1800

ggtgggattg cagagcgcgt gggcggaaac aacagtttag taccacctcg ctcacgcaac 1860

gacgcgacca cttgcttata agctgctgcg ctgaggctca gcaaggatcc cagccccgtg 1920

agtttcagag ctatgctgga aacagcatag caagttgaaa taaggctagt ccgttatcaa 1980

cttgaaaaag tggcaccgag tcggtgcttt ttttttagga atctttaaac atacgaacag 2040

atcacttaaa gttcttctga agcaacttaa agttatcagg catgcatgga tcttggagga 2100

atcagatgtg cagtcaggga ccatagcaca agacaggcgt cttctactgg tgctaccagc 2160

aaatgctgga agccgggaac actgggtacg ttggaaacca cgtgtgatgt gaaggagtaa 2220

gataaactgt aggagaaaag catttcgtag tgggccatga agcctttcag gacatgtatt 2280

gcagtatggg ccggcccatt acgcaattgg acgacaacaa agactagtat tagtaccacc 2340

tcggctatcc acatagatca aagctggttt aaaagagttg tgcagatgat ccgtggcctc 2400

atagcactca atgcggtgtt ttagagctag aaatagcaag ttaaaataag gctagtccgt 2460

tatcaacttg aaaaagtggc accgagtcgg tgcttttttt ttttaagctt acaaattcgg 2520

gtcaaggcgg aagccagcgc gccaccccac gtcagcaaat acggaggcgc ggggttgacg 2580

gcgtcacccg gtcctaacgg cgaccaacaa accagccaga agaaattaca gtaaaaaaaa 2640

agtaaattgc actttgatcc accttttatt acctaagtct caatttggat cacccttaaa 2700

cctatctttt caatttgggc cgggttgtgg tttggactac catgaacaac ttttcgtcat 2760

gtctaacttc cctttcagca aacatatgaa ccatatatag aggagatcgg ccgtatacta 2820

gagctgatgt gtttaaggtc gttgattgca cgagaaaaaa aaatccaaat cgcaacaata 2880

gcaaatttat ctggttcaaa gtgaaaagat atgtttaaag gtagtccaaa gtaaaactta 2940

tagataataa aatgtggtcc aaagcgtaat tcactcaaaa aaaatcaacg agacgtgtac 3000

caaacggaga caaacggcat cttctcgaaa tttcccaacc gctcgctcgc ccgcctcgtc 3060

ttcccggaaa ccgcggtggt ttcagcgtgg cggattctcc aagcagacgg agacgtcacg 3120

gcacgggact cctcccacca cccaaccgcc ataaatacca gccccctcat ctcctctcct 3180

cgcatcagct ccacccccga aaaatttctc cccaatctcg cgaggctctc gtcgtcgaat 3240

cgaatcctct cgcgtcctca aggtacgctg cttctcctct cctcgcttcg tttcgattcg 3300

atttcggacg ggtgaggttg ttttgttgct agatccgatt ggtggttagg gttgtcgatg 3360

tgattatcgt gagatgttta ggggttgtag atctgatggt tgtgatttgg gcacggttgg 3420

ttcgataggt ggaatcgtgg ttaggttttg ggattggatg ttggttctga tgattggggg 3480

gaatttttac ggttagatga attgttggat gattcgattg gggaaatcgg tgtagatctg 3540

ttggggaatt gtggaactag tcatgcctga gtgattggtg cgatttgtag cgtgttccat 3600

cttgtaggcc ttgttgcgag catgttcaga tctactgttc cgctcttgat tgagttattg 3660

gtgccatggg ttggtgcaaa cacaggcttt aatatgttat atctgttttg tgtttgatgt 3720

agatctgtag ggtagttctt cttagacatg gttcaattat gtagcttgtg cgtttcgatt 3780

tgatttcata tgttcacaga ttagataatg atgaactctt ttaattaatt gtcaatggta 3840

aataggaagt cttgtcgcta tatctgtcat aatgatctca tgttactatc tgccagtaat 3900

ttatgctaag aactatatta gaatatcatg ttacaatctg tagtaatatc atgttacaat 3960

ctgtagttca tctatataat ctattgtggt aatttctttt tactatctgt gtgaagatta 4020

ttgccactag ttcattctac ttatttctga agttcaggat acgtgtgctg ttactaccta 4080

tctgaataca tgtgtgatgt gcctgttact atctttttga atacatgtat gttctgttgg 4140

aatatgtttg ctgtttgatc cgttgttgtg tccttaatct tgtgctagtt cttaccctat 4200

ctgtttggtg attatttctt gcagtacgta agcatgtccg aggtggagtt ctcccacgag 4260

tactggatga ggcacgcact caccctcgca aagagggcat gggacgagag ggaggtgcct 4320

gtgggagcag tgctcgtgca caacaacagg gtgatcggag agggatggaa caggcctatc 4380

ggaaggcacg accctaccgc acacgcagag atcatggcac tcaggcaggg aggcctcgtg 4440

atgcagaact acaggctcat cgacgccacc ctctacgtga ccctcgagcc ttgcgtgatg 4500

tgcgcaggag ccatgatcca ctccaggatc ggaagggtgg tgttcggagc aagggacgca 4560

aagaccggag cagccggctc cctcatggac gtgctccacc acccgggcat gaaccacagg 4620

gtggagatca ccgagggaat cctcgcagac gagtgcgcag ccctcctctc cgacttcttc 4680

aggatgagga ggcaggagat caaggcccag aagaaggccc agtcctccac cgactccggc 4740

ggctcatcag gcggctcctc cggctccgag acaccgggca cctccgagtc cgccaccccg 4800

gagtcctccg gcggctcctc cggcggctcc tccgaggtgg agttctccca cgagtactgg 4860

atgaggcacg cactcaccct cgcaaagagg gcaagggacg agagggaggt gcctgtggga 4920

gcagtgctcg tgctcaacaa cagggtgatc ggagagggat ggaacagggc aatcggcctc 4980

cacgacccta ccgcacacgc agagatcatg gcactcaggc agggaggcct cgtgatgcag 5040

aactacaggc tcatcgacgc caccctctac gtgaccttcg agccttgcgt gatgtgcgca 5100

ggagccatga tccactccag gatcggcagg gtggtgttcg gcgtgaggaa cgcaaagacc 5160

ggagcagcag gctccctcat ggacgtgctc cactacccgg gcatgaacca cagggtggag 5220

atcaccgagg gaatcctcgc agacgagtgc gcagccctcc tctgctactt cttcaggatg 5280

ccgaggcagg tgttcaacgc ccagaagaag gcccagtcct ccaccgactc cggcggctca 5340

tcaggcggct cctccggctc cgagacaccg ggcacctccg agtccgccac cccggagtcc 5400

tccggcggct cctccggcgg ctccgacaag aagtactcca tcggcctcgc catcggcacc 5460

aacagcgtcg gctgggcggt gatcaccgac gagtacaagg tcccgtccaa gaagttcaag 5520

gtcctgggca acaccgaccg ccactccatc aagaagaacc tcatcggcgc cctcctcttc 5580

gactccggcg agacggcgga ggcgacccgc ctcaagcgca ccgcccgccg ccgctacacc 5640

cgccgcaaga accgcatctg ctacctccag gagatcttct ccaacgagat ggcgaaggtc 5700

gacgactcct tcttccaccg cctcgaggag tccttcctcg tggaggagga caagaagcac 5760

gagcgccacc ccatcttcgg caacatcgtc gacgaggtcg cctaccacga gaagtacccc 5820

actatctacc accttcgtaa gaagcttgtt gactctactg ataaggctga tcttcgtctc 5880

atctaccttg ctctcgctca catgatcaag ttccgtggtc acttccttat cgagggtgac 5940

cttaaccctg ataactccga cgtggacaag ctcttcatcc agctcgtcca gacctacaac 6000

cagctcttcg aggagaaccc tatcaacgct tccggtgtcg acgctaaggc gatcctttcc 6060

gctaggctct ccaagtccag gcgtctcgag aacctcatcg cccagctccc tggtgagaag 6120

aagaacggtc ttttcggtaa cctcatcgct ctctccctcg gtctgacccc taacttcaag 6180

tccaacttcg acctcgctga ggacgctaag cttcagctct ccaaggatac ctacgacgat 6240

gatctcgaca acctcctcgc tcagattgga gatcagtacg ctgatctctt ccttgctgct 6300

aagaacctct ccgatgctat cctcctttcg gatatcctta gggttaacac tgagatcact 6360

aaggctcctc tttctgcttc catgatcaag cgctacgacg agcaccacca ggacctcacc 6420

ctcctcaagg ctcttgttcg tcagcagctc cccgagaagt acaaggagat cttcttcgac 6480

cagtccaaga acggctacgc cggttacatt gacggtggag ctagccagga ggagttctac 6540

aagttcatca agccaatcct tgagaagatg gatggtactg aggagcttct cgttaagctt 6600

aaccgtgagg acctccttag gaagcagagg actttcgata acggctctat ccctcaccag 6660

atccaccttg gtgagcttca cgccatcctt cgtaggcagg aggacttcta ccctttcctc 6720

aaggacaacc gtgagaagat cgagaagatc cttactttcc gtattcctta ctacgttggt 6780

cctcttgctc gtggtaactc ccgtttcgct tggatgacta ggaagtccga ggagactatc 6840

accccttgga acttcgagga ggttgttgac aagggtgctt ccgcccagtc cttcatcgag 6900

cgcatgacca acttcgacaa gaacctcccc aacgagaagg tcctccccaa gcactccctc 6960

ctctacgagt acttcacggt ctacaacgag ctcaccaagg tcaagtacgt caccgagggt 7020

atgcgcaagc ctgccttcct ctccggcgag cagaagaagg ctatcgttga cctcctcttc 7080

aagaccaacc gcaaggtcac cgtcaagcag ctcaaggagg actacttcaa gaagatcgag 7140

tgcttcgact ccgtcgagat cagcggcgtt gaggaccgtt tcaacgcttc tctcggtacc 7200

taccacgatc tcctcaagat catcaaggac aaggacttcc tcgacaacga ggagaacgag 7260

gacatcctcg aggacatcgt cctcactctt actctcttcg aggataggga gatgatcgag 7320

gagaggctca agacttacgc tcatctcttc gatgacaagg ttatgaagca gctcaagcgt 7380

cgccgttaca ccggttgggg taggctctcc cgcaagctca tcaacggtat cagggataag 7440

cagagcggca agactatcct cgacttcctc aagtctgatg gtttcgctaa caggaacttc 7500

atgcagctca tccacgatga ctctcttacc ttcaaggagg atattcagaa ggctcaggtg 7560

tccggtcagg gcgactctct ccacgagcac attgctaacc ttgctggttc ccctgctatc 7620

aagaagggca tccttcagac tgttaaggtt gtcgatgagc ttgtcaaggt tatgggtcgt 7680

cacaagcctg agaacatcgt catcgagatg gctcgtgaga accagactac ccagaagggt 7740

cagaagaact cgagggagcg catgaagagg attgaggagg gtatcaagga gcttggttct 7800

cagatcctta aggagcaccc tgtcgagaac acccagctcc agaacgagaa gctctacctc 7860

tactacctcc agaacggtag ggatatgtac gttgaccagg agctcgacat caacaggctt 7920

tctgactacg acgtcgacca cattgttcct cagtctttcc ttaaggatga ctccatcgac 7980

aacaaggtcc tcacgaggtc cgacaagaac aggggtaagt cggacaacgt cccttccgag 8040

gaggttgtca agaagatgaa gaactactgg aggcagcttc tcaacgctaa gctcattacc 8100

cagaggaagt tcgacaacct cacgaaggct gagaggggtg gcctttccga gcttgacaag 8160

gctggtttca tcaagaggca gcttgttgag acgaggcaga ttaccaagca cgttgctcag 8220

atcctcgatt ctaggatgaa caccaagtac gacgagaacg acaagctcat ccgcgaggtc 8280

aaggtgatca ccctcaagtc caagctcgtc tccgacttcc gcaaggactt ccagttctac 8340

aaggtccgcg agatcaacaa ctaccaccac gctcacgatg cttaccttaa cgctgtcgtt 8400

ggtaccgctc ttatcaagaa gtaccctaag cttgagtccg agttcgtcta cggtgactac 8460

aaggtctacg acgttcgtaa gatgatcgcc aagtccgagc aggagatcgg caaggccacc 8520

gccaagtact tcttctactc caacatcatg aacttcttca agaccgagat caccctcgcc 8580

aacggcgaga tccgcaagcg ccctcttatc gagacgaacg gtgagactgg tgagatcgtt 8640

tgggacaagg gtcgcgactt cgctactgtt cgcaaggtcc tttctatgcc tcaggttaac 8700

atcgtcaaga agaccgaggt ccagaccggt ggcttctcca aggagtctat ccttccaaag 8760

agaaactcgg acaagctcat cgctaggaag aaggattggg accctaagaa gtacggtggt 8820

ttcgactccc ctactgtcgc ctactccgtc ctcgtggtcg ccaaggtgga gaagggtaag 8880

tcgaagaagc tcaagtccgt caaggagctc ctcggcatca ccatcatgga gcgctcctcc 8940

ttcgagaaga acccgatcga cttcctcgag gccaagggct acaaggaggt caagaaggac 9000

ctcatcatca agctccccaa gtactctctt ttcgagctcg agaacggtcg taagaggatg 9060

ctggcttccg ctggtgagct ccagaagggt aacgagcttg ctcttccttc caagtacgtg 9120

aacttcctct acctcgcctc ccactacgag aagctcaagg gttcccctga ggataacgag 9180

cagaagcagc tcttcgtgga gcagcacaag cactacctcg acgagatcat cgagcagatc 9240

tccgagttct ccaagcgcgt catcctcgct gacgctaacc tcgacaaggt cctctccgcc 9300

tacaacaagc accgcgacaa gcccatccgc gagcaggccg agaacatcat ccacctcttc 9360

acgctcacga acctcggcgc ccctgctgct ttcaagtact tcgacaccac catcgacagg 9420

aagcgttaca cgtccaccaa ggaggttctc gacgctactc tcatccacca gtccatcacc 9480

ggtctttacg agactcgtat cgacctttcc cagcttggtg gtgatgacga tgacaaaatg 9540

gcaccgaaga aaaaaaggaa ggtcggcggc tccccgaaga aaaaaaggaa ggtcggcggc 9600

tccccgaaga aaaaaaggaa ggtcggcggc tccccgaaga aaaaaaggaa ggtcggaatc 9660

catggcgttc catagactag ttcagccagt ttggtggagc tgccgatgtg cctggtcgtc 9720

ccgagcctct gttcgtcaag tatttgtggt gctgatgtct acttgtgtct ggtttaatgg 9780

accatcgagt ccgtatgata tgttagtttt atgaaacagt ttcctgtggg acagcagtat 9840

gctttatgaa taagttggat ttgaacctaa atatgtgctc aatttgctca tttgcatctc 9900

attcctgttg atgttttatc tgagttgcaa gtttgaaaat gctgcatatt cttattaaat 9960

cgtcatttac ttttatctta atgagctttg caatggccta tgggatataa aagagatcgt 10020

tcaaacattt ggcaataaag tttcttaaga ttgaatcctg ttgccggtct tgcgatgatt 10080

atcatataat ttctgttgaa ttacgttaag catgtaataa ttaacatgta atgcatgacg 10140

ttatttatga gatgggtttt tatgattaga gtcccgcaat tatacattta atacgcgata 10200

gaaaacaaaa tatagcgcgc aaactaggat aaattatcgc gcgcggtgtc atctatgtta 10260

ctagatcggc gcctgtccgg gcgcgcctgg tggatcgtcc gcctaggctg cagtgcagcg 10320

tgacccggtc gtgcccctct ctagagataa tgagcattgc atgtctaagt tataaaaaat 10380

taccacatat tttttttgtc acacttgttt gaagtgcagt ttatctatct ttatacatat 10440

atttaaactt tactctacga ataatataat ctatagtact acaataatat cagtgtttta 10500

gagaatcata taaatgaaca gttagacatg gtctaaagga caattgagta ttttgacaac 10560

aggactctac agttttatct ttttagtgtg catgtgttct cctttttttt tgcaaatagc 10620

ttcacctata taatacttca tccattttat tagtacatcc atttagggtt tagggttaat 10680

ggtttttata gactaatttt tttagtacat ctattttatt ctattttagc ctctaaatta 10740

agaaaactaa aactctattt tagttttttt atttaataat ttagatataa aatagaataa 10800

aataaagtga ctaaaaatta aacaaatacc ctttaagaaa ttaaaaaaac taaggaaaca 10860

tttttcttgt ttcgagtaga taatgccagc ctgttaaacg ccgtcgacga gtctaacgga 10920

caccaaccag cgaaccagca gcgtcgcgtc gggccaagcg aagcagacgg cacggcatct 10980

ctgtcgctgc ctctggaccc ctctcgagag ttccgctcca ccgttggact tgctccgctg 11040

tcggcatcca gaaattgcgt ggcggagcgg cagacgtgag ccggcacggc aggcggcctc 11100

ctcctcctct cacggcaccg gcagctacgg gggattcctt tcccaccgct ccttcgcttt 11160

cccttcctcg cccgccgtaa taaatagaca ccccctccac accctctttc cccaacctcg 11220

tgttgttcgg agcgcacaca cacacaacca gatctccccc aaatccaccc gtcggcacct 11280

ccgcttcaag gtacgccgct cgtcctcccc ccccccccct ctctaccttc tctagatcgg 11340

cgttccggtc catggttagg gcccggtagt tctacttctg ttcatgtttg tgttagatcc 11400

gtgtttgtgt tagatccgtg ctgctagcgt tcgtacacgg atgcgacctg tacgtcagac 11460

acgttctgat tgctaacttg ccagtgtttc tctttgggga atcctgggat ggctctagcc 11520

gttccgcaga cgggatcgat ttcatgattt tttttgtttc gttgcatagg gtttggtttg 11580

cccttttcct ttatttcaat atatgccgtg cacttgtttg tcgggtcatc ttttcatgct 11640

tttttttgtc ttggttgtga tgatgtggtc tggttgggcg gtcgttctag atcggagtag 11700

aattctgttt caaactacct ggtggattta ttaattttgg atctgtatgt gtgtgccata 11760

catattcata gttacgaatt gaagatgatg gatggaaata tcgatctagg ataggtatac 11820

atgttgatgc gggttttact gatgcatata cagagatgct ttttgttcgc ttggttgtga 11880

tgatgtggtg tggttgggcg gtcgttcatt cgttctagat cggagtagaa tactgtttca 11940

aactacctgg tgtatttatt aattttggaa ctgtatgtgt gtgtcataca tcttcatagt 12000

tacgagttta agatggatgg aaatatcgat ctaggatagg tatacatgtt gatgtgggtt 12060

ttactgatgc atatacatga tggcatatgc agcatctatt catatgctct aaccttgagt 12120

acctatctat tataataaac aagtatgttt tataattatt ttgatcttga tatacttgga 12180

tgatggcata tgcagcagct atatgtggat ttttttagcc ctgccttcat acgctattta 12240

tttgcttggt actgtttctt ttgtcgatgc tcaccctgtt gtttggtgtt acttctgcag 12300

gagctcctca tagcactcaa tgcggttggc aaaaagcctg aactcaccgc gacgtctgtc 12360

gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc tgatgcagct ctcggagggc 12420

gaagaatctc gtgctttcag cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat 12480

agctgcgccg atggtttcta caaagatcgt tatgtttatc ggcactttgc atcggccgcg 12540

ctcccgattc cggaagtgct tgacattggg gagtttagcg agagcctgac ctattgcatc 12600

tcccgccgtt cacagggtgt cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt 12660

ctacaaccgg tcgcggaggc tatggatgcg atcgctgcgg ccgatcttag ccagacgagc 12720

gggttcggcc cattcggacc gcaaggaatc ggtcaataca ctacatggcg tgatttcata 12780

tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg tgatggacga caccgtcagt 12840

gcgtccgtcg cgcaggctct cgatgagctg atgctttggg ccgaggactg ccccgaagtc 12900

cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa tggccgcata 12960

acagcggtca ttgactggag cgaggcgatg ttcggggatt cccaatacga ggtcgccaac 13020

atcttcttct ggaggccgtg gttggcttgt atggagcagc agacgcgcta cttcgagcgg 13080

aggcatccgg agcttgcagg atcgccacga ctccgggcgt atatgctccg cattggtctt 13140

gaccaactct atcagagctt ggttgacggc aatttcgatg atgcagcttg ggcgcagggt 13200

cgatgcgacg caatcgtccg atccggagcc gggactgtcg ggcgtacaca aatcgcccgc 13260

agaagcgcgg ccgtctggac cgatggctgt gtagaagtac tcgccgatag tggaaaccga 13320

cgccccagca ctcgtccgag ggcaaagaaa tagagtagat gccgaccggg atctgtcgat 13380

cgacaagctc gagtttctcc ataataatgt gtgagtagtt cccagataag ggaattaggg 13440

ttcctatagg gtttcgctca tgtgttgagc atataagaaa cccttagtat gtatttgtat 13500

ttgtaaaata cttctatcaa taaaatttct aattcctaaa accaaaatcc agtactaaaa 13560

tccagatccc ccgaattaat tcggcgttaa ttcagcctgc aggacgcgtt taattaagtg 13620

cacgcggccg cctacttagt caagagcctc gcacgcgact gtcacgcggc caggatcgcc 13680

tcgtgagcct cgcaatctgt acctagtgtt taaactatca gtgtttgaca ggatatattg 13740

gcgggtaaac ctaagagaaa agagcgttta ttagaataac ggatatttaa aagggcgtga 13800

aaaggtttat ccgttcgtcc atttgtatgt gcatgccaac cacagggttc ccctcgggat 13860

caaagtactt tgatccaacc cctccgctgc tatagtgcag tcggcttctg acgttcagtg 13920

cagccgtctt ctgaaaacga catgtcgcac aagtcctaag ttacgcgaca ggctgccgcc 13980

ctgccctttt cctggcgttt tcttgtcgcg tgttttagtc gcataaagta gaatacttgc 14040

gactagaacc ggagacatta cgccatgaac aagagcgccg ccgctggcct gctgggctat 14100

gcccgcgtca gcaccgacga ccaggacttg accaaccaac gggccgaact gcacgcggcc 14160

ggctgcacca agctgttttc cgagaagatc accggcacca ggcgcgaccg cccggagctg 14220

gccaggatgc ttgaccacct acgccctggc gacgttgtga cagtgaccag gctagaccgc 14280

ctggcccgca gcacccgcga cctactggac attgccgagc gcatccagga ggccggcgcg 14340

ggcctgcgta gcctggcaga gccgtgggcc gacaccacca cgccggccgg ccgcatggtg 14400

ttgaccgtgt tcgccggcat tgccgagttc gagcgttccc taatcatcga ccgcacccgg 14460

agcgggcgcg aggccgccaa ggcccgaggc gtgaagtttg gcccccgccc taccctcacc 14520

ccggcacaga tcgcgcacgc ccgcgagctg atcgaccagg aaggccgcac cgtgaaagag 14580

gcggctgcac tgcttggcgt gcatcgctcg accctgtacc gcgcacttga gcgcagcgag 14640

gaagtgacgc ccaccgaggc caggcggcgc ggtgccttcc gtgaggacgc attgaccgag 14700

gccgacgccc tggcggccgc cgagaatgaa cgccaagagg aacaagcatg aaaccgcacc 14760

aggacggcca ggacgaaccg tttttcatta ccgaagagat cgaggcggag atgatcgcgg 14820

ccgggtacgt gttcgagccg cccgcgcacg tctcaaccgt gcggctgcat gaaatcctgg 14880

ccggtttgtc tgatgccaag ctggcggcct ggccggccag cttggccgct gaagaaaccg 14940

agcgccgccg tctaaaaagg tgatgtgtat ttgagtaaaa cagcttgcgt catgcggtcg 15000

ctgcgtatat gatgcgatga gtaaataaac aaatacgcaa ggggaacgca tgaaggttat 15060

cgctgtactt aaccagaaag gcgggtcagg caagacgacc atcgcaaccc atctagcccg 15120

cgccctgcaa ctcgccgggg ccgatgttct gttagtcgat tccgatcccc agggcagtgc 15180

ccgcgattgg gcggccgtgc gggaagatca accgctaacc gttgtcggca tcgaccgccc 15240

gacgattgac cgcgacgtga aggccatcgg ccggcgcgac ttcgtagtga tcgacggagc 15300

gccccaggcg gcggacttgg ctgtgtccgc gatcaaggca gccgacttcg tgctgattcc 15360

ggtgcagcca agcccttacg acatatgggc caccgccgac ctggtggagc tggttaagca 15420

gcgcattgag gtcacggatg gaaggctaca agcggccttt gtcgtgtcgc gggcgatcaa 15480

aggcacgcgc atcggcggtg aggttgccga ggcgctggcc gggtacgagc tgcccattct 15540

tgagtcccgt atcacgcagc gcgtgagcta cccaggcact gccgccgccg gcacaaccgt 15600

tcttgaatca gaacccgagg gcgacgctgc ccgcgaggtc caggcgctgg ccgctgaaat 15660

taaatcaaaa ctcatttgag ttaatgaggt aaagagaaaa tgagcaaaag cacaaacacg 15720

ctaagtgccg gccgtccgag cgcacgcagc agcaaggctg caacgttggc cagcctggca 15780

gacacgccag ccatgaagcg ggtcaacttt cagttgccgg cggaggatca caccaagctg 15840

aagatgtacg cggtacgcca aggcaagacc attaccgagc tgctatctga atacatcgcg 15900

cagctaccag agtaaatgag caaatgaata aatgagtaga tgaattttag cggctaaagg 15960

aggcggcatg gaaaatcaag aacaaccagg caccgacgcc gtggaatgcc ccatgtgtgg 16020

aggaacgggc ggttggccag gcgtaagcgg ctgggttgtc tgccggccct gcaatggcac 16080

tggaaccccc aagcccgagg aatcggcgtg acggtcgcaa accatccggc ccggtacaaa 16140

tcggcgcggc gctgggtgat gacctggtgg agaagttgaa ggccgcgcag gccgcccagc 16200

ggcaacgcat cgaggcagaa gcacgccccg gtgaatcgtg gcaagcggcc gctgatcgaa 16260

tccgcaaaga atcccggcaa ccgccggcag ccggtgcgcc gtcgattagg aagccgccca 16320

agggcgacga gcaaccagat tttttcgttc cgatgctcta tgacgtgggc acccgcgata 16380

gtcgcagcat catggacgtg gccgttttcc gtctgtcgaa gcgtgaccga cgagctggcg 16440

aggtgatccg ctacgagctt ccagacgggc acgtagaggt ttccgcaggg ccggccggca 16500

tggccagtgt gtgggattac gacctggtac tgatggcggt ttcccatcta accgaatcca 16560

tgaaccgata ccgggaaggg aagggagaca agcccggccg cgtgttccgt ccacacgttg 16620

cggacgtact caagttctgc cggcgagccg atggcggaaa gcagaaagac gacctggtag 16680

aaacctgcat tcggttaaac accacgcacg ttgccatgca gcgtacgaag aaggccaaga 16740

acggccgcct ggtgacggta tccgagggtg aagccttgat tagccgctac aagatcgtaa 16800

agagcgaaac cgggcggccg gagtacatcg agatcgagct agctgattgg atgtaccgcg 16860

agatcacaga aggcaagaac ccggacgtgc tgacggttca ccccgattac tttttgatcg 16920

atcccggcat cggccgtttt ctctaccgcc tggcacgccg cgccgcaggc aaggcagaag 16980

ccagatggtt gttcaagacg atctacgaac gcagtggcag cgccggagag ttcaagaagt 17040

tctgtttcac cgtgcgcaag ctgatcgggt caaatgacct gccggagtac gatttgaagg 17100

aggaggcggg gcaggctggc ccgatcctag tcatgcgcta ccgcaacctg atcgagggcg 17160

aagcatccgc cggttcctaa tgtacggagc agatgctagg gcaaattgcc ctagcagggg 17220

aaaaaggtcg aaaaggtctc tttcctgtgg atagcacgta cattgggaac ccaaagccgt 17280

acattgggaa ccggaacccg tacattggga acccaaagcc gtacattggg aaccggtcac 17340

acatgtaagt gactgatata aaagagaaaa aaggcgattt ttccgcctaa aactctttaa 17400

aacttattaa aactcttaaa acccgcctgg cctgtgcata actgtctggc cagcgcacag 17460

ccgaagagct gcaaaaagcg cctacccttc ggtcgctgcg ctccctacgc cccgccgctt 17520

cgcgtcggcc tatcgcggcc gctggccgct caaaaatggc tggcctacgg ccaggcaatc 17580

taccagggcg cggacaagcc gcgccgtcgc cactcgaccg ccggcgccca catcaaggca 17640

ccctgcctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag 17700

acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca 17760

gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg 17820

tatactggct taactatgcg gcatcagagc agattgtact gagagtgcac catatgcggt 17880

gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgctct tccgcttcct 17940

cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa 18000

aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa 18060

aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc 18120

tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 18180

caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 18240

cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 18300

ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 18360

gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 18420

agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 18480

gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 18540

acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 18600

gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt 18660

gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 18720

cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgcattcta 18780

ggtactaaaa caattcatcc agtaaaatat aatattttat tttctcccaa tcaggcttga 18840

tccccagtaa gtcaaaaaat agctcgacat actgttcttc cccgatatcc tccctgatcg 18900

accggacgca gaaggcaatg tcataccact tgtccgccct gccgcttctc ccaagatcaa 18960

taaagccact tactttgcca tctttcacaa agatgttgct gtctcccagg tcgccgtggg 19020

aaaagacaag ttcctcttcg ggcttttccg tctttaaaaa atcatacagc tcgcgcggat 19080

ctttaaatgg agtgtcttct tcccagtttt cgcaatccac atcggccaga tcgttattca 19140

gtaagtaatc caattcggct aagcggctgt ctaagctatt cgtataggga caatccgata 19200

tgtcgatgga gtgaaagagc ctgatgcact ccgcatacag ctcgataatc ttttcagggc 19260

tttgttcatc ttcatactct tccgagcaaa ggacgccatc ggcctcactc atgagcagat 19320

tgctccagcc atcatgccgt tcaaagtgca ggacctttgg aacaggcagc tttccttcca 19380

gccatagcat catgtccttt tcccgttcca catcataggt ggtcccttta taccggctgt 19440

ccgtcatttt taaatatagg ttttcatttt ctcccaccag cttatatacc ttagcaggag 19500

acattccttc cgtatctttt acgcagcggt atttttcgat cagttttttc aattccggtg 19560

atattctcat tttagccatt tattatttcc ttcctctttt ctacagtatt taaagatacc 19620

ccaagaagct aattataaca agacgaactc caattcactg ttccttgcat tctaaaacct 19680

taaataccag aaaacagctt tttcaaagtt gttttcaaag ttggcgtata acatagtatc 19740

gacggagccg attttgaaac cgcggtgatc acaggcagca acgctctgtc atcgttacaa 19800

tcaacatgct accctccgcg agatcatccg tgtttcaaac ccggcagctt agttgccgtt 19860

cttccgaata gcatcggtaa catgagcaaa gtctgccgcc ttacaacggc tctcccgctg 19920

acgccgtccc ggactgatgg gctgcctgta tcgagtggtg attttgtgcc gagctgccgg 19980

tcggggagct gttggctggc t 20001

<210>2

<211>167

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>2

Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu

1 5 10 15

Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala

20 25 30

Val Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro

35 40 45

Ile Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg

50 55 60

Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu

65 70 75 80

Tyr Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His

85 90 95

Ser Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly

100 105 110

Ala Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His

115 120 125

Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu

130 135 140

Leu Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys

145 150 155 160

Lys Ala Gln Ser Ser Thr Asp

165

<210>3

<211>1367

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>3

Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val Gly

1 5 10 15

Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys

20 25 30

Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly

35 40 45

Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys

50 55 60

Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr

65 70 75 80

Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe

85 90 95

Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His

100 105 110

Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His

115 120 125

Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser

130 135 140

Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met

145 150 155 160

Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp

165 170 175

Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn

180 185 190

Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys

195 200 205

Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu

210 215 220

Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu

225 230 235 240

Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp

245 250 255

Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp

260 265 270

Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu

275 280 285

Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile

290 295 300

Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met

305 310 315 320

Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala

325 330 335

Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp

340 345 350

Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln

355 360 365

Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly

370 375 380

Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys

385 390 395 400

Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly

405 410 415

Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu

420 425 430

Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro

435 440 445

Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met

450 455 460

Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val

465 470 475 480

Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn

485 490 495

Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu

500 505 510

Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr

515 520 525

Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys

530 535 540

Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val

545 550 555 560

Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser

565 570 575

Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr

580 585 590

Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn

595 600 605

Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu

610 615 620

Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His

625 630 635 640

Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr

645 650 655

Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys

660 665 670

Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala

675 680 685

Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys

690 695 700

Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His

705 710 715 720

Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile

725 730 735

Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg

740 745 750

His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr

755 760 765

Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu

770 775 780

Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val

785 790 795 800

Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln

805 810 815

Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu

820 825 830

Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp

835 840 845

Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly

850 855 860

Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn

865 870 875 880

Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe

885 890 895

Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys

900 905 910

Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys

915 920 925

His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu

930 935 940

Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys

945 950 955 960

Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu

965 970 975

Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val

980 985 990

Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val

995 1000 1005

Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys

1010 1015 1020

Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr

1025 1030 1035

Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn

1040 1045 1050

Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr

1055 1060 1065

Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg

1070 1075 1080

Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu

1085 1090 1095

Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg

1100 1105 1110

Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys

1115 1120 1125

Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu

1130 1135 1140

Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser

1145 1150 1155

Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe

1160 1165 1170

Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu

1175 1180 1185

Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe

1190 1195 1200

Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu

1205 1210 1215

Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn

1220 1225 1230

Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro

1235 1240 1245

Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His

1250 1255 1260

Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg

1265 1270 1275

Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr

1280 1285 1290

Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile

1295 1300 1305

Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe

1310 1315 1320

Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr

1325 1330 1335

Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly

1340 1345 1350

Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp

1355 1360 1365

<210>4

<211>1026

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

atgaaaaagc ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac 60

agcgtctccg acctgatgca gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat 120

gtaggagggc gtggatatgt cctgcgggta aatagctgcg ccgatggttt ctacaaagat 180

cgttatgttt atcggcactt tgcatcggcc gcgctcccga ttccggaagt gcttgacatt 240

ggggagttta gcgagagcct gacctattgc atctcccgcc gttcacaggg tgtcacgttg 300

caagacctgc ctgaaaccga actgcccgct gttctacaac cggtcgcgga ggctatggat 360

gcgatcgctg cggccgatct tagccagacg agcgggttcg gcccattcgg accgcaagga 420

atcggtcaat acactacatg gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat 480

cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag 540

ctgatgcttt gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc 600

tccaacaatg tcctgacgga caatggccgc ataacagcgg tcattgactg gagcgaggcg 660

atgttcgggg attcccaata cgaggtcgcc aacatcttct tctggaggcc gtggttggct 720

tgtatggagc agcagacgcg ctacttcgag cggaggcatc cggagcttgc aggatcgcca 780

cgactccggg cgtatatgct ccgcattggt cttgaccaac tctatcagag cttggttgac 840

ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga 900

gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc 960

tgtgtagaag tactcgccga tagtggaaac cgacgcccca gcactcgtcc gagggcaaag 1020

aaatag 1026

<210>5

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

ctcatagcac tcaatgcggt tgg 23

Claims

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

the sgRNA framework is an RNA molecule obtained by replacing T in the 2418 th 2493 th site of the sequence 1 with U;

the esgRNA framework is an RNA molecule obtained by replacing T in the 617-702 th site of the sequence 1 with U;

the biological material related to the Cas9 nuclease is a nucleic acid molecule encoding the Cas9 nuclease or an expression cassette, a recombinant vector, a recombinant microorganism, a transgenic cell line containing the nucleic acid molecule;

the biological material related to the adenine deaminase is a nucleic acid molecule encoding the adenine deaminase or an expression cassette, a recombinant vector, a recombinant microorganism and a transgenic cell line containing the nucleic acid molecule;

the resistance gene of the screening agent is an exogenous resistance gene;

the biological material related to the screening agent resistance gene with the loss of function is a nucleic acid molecule encoding the screening agent resistance gene with the loss of function or an expression cassette, a recombinant vector, a recombinant microorganism and a transgenic cell line containing the nucleic acid molecule;

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 screening 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 screening agent resistance gene target sequence with the function lost;

the surrogate target sequence is sequence 5.

2. The kit of claim 1, wherein: the screening agent resistance gene is a hygromycin resistance gene.

3. The kit of claim 1, wherein: the Cas9 nuclease is SpCas9n protein;

the adenine deaminase is an ecTadA protein;

the SpCas9n protein is a protein shown as a sequence 3;

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);

the ecTadA protein is a protein shown in sequence 2;

F1) a nucleic acid molecule encoding said ecTadA protein;

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

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

4. Use of the kit of any one of claims 1 to 3 in any one of M1) -M6):

5, 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 1-3, 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 1-3, 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.

6. The use according to claim 4 or the method according to claim 5, characterized in that: the organism is a plant or an animal; the biological cell is a plant cell or an animal cell.

7. The use or method according to claim 6, wherein: the plant is a monocotyledon or a dicotyledon; the plant cell is a monocotyledon cell or a dicotyledon cell.

8. The use or method according to claim 7, wherein: the monocotyledon is a gramineous plant; the monocotyledon cell is a gramineae plant cell.

9. The use or method according to claim 8, wherein: the gramineous plant is rice; the gramineous plant cell is a rice cell.