CN110628795A - Cell enrichment technology using inactivated screening agent resistance gene as report system for A.G base substitution and application thereof - Google Patents

Cell enrichment technology using inactivated screening agent resistance gene as report system for A.G base substitution and application thereof Download PDF

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CN110628795A
CN110628795A CN201910938696.5A CN201910938696A CN110628795A CN 110628795 A CN110628795 A CN 110628795A CN 201910938696 A CN201910938696 A CN 201910938696A CN 110628795 A CN110628795 A CN 110628795A
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resistance gene
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杨永星
杨进孝
康桂婷
王飞鹏
宋金岭
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Beijing Academy of Agriculture and Forestry Sciences
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Abstract

The invention discloses a cell enrichment technology of A.G base substitution by taking an inactivated screening agent resistance gene as a report system and application thereof. The cell enrichment technology carrier comprises the following reagents: a sgRNA of a target gene target sequence, a sgRNA of a screening agent resistance gene target sequence with lost target function, an A.G base substitution system and a screening agent resistance gene with lost function; the A.G base substitution system can restore the function of a selection agent resistance gene with a loss of function by carrying out A.G base substitution on a selection agent resistance gene target sequence with a loss of function under the guidance of sgRNA targeting the selection agent resistance gene target sequence with a 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

Cell enrichment technology using inactivated screening agent resistance gene as report system for A.G base substitution and application thereof
Technical Field
The invention relates to the field of biotechnology, in particular to a cell enrichment technology of A.G base substitution by taking an inactivated screening agent resistance gene as a report system and application thereof.
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 following repair process, so that A-G conversion is realized.
At present, researches for enriching A.G base-substituted cells in plants by reporter gene-mediated cell enrichment technology are very limited, and at present, reports for enriching A.G base-substituted cells at the cellular level and further improving A.G base substitution efficiency by using a selection marker in the transformation process are not available.
Disclosure of Invention
The invention aims to provide a cell enrichment technology for A.G base substitution by taking an inactivated screening agent resistance gene as a reporter system, which can realize the enrichment of A.G base substituted cells on the cell level and further improve the A.G base substitution efficiency of a target spot.
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 sgRNA targeting a target gene target sequence and sgRNA targeting the loss-of-function screening agent resistance gene target sequence;
the sgRNA structure is as follows: an RNA-sgRNA backbone transcribed from the 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 617-692 position 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.
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 (denoted as a surrogate target sequence) is additionally added to the sequence of the selection agent resistance gene whose function is lost in order that the gene can recover its function after the substitution of A.G bases, the sequence of the selection agent resistance gene whose function is lost includes not only the selection agent resistance gene itself whose function is lost 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 can satisfy 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 target sequence of the screening agent resistance gene with the loss of function is 1 st-20 th site 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 of the target sequence of the screening agent resistance gene with the target of losing functions, 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 for initiating the transcription of the ecTadA gene but also a terminator for terminating the transcription of the ecTadA gene. Further, the expression cassette may also include an enhancer sequence. Furthermore, 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 particular embodiment of the invention, the recombinant vector is in particular a sgRNA-ATG-Hyg-ATG/sgRNA-GT-1 recombinant expression vector and sgRNA-ATG-Hyg-ATG/sgRNA-GT-2 recombinant expression vector or sgRNA-ATG-Hyg-ATGThe sgRNA-GT-3 recombinant expression vector.
The sgRNA-ATG-Hyg-ATGThe sequence of the/sgRNA-GT-1 recombinant expression vector is sequence 1. The sgRNA-ATG-Hyg-ATGthe/sgRNA-GT-1 recombinant expression vector contains four target sequences, and the sequences are shown in a table 1.
The sgRNA-ATG-Hyg-ATGThe sequence of the/sgRNA-GT-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 sgRNA-ATG-Hyg-ATGThe sequence of the/sgRNA-GT-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 invention, the recombinant microorganism specifically comprises the sgRNA-ATG-Hyg-ATG/sgRNA-GT-1 recombinant expression vector or sgRNA-ATG-Hyg-ATG/sgRNA-GT-2 recombinant expression vector or sgRNA-ATG-Hyg-ATGThe agrobacterium EHA105 of the sgRNA-GT-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 above-mentioned non-functional selection agent resistance gene or biological material related to the non-functional selection agent resistance gene also falls within the scope of the present invention.
In order to achieve the above object, the present invention also provides a novel use of the above-mentioned kit or the above-mentioned loss-of-function screening agent resistance gene or a biological material related to the loss-of-function screening agent resistance gene.
The present invention provides the use of the above kit or the above loss-of-function screener resistance gene or a biological material related to the loss-of-function screener resistance gene 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.
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 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 the coding gene of the Cas9 nuclease, the DNA molecule of the sgRNA of the transcription target gene target sequence of the loss-of-function screening agent resistance gene, the coding gene of the adenine deaminase and the loss-of-function screening agent resistance gene 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 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 the Cas9 nuclease, sgRNA 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 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.
In the above method, the gene encoding Cas9 nuclease, the DNA molecule of sgRNA 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 sgRNA 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 biological cell by the same recombinant expression vector, or may be introduced into an organism or a biological cell by two or more recombinant expression vectors together.
In a specific embodiment of the 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 adenine deaminase coding genes. The recombinant expression vector is specifically the sgRNA-ATG-Hyg-ATG/sgRNA-GT-1 recombinant expression vector or sgRNA described above-ATG-Hyg-ATG/sgRNA-GT-2 recombinant expression vector or sgRNA described above-ATG-Hyg-ATGThe sgRNA-GT-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 cell enrichment technology principle of the invention is as follows: a cell enrichment technique using the inactivated screening agent resistance gene as a reporter gene to replace A.G bases is established, so that cells with A.G bases replaced on the reporter gene can grow in a medium containing the screening agent, and cells without A.G bases replaced can not grow in the 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 carrying out A.G base replacement on the endogenous target gene target spot, so that enrichment of the cells carrying out A.G base replacement on 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 cell enrichment technology realizes the enrichment of A.G base replacement cells on the cellular level for different deaminase mediated base editors or different Cas9 enzyme mediated base editors, and greatly improves the A.G base replacement efficiency.
Drawings
FIG. 1 is a schematic diagram of the structure of a cell enrichment technology carrier and a non-cell enrichment technology carrier.
FIG. 2 is a schematic diagram of the operation principle of enriching cells with A.G base substitutions by cell enrichment technology.
FIG. 3 is a schematic structural diagram of a recombinant vector.
FIG. 4 is a comparison of the efficiency of A.G base substitution on target in the healing of rice resistance by cell enrichment and non-cell enrichment.
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 rice seedlings and the expression of 5 hormone marker genes [ J ]. proceedings of university of Master Henan (Nature edition), 2017(2): 48-52.; the public is available from the agroforestry academy of sciences of Beijing.
Recovering the culture medium: n6 solid medium containing 200mg/L timentin.
Screening a culture medium: n6 solid medium containing 50mg/L hygromycin.
Example 1 establishment of EcTadA & ecTadA & Cas9 n-mediated cell enrichment technique for A.G base substitutions
Establishment of EcTadA & ecTadA & Cas9 n-mediated cell enrichment technology vector with A.G base substitution
The common technology (non-cell enrichment technology) vector for EcTadA & ecTadA & Cas9n (ABE) -mediated A.G base replacement was named sgRNA-GT.
Mixing EcTadA&ecTadA&Cas9n (ABE) mediated cell enrichment technology vector for A.G base replacement is named sgRNA-ATG-Hyg-ATG/sgRNA-GT。
sgRNA-GT and sgRNA-ATG-Hyg-ATGThe structure schematic diagram of the/sgRNA-GT vector is shown in FIG. 1.
The cell enrichment technology vector is obtained by modifying a screening agent resistance gene on the basis of a non-cell enrichment technology vector to lose the function of the screening agent resistance gene and adding a corresponding proxy target point to the sgRNA part.
Taking the screening agent resistance gene as Hygromycin resistance gene Hygromycin as an example: the screening agent resistance gene in the carrier of the non-cell enrichment technology is the complete Hygromycin resistance gene Hygromycin. The screening agent resistance gene in the cell enrichment technical carrier is a Hygromycin resistance gene Hygromycin (Hygromycin) with lost 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 (containing PAM) at the 5' end. Wherein, the sequence of the proxy target point is as follows: ctcatagcactcaatgcggtTGG (capital letters are PAM sequences).
Second, the operational principle of the EcTadA & ecTadA & Cas9n mediated cell enrichment technology of A.G base substitution
The operation principle of the cell enrichment technique by A.G base substitution is shown in FIG. 2. Taking the screening agent resistance gene as Hygromycin resistance gene Hygromycin as an example: in the cell enrichment 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 cell enrichment technology mutates A6 in a surrogate 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 and the resistance function is recovered, so that 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 cell enrichment 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: sgRNA-ATG-Hyg-ATGA sgRNA-GT recombinant expression vector and a sgRNA-GT recombinant expression vector. Schematic diagrams of two recombinant expression vectors are shown in FIG. 3. 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: sgRNA-ATG-Hyg-ATG/sgRNA-GT-1 recombinant expression vector and sgRNA-ATG-Hyg-ATG/sgRNA-GT-2 recombinant expression vector and sgRNA-ATG-Hyg-ATGThe expression vector comprises a/sgRNA-GT-3 recombinant expression vector, a sgRNA-GT-1 recombinant expression vector, a sgRNA-GT-2 recombinant expression vector and a 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:
sgRNA-ATG-Hyg-ATGthe sequence of the/sgRNA-GT-1 recombinant expression vector is sequence 1 in a sequence table. The nucleotide sequence of OsU6a promoter at the 131-596 position, the nucleotide sequence of OsU6b promoter at the 702-1034 position, the nucleotide sequence of OsU6c promoter at the 1140-1881 position and the nucleotide sequence of OsU3 promoter at the 1987-2367 position of the sequence 1; the 597-position 616, the 1035-position 1054 and the 1882-position 1901 are ALS-T4, CDC48-T3 and AAT target sequences respectively, and the 2368-position 2387 is a reporter gene surrogate target sequence; the nucleotide sequences of sgRNA are 617-692, 1055-1130, 1902-1977 and 2388-2463. The 2482-4195 th site of the sequence 1 is the nucleotide sequence of the OsUbq3 promoter, and the 4205-4705 th site and the 4802-5299 th site are the ecTadAThe coding sequences of (1), all coding sequences encode the ecTadA protein shown in sequence 2; no. 5396-9496 of the sequence 1 is a coding sequence of SpCas9n protein, and the SpCas9n protein shown in the coding sequence 3; the 9653-9985 th site of the sequence 1 is a 3' UTR sequence of OsUbq 3; the nucleotide sequence of Nos terminator at positions 9986-10237 of the sequence 1. The 10279-12271 site of the sequence 1 is the nucleotide sequence of ZmUbi1 promoter, the 12278-12300 site is the target sequence of surrogate target, the 12302-13324 site is the nucleotide sequence of hygromycin phosphotransferase with the initiation codon removed, and the 13351-13566 site is the nucleotide sequence of CaMV35S terminator. sgRNA-ATG-Hyg-ATGFour target sequences in the/sgRNA-GT-1 recombinant expression vector are shown in Table 1, and the targets are ALS-T4, CDC48-T3, AAT and ST1152 surrogate targets respectively.
sgRNA-ATG-Hyg-ATGThe sequence of the/sgRNA-GT-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.
sgRNA-ATG-Hyg-ATGThe sequence of the/sgRNA-GT-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 sequence of the sgRNA-GT-1 recombinant expression vector is obtained by replacing the 12278-position 13324 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 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 sgRNA-GT-3 recombinant expression vector is that the first three target sequences of the sgRNA-GT-1 recombinant expression vector 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 target nucleotide sequence of sgRNA and the corresponding PAM sequence of each vector are shown in table 1.
TABLE 1
II, obtaining the rice positive resistance callus
The sgRNA obtained in the step one-ATG-Hyg-ATG/sgRNA-GT-1 vector, sgRNA-ATG-Hyg-ATG/sgRNA-GT-2 vector, sgRNA-ATG-Hyg-ATGThe method comprises the following steps of 1-8 respectively operating an sgRNA-GT-3 vector, an sgRNA-GT-1 vector, an sgRNA-GT-2 vector and an sgRNA-GT-3 vector:
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 by sterile water for 3 times, sucked by filter paper to remove water, then placed on an N6 solid culture medium with the embryo of the seeds facing downwards, and cultured in dark at 28 ℃ for 4-6 weeks to obtain the callus of the rice.
4. After the step 3 is completed, soaking the rice callus 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), soaking for 10min, then placing the rice callus on a culture dish (containing about 200ml of the agrobacterium-free infection solution) paved with two layers of sterilization filter paper, and performing dark culture 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 the 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, carrying out PCR amplification on T3 by adopting a primer pair 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 4.
The results show that: by using a cell enrichment technology, the A.G base replacement efficiency of the ALS-T4 target point is increased from 34% to 98% in rice resistance healing; the A.G base replacement efficiency of the CDC48-T3 target point is increased from 36% to 91%; the A.G base replacement efficiency of the AAT target point is increased from 23 percent to 70 percent; the A.G base replacement efficiency of the NRT1.1B-T4 target point is increased from 0% to 16%; the A.G base substitution efficiency of the SPL14 target increased from 20% to 95%; the A.G base replacement efficiency of the WRKY45 target point is increased from 93% to 100%; the A.G base substitution efficiency of DELLA targets increased from 32% to 100%. In conclusion, the A.G base replacement efficiency of most targets is improved to 2.5-3 times of that of the common technology by using the cell enrichment 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> cell enrichment technique by A.G base substitution using inactivated screening agent resistance gene as reporter system and use thereof
<160>5
<170>PatentIn version 3.5
<210>1
<211>19972
<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 tagagctaga aatagcaagt taaaataagg ctagtccgtt 660
atcaacttga aaaagtggca ccgagtcggt gctttttttt ttgcaagaac gaactaagcc 720
ggacaaaaaa aaaaggagca catatacaaa ccggttttat tcatgaatgg tcacgatgga 780
tgatggggct cagacttgag ctacgaggcc gcaggcgaga gaagcctagt gtgctctctg 840
cttgtttggg ccgtaacgga ggatacggcc gacgagcgtg tactaccgcg cgggatgccg 900
ctgggcgctg cgggggccgt tggatgggga tcggtgggtc gcgggagcgt tgaggggaga 960
caggtttagt accacctcgc ctaccgaaca atgaagaacc caccttataa ccccgcgcgc 1020
tgccgcttgt gttgtagcac ccatgacaat gacagtttta gagctagaaa tagcaagtta 1080
aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc tttttttttc 1140
tcattagcgg tatgcatgtt ggtagaagtc ggagatgtaa ataattttca ttatataaaa 1200
aaggtacttc gagaaaaata aatgcatacg aattaattct ttttatgttt tttaaaccaa 1260
gtatatagaa tttattgatg gttaaaattt caaaaatatg acgagagaaa ggttaaacgt 1320
acggcatata cttctgaaca gagagggaat atggggtttt tgttgctccc aacaattctt 1380
aagcacgtaa aggaaaaaag cacattatcc acattgtact tccagagata tgtacagcat 1440
tacgtaggta cgttttcttt ttcttcccgg agagatgata caataatcat gtaaacccag 1500
aatttaaaaa atattcttta ctataaaaat tttaattagg gaacgtatta ttttttacat 1560
gacacctttt gagaaagagg gacttgtaat atgggacaaa tgaacaattt ctaagaaatg 1620
ggcatatgac tctcagtaca atggaccaaa ttccctccag tcggcccagc aatacaaagg 1680
gaaagaaatg agggggccca caggccacgg cccacttttc tccgtggtgg ggagatccag 1740
ctagaggtcc ggcccacaag tggcccttgc cccgtgggac ggtgggattg cagagcgcgt 1800
gggcggaaac aacagtttag taccacctcg ctcacgcaac gacgcgacca cttgcttata 1860
agctgctgcg ctgaggctca gcaaggatcc cagccccgtg agttttagag ctagaaatag 1920
caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt 1980
ttttttagga atctttaaac atacgaacag atcacttaaa gttcttctga agcaacttaa 2040
agttatcagg catgcatgga tcttggagga atcagatgtg cagtcaggga ccatagcaca 2100
agacaggcgt cttctactgg tgctaccagc aaatgctgga agccgggaac actgggtacg 2160
ttggaaacca cgtgtgatgt gaaggagtaa gataaactgt aggagaaaag catttcgtag 2220
tgggccatga agcctttcag gacatgtatt gcagtatggg ccggcccatt acgcaattgg 2280
acgacaacaa agactagtat tagtaccacc tcggctatcc acatagatca aagctggttt 2340
aaaagagttg tgcagatgat ccgtggcctc atagcactca atgcggtgtt ttagagctag 2400
aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg 2460
tgcttttttt tttttaagct tacaaattcg ggtcaaggcg gaagccagcg cgccacccca 2520
cgtcagcaaa tacggaggcg cggggttgac ggcgtcaccc ggtcctaacg gcgaccaaca 2580
aaccagccag aagaaattac agtaaaaaaa aagtaaattg cactttgatc caccttttat 2640
tacctaagtc tcaatttgga tcacccttaa acctatcttt tcaatttggg ccgggttgtg 2700
gtttggacta ccatgaacaa cttttcgtca tgtctaactt ccctttcagc aaacatatga 2760
accatatata gaggagatcg gccgtatact agagctgatg tgtttaaggt cgttgattgc 2820
acgagaaaaa aaaatccaaa tcgcaacaat agcaaattta tctggttcaa agtgaaaaga 2880
tatgtttaaa ggtagtccaa agtaaaactt atagataata aaatgtggtc caaagcgtaa 2940
ttcactcaaa aaaaatcaac gagacgtgta ccaaacggag acaaacggca tcttctcgaa 3000
atttcccaac cgctcgctcg cccgcctcgt cttcccggaa accgcggtgg tttcagcgtg 3060
gcggattctc caagcagacg gagacgtcac ggcacgggac tcctcccacc acccaaccgc 3120
cataaatacc agccccctca tctcctctcc tcgcatcagc tccacccccg aaaaatttct 3180
ccccaatctc gcgaggctct cgtcgtcgaa tcgaatcctc tcgcgtcctc aaggtacgct 3240
gcttctcctc tcctcgcttc gtttcgattc gatttcggac gggtgaggtt gttttgttgc 3300
tagatccgat tggtggttag ggttgtcgat gtgattatcg tgagatgttt aggggttgta 3360
gatctgatgg ttgtgatttg ggcacggttg gttcgatagg tggaatcgtg gttaggtttt 3420
gggattggat gttggttctg atgattgggg ggaattttta cggttagatg aattgttgga 3480
tgattcgatt ggggaaatcg gtgtagatct gttggggaat tgtggaacta gtcatgcctg 3540
agtgattggt gcgatttgta gcgtgttcca tcttgtaggc cttgttgcga gcatgttcag 3600
atctactgtt ccgctcttga ttgagttatt ggtgccatgg gttggtgcaa acacaggctt 3660
taatatgtta tatctgtttt gtgtttgatg tagatctgta gggtagttct tcttagacat 3720
ggttcaatta tgtagcttgt gcgtttcgat ttgatttcat atgttcacag attagataat 3780
gatgaactct tttaattaat tgtcaatggt aaataggaag tcttgtcgct atatctgtca 3840
taatgatctc atgttactat ctgccagtaa tttatgctaa gaactatatt agaatatcat 3900
gttacaatct gtagtaatat catgttacaa tctgtagttc atctatataa tctattgtgg 3960
taatttcttt ttactatctg tgtgaagatt attgccacta gttcattcta cttatttctg 4020
aagttcagga tacgtgtgct gttactacct atctgaatac atgtgtgatg tgcctgttac 4080
tatctttttg aatacatgta tgttctgttg gaatatgttt gctgtttgat ccgttgttgt 4140
gtccttaatc ttgtgctagt tcttacccta tctgtttggt gattatttct tgcagtacgt 4200
aagcatgtcc gaggtggagt tctcccacga gtactggatg aggcacgcac tcaccctcgc 4260
aaagagggca tgggacgaga gggaggtgcc tgtgggagca gtgctcgtgc acaacaacag 4320
ggtgatcgga gagggatgga acaggcctat cggaaggcac gaccctaccg cacacgcaga 4380
gatcatggca ctcaggcagg gaggcctcgt gatgcagaac tacaggctca tcgacgccac 4440
cctctacgtg accctcgagc cttgcgtgat gtgcgcagga gccatgatcc actccaggat 4500
cggaagggtg gtgttcggag caagggacgc aaagaccgga gcagccggct ccctcatgga 4560
cgtgctccac cacccgggca tgaaccacag ggtggagatc accgagggaa tcctcgcaga 4620
cgagtgcgca gccctcctct ccgacttctt caggatgagg aggcaggaga tcaaggccca 4680
gaagaaggcc cagtcctcca ccgactccgg cggctcatca ggcggctcct ccggctccga 4740
gacaccgggc acctccgagt ccgccacccc ggagtcctcc ggcggctcct ccggcggctc 4800
ctccgaggtg gagttctccc acgagtactg gatgaggcac gcactcaccc tcgcaaagag 4860
ggcaagggac gagagggagg tgcctgtggg agcagtgctc gtgctcaaca acagggtgat 4920
cggagaggga tggaacaggg caatcggcct ccacgaccct accgcacacg cagagatcat 4980
ggcactcagg cagggaggcc tcgtgatgca gaactacagg ctcatcgacg ccaccctcta 5040
cgtgaccttc gagccttgcg tgatgtgcgc aggagccatg atccactcca ggatcggcag 5100
ggtggtgttc ggcgtgagga acgcaaagac cggagcagca ggctccctca tggacgtgct 5160
ccactacccg ggcatgaacc acagggtgga gatcaccgag ggaatcctcg cagacgagtg 5220
cgcagccctc ctctgctact tcttcaggat gccgaggcag gtgttcaacg cccagaagaa 5280
ggcccagtcc tccaccgact ccggcggctc atcaggcggc tcctccggct ccgagacacc 5340
gggcacctcc gagtccgcca ccccggagtc ctccggcggc tcctccggcg gctccgacaa 5400
gaagtactcc atcggcctcg ccatcggcac caacagcgtc ggctgggcgg tgatcaccga 5460
cgagtacaag gtcccgtcca agaagttcaa ggtcctgggc aacaccgacc gccactccat 5520
caagaagaac ctcatcggcg ccctcctctt cgactccggc gagacggcgg aggcgacccg 5580
cctcaagcgc accgcccgcc gccgctacac ccgccgcaag aaccgcatct gctacctcca 5640
ggagatcttc tccaacgaga tggcgaaggt cgacgactcc ttcttccacc gcctcgagga 5700
gtccttcctc gtggaggagg acaagaagca cgagcgccac cccatcttcg gcaacatcgt 5760
cgacgaggtc gcctaccacg agaagtaccc cactatctac caccttcgta agaagcttgt 5820
tgactctact gataaggctg atcttcgtct catctacctt gctctcgctc acatgatcaa 5880
gttccgtggt cacttcctta tcgagggtga ccttaaccct gataactccg acgtggacaa 5940
gctcttcatc cagctcgtcc agacctacaa ccagctcttc gaggagaacc ctatcaacgc 6000
ttccggtgtc gacgctaagg cgatcctttc cgctaggctc tccaagtcca ggcgtctcga 6060
gaacctcatc gcccagctcc ctggtgagaa gaagaacggt cttttcggta acctcatcgc 6120
tctctccctc ggtctgaccc ctaacttcaa gtccaacttc gacctcgctg aggacgctaa 6180
gcttcagctc tccaaggata cctacgacga tgatctcgac aacctcctcg ctcagattgg 6240
agatcagtac gctgatctct tccttgctgc taagaacctc tccgatgcta tcctcctttc 6300
ggatatcctt agggttaaca ctgagatcac taaggctcct ctttctgctt ccatgatcaa 6360
gcgctacgac gagcaccacc aggacctcac cctcctcaag gctcttgttc gtcagcagct 6420
ccccgagaag tacaaggaga tcttcttcga ccagtccaag aacggctacg ccggttacat 6480
tgacggtgga gctagccagg aggagttcta caagttcatc aagccaatcc ttgagaagat 6540
ggatggtact gaggagcttc tcgttaagct taaccgtgag gacctcctta ggaagcagag 6600
gactttcgat aacggctcta tccctcacca gatccacctt ggtgagcttc acgccatcct 6660
tcgtaggcag gaggacttct accctttcct caaggacaac cgtgagaaga tcgagaagat 6720
ccttactttc cgtattcctt actacgttgg tcctcttgct cgtggtaact cccgtttcgc 6780
ttggatgact aggaagtccg aggagactat caccccttgg aacttcgagg aggttgttga 6840
caagggtgct tccgcccagt ccttcatcga gcgcatgacc aacttcgaca agaacctccc 6900
caacgagaag gtcctcccca agcactccct cctctacgag tacttcacgg tctacaacga 6960
gctcaccaag gtcaagtacg tcaccgaggg tatgcgcaag cctgccttcc tctccggcga 7020
gcagaagaag gctatcgttg acctcctctt caagaccaac cgcaaggtca ccgtcaagca 7080
gctcaaggag gactacttca agaagatcga gtgcttcgac tccgtcgaga tcagcggcgt 7140
tgaggaccgt ttcaacgctt ctctcggtac ctaccacgat ctcctcaaga tcatcaagga 7200
caaggacttc ctcgacaacg aggagaacga ggacatcctc gaggacatcg tcctcactct 7260
tactctcttc gaggataggg agatgatcga ggagaggctc aagacttacg ctcatctctt 7320
cgatgacaag gttatgaagc agctcaagcg tcgccgttac accggttggg gtaggctctc 7380
ccgcaagctc atcaacggta tcagggataa gcagagcggc aagactatcc tcgacttcct 7440
caagtctgat ggtttcgcta acaggaactt catgcagctc atccacgatg actctcttac 7500
cttcaaggag gatattcaga aggctcaggt gtccggtcag ggcgactctc tccacgagca 7560
cattgctaac cttgctggtt cccctgctat caagaagggc atccttcaga ctgttaaggt 7620
tgtcgatgag cttgtcaagg ttatgggtcg tcacaagcct gagaacatcg tcatcgagat 7680
ggctcgtgag aaccagacta cccagaaggg tcagaagaac tcgagggagc gcatgaagag 7740
gattgaggag ggtatcaagg agcttggttc tcagatcctt aaggagcacc ctgtcgagaa 7800
cacccagctc cagaacgaga agctctacct ctactacctc cagaacggta gggatatgta 7860
cgttgaccag gagctcgaca tcaacaggct ttctgactac gacgtcgacc acattgttcc 7920
tcagtctttc cttaaggatg actccatcga caacaaggtc ctcacgaggt ccgacaagaa 7980
caggggtaag tcggacaacg tcccttccga ggaggttgtc aagaagatga agaactactg 8040
gaggcagctt ctcaacgcta agctcattac ccagaggaag ttcgacaacc tcacgaaggc 8100
tgagaggggt ggcctttccg agcttgacaa ggctggtttc atcaagaggc agcttgttga 8160
gacgaggcag attaccaagc acgttgctca gatcctcgat tctaggatga acaccaagta 8220
cgacgagaac gacaagctca tccgcgaggt caaggtgatc accctcaagt ccaagctcgt 8280
ctccgacttc cgcaaggact tccagttcta caaggtccgc gagatcaaca actaccacca 8340
cgctcacgat gcttacctta acgctgtcgt tggtaccgct cttatcaaga agtaccctaa 8400
gcttgagtcc gagttcgtct acggtgacta caaggtctac gacgttcgta agatgatcgc 8460
caagtccgag caggagatcg gcaaggccac cgccaagtac ttcttctact ccaacatcat 8520
gaacttcttc aagaccgaga tcaccctcgc caacggcgag atccgcaagc gccctcttat 8580
cgagacgaac ggtgagactg gtgagatcgt ttgggacaag ggtcgcgact tcgctactgt 8640
tcgcaaggtc ctttctatgc ctcaggttaa catcgtcaag aagaccgagg tccagaccgg 8700
tggcttctcc aaggagtcta tccttccaaa gagaaactcg gacaagctca tcgctaggaa 8760
gaaggattgg gaccctaaga agtacggtgg tttcgactcc cctactgtcg cctactccgt 8820
cctcgtggtc gccaaggtgg agaagggtaa gtcgaagaag ctcaagtccg tcaaggagct 8880
cctcggcatc accatcatgg agcgctcctc cttcgagaag aacccgatcg acttcctcga 8940
ggccaagggc tacaaggagg tcaagaagga cctcatcatc aagctcccca agtactctct 9000
tttcgagctc gagaacggtc gtaagaggat gctggcttcc gctggtgagc tccagaaggg 9060
taacgagctt gctcttcctt ccaagtacgt gaacttcctc tacctcgcct cccactacga 9120
gaagctcaag ggttcccctg aggataacga gcagaagcag ctcttcgtgg agcagcacaa 9180
gcactacctc gacgagatca tcgagcagat ctccgagttc tccaagcgcg tcatcctcgc 9240
tgacgctaac ctcgacaagg tcctctccgc ctacaacaag caccgcgaca agcccatccg 9300
cgagcaggcc gagaacatca tccacctctt cacgctcacg aacctcggcg cccctgctgc 9360
tttcaagtac ttcgacacca ccatcgacag gaagcgttac acgtccacca aggaggttct 9420
cgacgctact ctcatccacc agtccatcac cggtctttac gagactcgta tcgacctttc 9480
ccagcttggt ggtgatgacg atgacaaaat ggcaccgaag aaaaaaagga aggtcggcgg 9540
ctccccgaag aaaaaaagga aggtcggcgg ctccccgaag aaaaaaagga aggtcggcgg 9600
ctccccgaag aaaaaaagga aggtcggaat ccatggcgtt ccatagacta gttcagccag 9660
tttggtggag ctgccgatgt gcctggtcgt cccgagcctc tgttcgtcaa gtatttgtgg 9720
tgctgatgtc tacttgtgtc tggtttaatg gaccatcgag tccgtatgat atgttagttt 9780
tatgaaacag tttcctgtgg gacagcagta tgctttatga ataagttgga tttgaaccta 9840
aatatgtgct caatttgctc atttgcatct cattcctgtt gatgttttat ctgagttgca 9900
agtttgaaaa tgctgcatat tcttattaaa tcgtcattta cttttatctt aatgagcttt 9960
gcaatggcct atgggatata aaagagatcg ttcaaacatt tggcaataaa gtttcttaag 10020
attgaatcct gttgccggtc ttgcgatgat tatcatataa tttctgttga attacgttaa 10080
gcatgtaata attaacatgt aatgcatgac gttatttatg agatgggttt ttatgattag 10140
agtcccgcaa ttatacattt aatacgcgat agaaaacaaa atatagcgcg caaactagga 10200
taaattatcg cgcgcggtgt catctatgtt actagatcgg cgcctgtccg ggcgcgcctg 10260
gtggatcgtc cgcctaggct gcagtgcagc gtgacccggt cgtgcccctc tctagagata 10320
atgagcattg catgtctaag ttataaaaaa ttaccacata ttttttttgt cacacttgtt 10380
tgaagtgcag tttatctatc tttatacata tatttaaact ttactctacg aataatataa 10440
tctatagtac tacaataata tcagtgtttt agagaatcat ataaatgaac agttagacat 10500
ggtctaaagg acaattgagt attttgacaa caggactcta cagttttatc tttttagtgt 10560
gcatgtgttc tccttttttt ttgcaaatag cttcacctat ataatacttc atccatttta 10620
ttagtacatc catttagggt ttagggttaa tggtttttat agactaattt ttttagtaca 10680
tctattttat tctattttag cctctaaatt aagaaaacta aaactctatt ttagtttttt 10740
tatttaataa tttagatata aaatagaata aaataaagtg actaaaaatt aaacaaatac 10800
cctttaagaa attaaaaaaa ctaaggaaac atttttcttg tttcgagtag ataatgccag 10860
cctgttaaac gccgtcgacg agtctaacgg acaccaacca gcgaaccagc agcgtcgcgt 10920
cgggccaagc gaagcagacg gcacggcatc tctgtcgctg cctctggacc cctctcgaga 10980
gttccgctcc accgttggac ttgctccgct gtcggcatcc agaaattgcg tggcggagcg 11040
gcagacgtga gccggcacgg caggcggcct cctcctcctc tcacggcacc ggcagctacg 11100
ggggattcct ttcccaccgc tccttcgctt tcccttcctc gcccgccgta ataaatagac 11160
accccctcca caccctcttt ccccaacctc gtgttgttcg gagcgcacac acacacaacc 11220
agatctcccc caaatccacc cgtcggcacc tccgcttcaa ggtacgccgc tcgtcctccc 11280
cccccccccc tctctacctt ctctagatcg gcgttccggt ccatggttag ggcccggtag 11340
ttctacttct gttcatgttt gtgttagatc cgtgtttgtg ttagatccgt gctgctagcg 11400
ttcgtacacg gatgcgacct gtacgtcaga cacgttctga ttgctaactt gccagtgttt 11460
ctctttgggg aatcctggga tggctctagc cgttccgcag acgggatcga tttcatgatt 11520
ttttttgttt cgttgcatag ggtttggttt gcccttttcc tttatttcaa tatatgccgt 11580
gcacttgttt gtcgggtcat cttttcatgc ttttttttgt cttggttgtg atgatgtggt 11640
ctggttgggc ggtcgttcta gatcggagta gaattctgtt tcaaactacc tggtggattt 11700
attaattttg gatctgtatg tgtgtgccat acatattcat agttacgaat tgaagatgat 11760
ggatggaaat atcgatctag gataggtata catgttgatg cgggttttac tgatgcatat 11820
acagagatgc tttttgttcg cttggttgtg atgatgtggt gtggttgggc ggtcgttcat 11880
tcgttctaga tcggagtaga atactgtttc aaactacctg gtgtatttat taattttgga 11940
actgtatgtg tgtgtcatac atcttcatag ttacgagttt aagatggatg gaaatatcga 12000
tctaggatag gtatacatgt tgatgtgggt tttactgatg catatacatg atggcatatg 12060
cagcatctat tcatatgctc taaccttgag tacctatcta ttataataaa caagtatgtt 12120
ttataattat tttgatcttg atatacttgg atgatggcat atgcagcagc tatatgtgga 12180
tttttttagc cctgccttca tacgctattt atttgcttgg tactgtttct tttgtcgatg 12240
ctcaccctgt tgtttggtgt tacttctgca ggagctcctc atagcactca atgcggttgg 12300
caaaaagcct gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag 12360
cgtctccgac ctgatgcagc tctcggaggg cgaagaatct cgtgctttca gcttcgatgt 12420
aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg 12480
ttatgtttat cggcactttg catcggccgc gctcccgatt ccggaagtgc ttgacattgg 12540
ggagtttagc gagagcctga cctattgcat ctcccgccgt tcacagggtg tcacgttgca 12600
agacctgcct gaaaccgaac tgcccgctgt tctacaaccg gtcgcggagg ctatggatgc 12660
gatcgctgcg gccgatctta gccagacgag cgggttcggc ccattcggac cgcaaggaat 12720
cggtcaatac actacatggc gtgatttcat atgcgcgatt gctgatcccc atgtgtatca 12780
ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct 12840
gatgctttgg gccgaggact gccccgaagt ccggcacctc gtgcacgcgg atttcggctc 12900
caacaatgtc ctgacggaca atggccgcat aacagcggtc attgactgga gcgaggcgat 12960
gttcggggat tcccaatacg aggtcgccaa catcttcttc tggaggccgt ggttggcttg 13020
tatggagcag cagacgcgct acttcgagcg gaggcatccg gagcttgcag gatcgccacg 13080
actccgggcg tatatgctcc gcattggtct tgaccaactc tatcagagct tggttgacgg 13140
caatttcgat gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc 13200
cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg 13260
tgtagaagta ctcgccgata gtggaaaccg acgccccagc actcgtccga gggcaaagaa 13320
atagagtaga tgccgaccgg gatctgtcga tcgacaagct cgagtttctc cataataatg 13380
tgtgagtagt tcccagataa gggaattagg gttcctatag ggtttcgctc atgtgttgag 13440
catataagaa acccttagta tgtatttgta tttgtaaaat acttctatca ataaaatttc 13500
taattcctaa aaccaaaatc cagtactaaa atccagatcc cccgaattaa ttcggcgtta 13560
attcagcctg caggacgcgt ttaattaagt gcacgcggcc gcctacttag tcaagagcct 13620
cgcacgcgac tgtcacgcgg ccaggatcgc ctcgtgagcc tcgcaatctg tacctagtgt 13680
ttaaactatc agtgtttgac aggatatatt ggcgggtaaa cctaagagaa aagagcgttt 13740
attagaataa cggatattta aaagggcgtg aaaaggttta tccgttcgtc catttgtatg 13800
tgcatgccaa ccacagggtt cccctcggga tcaaagtact ttgatccaac ccctccgctg 13860
ctatagtgca gtcggcttct gacgttcagt gcagccgtct tctgaaaacg acatgtcgca 13920
caagtcctaa gttacgcgac aggctgccgc cctgcccttt tcctggcgtt ttcttgtcgc 13980
gtgttttagt cgcataaagt agaatacttg cgactagaac cggagacatt acgccatgaa 14040
caagagcgcc gccgctggcc tgctgggcta tgcccgcgtc agcaccgacg accaggactt 14100
gaccaaccaa cgggccgaac tgcacgcggc cggctgcacc aagctgtttt ccgagaagat 14160
caccggcacc aggcgcgacc gcccggagct ggccaggatg cttgaccacc tacgccctgg 14220
cgacgttgtg acagtgacca ggctagaccg cctggcccgc agcacccgcg acctactgga 14280
cattgccgag cgcatccagg aggccggcgc gggcctgcgt agcctggcag agccgtgggc 14340
cgacaccacc acgccggccg gccgcatggt gttgaccgtg ttcgccggca ttgccgagtt 14400
cgagcgttcc ctaatcatcg accgcacccg gagcgggcgc gaggccgcca aggcccgagg 14460
cgtgaagttt ggcccccgcc ctaccctcac cccggcacag atcgcgcacg cccgcgagct 14520
gatcgaccag gaaggccgca ccgtgaaaga ggcggctgca ctgcttggcg tgcatcgctc 14580
gaccctgtac cgcgcacttg agcgcagcga ggaagtgacg cccaccgagg ccaggcggcg 14640
cggtgccttc cgtgaggacg cattgaccga ggccgacgcc ctggcggccg ccgagaatga 14700
acgccaagag gaacaagcat gaaaccgcac caggacggcc aggacgaacc gtttttcatt 14760
accgaagaga tcgaggcgga gatgatcgcg gccgggtacg tgttcgagcc gcccgcgcac 14820
gtctcaaccg tgcggctgca tgaaatcctg gccggtttgt ctgatgccaa gctggcggcc 14880
tggccggcca gcttggccgc tgaagaaacc gagcgccgcc gtctaaaaag gtgatgtgta 14940
tttgagtaaa acagcttgcg tcatgcggtc gctgcgtata tgatgcgatg agtaaataaa 15000
caaatacgca aggggaacgc atgaaggtta tcgctgtact taaccagaaa ggcgggtcag 15060
gcaagacgac catcgcaacc catctagccc gcgccctgca actcgccggg gccgatgttc 15120
tgttagtcga ttccgatccc cagggcagtg cccgcgattg ggcggccgtg cgggaagatc 15180
aaccgctaac cgttgtcggc atcgaccgcc cgacgattga ccgcgacgtg aaggccatcg 15240
gccggcgcga cttcgtagtg atcgacggag cgccccaggc ggcggacttg gctgtgtccg 15300
cgatcaaggc agccgacttc gtgctgattc cggtgcagcc aagcccttac gacatatggg 15360
ccaccgccga cctggtggag ctggttaagc agcgcattga ggtcacggat ggaaggctac 15420
aagcggcctt tgtcgtgtcg cgggcgatca aaggcacgcg catcggcggt gaggttgccg 15480
aggcgctggc cgggtacgag ctgcccattc ttgagtcccg tatcacgcag cgcgtgagct 15540
acccaggcac tgccgccgcc ggcacaaccg ttcttgaatc agaacccgag ggcgacgctg 15600
cccgcgaggt ccaggcgctg gccgctgaaa ttaaatcaaa actcatttga gttaatgagg 15660
taaagagaaa atgagcaaaa gcacaaacac gctaagtgcc ggccgtccga gcgcacgcag 15720
cagcaaggct gcaacgttgg ccagcctggc agacacgcca gccatgaagc gggtcaactt 15780
tcagttgccg gcggaggatc acaccaagct gaagatgtac gcggtacgcc aaggcaagac 15840
cattaccgag ctgctatctg aatacatcgc gcagctacca gagtaaatga gcaaatgaat 15900
aaatgagtag atgaatttta gcggctaaag gaggcggcat ggaaaatcaa gaacaaccag 15960
gcaccgacgc cgtggaatgc cccatgtgtg gaggaacggg cggttggcca ggcgtaagcg 16020
gctgggttgt ctgccggccc tgcaatggca ctggaacccc caagcccgag gaatcggcgt 16080
gacggtcgca aaccatccgg cccggtacaa atcggcgcgg cgctgggtga tgacctggtg 16140
gagaagttga aggccgcgca ggccgcccag cggcaacgca tcgaggcaga agcacgcccc 16200
ggtgaatcgt ggcaagcggc cgctgatcga atccgcaaag aatcccggca accgccggca 16260
gccggtgcgc cgtcgattag gaagccgccc aagggcgacg agcaaccaga ttttttcgtt 16320
ccgatgctct atgacgtggg cacccgcgat agtcgcagca tcatggacgt ggccgttttc 16380
cgtctgtcga agcgtgaccg acgagctggc gaggtgatcc gctacgagct tccagacggg 16440
cacgtagagg tttccgcagg gccggccggc atggccagtg tgtgggatta cgacctggta 16500
ctgatggcgg tttcccatct aaccgaatcc atgaaccgat accgggaagg gaagggagac 16560
aagcccggcc gcgtgttccg tccacacgtt gcggacgtac tcaagttctg ccggcgagcc 16620
gatggcggaa agcagaaaga cgacctggta gaaacctgca ttcggttaaa caccacgcac 16680
gttgccatgc agcgtacgaa gaaggccaag aacggccgcc tggtgacggt atccgagggt 16740
gaagccttga ttagccgcta caagatcgta aagagcgaaa ccgggcggcc ggagtacatc 16800
gagatcgagc tagctgattg gatgtaccgc gagatcacag aaggcaagaa cccggacgtg 16860
ctgacggttc accccgatta ctttttgatc gatcccggca tcggccgttt tctctaccgc 16920
ctggcacgcc gcgccgcagg caaggcagaa gccagatggt tgttcaagac gatctacgaa 16980
cgcagtggca gcgccggaga gttcaagaag ttctgtttca ccgtgcgcaa gctgatcggg 17040
tcaaatgacc tgccggagta cgatttgaag gaggaggcgg ggcaggctgg cccgatccta 17100
gtcatgcgct accgcaacct gatcgagggc gaagcatccg ccggttccta atgtacggag 17160
cagatgctag ggcaaattgc cctagcaggg gaaaaaggtc gaaaaggtct ctttcctgtg 17220
gatagcacgt acattgggaa cccaaagccg tacattggga accggaaccc gtacattggg 17280
aacccaaagc cgtacattgg gaaccggtca cacatgtaag tgactgatat aaaagagaaa 17340
aaaggcgatt tttccgccta aaactcttta aaacttatta aaactcttaa aacccgcctg 17400
gcctgtgcat aactgtctgg ccagcgcaca gccgaagagc tgcaaaaagc gcctaccctt 17460
cggtcgctgc gctccctacg ccccgccgct tcgcgtcggc ctatcgcggc cgctggccgc 17520
tcaaaaatgg ctggcctacg gccaggcaat ctaccagggc gcggacaagc cgcgccgtcg 17580
ccactcgacc gccggcgccc acatcaaggc accctgcctc gcgcgtttcg gtgatgacgg 17640
tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc 17700
cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc 17760
catgacccag tcacgtagcg atagcggagt gtatactggc ttaactatgc ggcatcagag 17820
cagattgtac tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga 17880
aaataccgca tcaggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt 17940
cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca 18000
ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa 18060
aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat 18120
cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc 18180
cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc 18240
gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt 18300
tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac 18360
cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg 18420
ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca 18480
gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc 18540
gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa 18600
accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa 18660
ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac 18720
tcacgttaag ggattttggt catgcattct aggtactaaa acaattcatc cagtaaaata 18780
taatatttta ttttctccca atcaggcttg atccccagta agtcaaaaaa tagctcgaca 18840
tactgttctt ccccgatatc ctccctgatc gaccggacgc agaaggcaat gtcataccac 18900
ttgtccgccc tgccgcttct cccaagatca ataaagccac ttactttgcc atctttcaca 18960
aagatgttgc tgtctcccag gtcgccgtgg gaaaagacaa gttcctcttc gggcttttcc 19020
gtctttaaaa aatcatacag ctcgcgcgga tctttaaatg gagtgtcttc ttcccagttt 19080
tcgcaatcca catcggccag atcgttattc agtaagtaat ccaattcggc taagcggctg 19140
tctaagctat tcgtataggg acaatccgat atgtcgatgg agtgaaagag cctgatgcac 19200
tccgcataca gctcgataat cttttcaggg ctttgttcat cttcatactc ttccgagcaa 19260
aggacgccat cggcctcact catgagcaga ttgctccagc catcatgccg ttcaaagtgc 19320
aggacctttg gaacaggcag ctttccttcc agccatagca tcatgtcctt ttcccgttcc 19380
acatcatagg tggtcccttt ataccggctg tccgtcattt ttaaatatag gttttcattt 19440
tctcccacca gcttatatac cttagcagga gacattcctt ccgtatcttt tacgcagcgg 19500
tatttttcga tcagtttttt caattccggt gatattctca ttttagccat ttattatttc 19560
cttcctcttt tctacagtat ttaaagatac cccaagaagc taattataac aagacgaact 19620
ccaattcact gttccttgca ttctaaaacc ttaaatacca gaaaacagct ttttcaaagt 19680
tgttttcaaa gttggcgtat aacatagtat cgacggagcc gattttgaaa ccgcggtgat 19740
cacaggcagc aacgctctgt catcgttaca atcaacatgc taccctccgc gagatcatcc 19800
gtgtttcaaa cccggcagct tagttgccgt tcttccgaat agcatcggta acatgagcaa 19860
agtctgccgc cttacaacgg ctctcccgct gacgccgtcc cggactgatg ggctgcctgt 19920
atcgagtggt gattttgtgc cgagctgccg gtcggggagc tgttggctgg ct 19972
<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 (10)

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 sgRNA targeting a target gene target sequence and sgRNA targeting the loss-of-function screening agent resistance gene target sequence;
the sgRNA structure is as follows: an RNA-sgRNA backbone transcribed from the 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 617-692 position 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.
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 loss-of-function screener resistance gene or biological material associated with the loss-of-function screener resistance gene of any one of claims 1-5.
7. Use of the kit of any one of claims 1 to 5 or the loss-of-function screener resistance gene of claim 6 or a biological material associated with said loss-of-function screener resistance gene 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 a sgRNA targeting a target gene target sequence, a DNA molecule transcribing a 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 the Cas9 nuclease of any of claims 1-5, sgRNA targeting a target gene sequence, sgRNA targeting the loss-of-function screener resistance gene target sequence, adenine deaminase, and a loss-of-function screener resistance gene into an organism or 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 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.
CN201910938696.5A 2019-09-30 2019-09-30 Cell enrichment technology using inactivated screening agent resistance gene as report system for A.G base substitution and application thereof Active CN110628795B (en)

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