Cell enrichment technology of C.T base substitution by taking inactivated screening agent resistance gene as report system and application thereof
阅读说明：本技术 以失活的筛选剂抗性基因为报告体系的c·t碱基替换的细胞富集技术及其应用 (Cell enrichment technology of C.T base substitution by taking inactivated screening agent resistance gene as report system and application thereof ) 是由 徐雯 杨进孝 张成伟 赵思 冯峰 于 2019-09-30 设计创作，主要内容包括：本发明公开了以失活的筛选剂抗性基因为报告体系的C·T碱基替换的细胞富集技术及其应用。所述细胞富集技术载体包括如下试剂：sgRNA、C·T碱基替换系统和功能丧失的筛选剂抗性基因；sgRNA由靶向目标基因靶点序列的tRNA-sgRNA和靶向功能丧失的筛选剂抗性基因靶点序列的tRNA-sgRNA组成；C·T碱基替换系统在靶向功能丧失的筛选剂抗性基因靶点序列的tRNA-sgRNA的向导下,可通过对功能丧失的筛选剂抗性基因靶点序列进行C·T碱基替换使功能丧失的筛选剂抗性基因功能恢复。本发明实现了细胞水平上C·T碱基替换细胞富集,大大提高C·T碱基替换效率。(The invention discloses a cell enrichment technology of C.T 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: sgRNA, C.T base substitution system, and a selection agent resistance gene with function loss; the sgRNA consists of tRNA-sgRNA targeting a target gene target sequence and tRNA-sgRNA targeting a loss-of-function screening agent resistance gene target sequence; the C.T base substitution system can restore the function of the selection agent resistance gene with the loss of function by carrying out C.T base substitution on the selection agent resistance gene target sequence with the loss of function under the guidance of tRNA-sgRNA of the target gene sequence of the selection agent resistance gene with the loss of function. The invention realizes the enrichment of C.T base substitution cells on the cell level and greatly improves the C.T base substitution efficiency.)
1. A kit comprising a sgRNA or a biological material related to the sgRNA, a c.t 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 c.t base substitution system comprises Cas9 nuclease or a biological material associated with the Cas9 nuclease and a cytosine deaminase or a biological material associated with the cytosine deaminase;
the C.T base replacement system can restore the function of the screening agent resistance gene with the loss of function by carrying out C.T base replacement 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 targeted loss of function;
the sgRNA backbone is S1) or S2) or S3):
s1) replacing T in the 571-646 th site of the sequence 1 with U to obtain an RNA molecule;
s2) carrying out substitution and/or deletion and/or addition of one or more nucleotides on the RNA molecule shown in S1) and having the same function;
s3) and S1) or S2) and has the same function.
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 C.T base replacement system can restore the function of the screening agent resistance gene with the loss of function by carrying out C.T base replacement on the surrogate target sequence under the guidance of the sgRNA of the screening agent resistance gene target sequence with the targeted loss of function;
and/or, the surrogate target sequence is 11305 th-11327 th of the sequence 1 or the sequence 10.
3. The kit of claim 1 or 2, wherein: the screening agent resistance gene is a hygromycin resistance gene.
4. The kit of any one of claims 1 to 3, wherein: the sgRNA is tRNA-sgRNA;
the tRNA-sgRNA consists of tRNA-sgRNA targeting a target gene target sequence and tRNA-sgRNA targeting the loss-of-function screening agent resistance gene target sequence;
the tRNA-sgRNA structure is as follows: tRNA-RNA transcribed from the target sequence-sgRNA backbone;
the tRNA is R1) or R2) or R3):
r1) is replaced by U in the 474-550 th position of the sequence 1;
r2) the RNA molecule shown in R1) is substituted and/or deleted and/or added by one or more nucleotides and has the same function;
r3) and R1) or R2) have more than 75 percent of identity or 75 percent of identity and have the same function.
5. The kit of any one of claims 1 to 4, wherein: the C.T base substitution system further comprises UGI or biological material associated with the UGI;
and/or, the Cas9 nuclease is SpCas9n protein or HypaCas9n protein;
and/or the cytosine deaminase is PmCDA1 protein or rAPOBEC1 protein;
and/or, the SpCas9n protein is a1) or a2) or A3):
A1) the amino acid sequence is a protein shown in a sequence 2;
A2) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 2 in the sequence table and has the same function;
A3) a fusion protein obtained by connecting a label to the N terminal or/and the C terminal of A1) or A2);
the 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 HypaCas9n protein is C1) or C2) or C3):
C1) the amino acid sequence is a protein shown in a sequence 7;
C2) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 7 in the sequence table and has the same function;
C3) a fusion protein obtained by connecting a label to the N terminal or/and the C terminal of C1) or C2);
the biological material related to the HypaCas9n is any one of D1) to D5):
D1) a nucleic acid molecule encoding the HypaCas9 n;
D2) an expression cassette comprising the nucleic acid molecule of D1);
D3) a recombinant vector containing the nucleic acid molecule of D1) or a recombinant vector containing the expression cassette of D2);
D4) a recombinant microorganism containing D1) the nucleic acid molecule, or a recombinant microorganism containing D2) the expression cassette, or a recombinant microorganism containing D3) the recombinant vector;
D5) a transgenic cell line comprising D1) the nucleic acid molecule or a transgenic cell line comprising the expression cassette of D2);
the PmCDA1 protein is E1) or E2) or E3):
E1) the amino acid sequence is a protein shown in a sequence 3;
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 3 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 the PmCDA1 protein is any one of F1) to F5):
F1) a nucleic acid molecule encoding the PmCDA1 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 rAPOBEC1 protein is G1) or G2) or G3):
G1) the amino acid sequence is a protein shown in a sequence 12;
G2) 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 as the sequence 12 in the sequence table and has the same function;
G3) a fusion protein obtained by connecting a tag to the N-terminus or/and the C-terminus of G1) or G2);
the biological material related to the rAPOBEC1 protein is any one of H1) to H5):
H1) a nucleic acid molecule encoding said rAPOBEC1 protein;
H2) an expression cassette comprising the nucleic acid molecule of H1);
H3) a recombinant vector containing H1) the nucleic acid molecule or a recombinant vector containing H2) the expression cassette;
H4) a recombinant microorganism containing H1) the nucleic acid molecule, or a recombinant microorganism containing H2) the expression cassette, or a recombinant microorganism containing H3) the recombinant vector;
H5) a transgenic cell line comprising H1) the nucleic acid molecule or a transgenic cell line comprising H2) the expression cassette;
the UGI protein is I1) or I2) or I3):
I1) the amino acid sequence is a protein shown in a sequence 4;
I2) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 4 in the sequence table and has the same function;
I3) a fusion protein obtained by connecting labels at the N terminal or/and the C terminal of I1) or I2);
the biological material related to the UGI protein is any one of J1) to J5):
J1) a nucleic acid molecule encoding the UGI protein;
J2) an expression cassette comprising the nucleic acid molecule of J1);
J3) a recombinant vector comprising J1) said nucleic acid molecule, or a recombinant vector comprising J2) said expression cassette;
J4) a recombinant microorganism containing J1) the nucleic acid molecule, or a recombinant microorganism containing J2) the expression cassette, or a recombinant microorganism containing J3) the recombinant vector;
J5) a transgenic cell line comprising J1) the nucleic acid molecule or a transgenic cell line comprising J2) the expression cassette;
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 C.T base substitution of the genome target sequence of the organism or the organism cells;
m2) preparing products for enriching the cells with the C.T base substitution of the genome target sequences of organisms or organism cells;
m3) improving the efficiency of C.T base substitution of the genome target sequence of an organism or an organism cell;
m4) preparing a product for improving the replacement efficiency of the C.T base of the genome target sequence of the organism or the organism cell;
m5) a c.t base substitution in a target sequence of a genome of an organism or a cell of an organism;
m6) preparation of products for C.T base substitution in target sequences of organisms or biological cells.
8, N1) or N2) or N3) or N4) or N5):
n1) A method for enriching cells in which a C.T base substitution has occurred for a target sequence in the genome of an organism or a cell of an organism or a method for increasing the efficiency of a C.T base substitution for a target sequence in the genome of an organism or a cell of an organism, comprising the steps of: introducing into an organism or cell of an organism a gene encoding a Cas9 nuclease, a DNA molecule transcribing a sgRNA targeted to a target gene target sequence, a DNA molecule transcribing a sgRNA targeted to the loss-of-function screener resistance gene target sequence, a gene encoding a cytosine deaminase, a gene encoding UGI, and a loss-of-function screener resistance gene of any one of claims 1-5, such that the Cas9 nuclease, the sgRNA, the cytosine deaminase, and UGI are all expressed; under the guidance of sgRNA of the target sequence of the screening agent resistance gene with targeted function loss, the Cas9 nuclease, the cytosine deaminase and the UGI can restore the function of the screening agent resistance gene with function loss by carrying out C.T base substitution on the target sequence of the screening agent resistance gene with function loss, thereby realizing the enrichment of cells with C.T base substitution of the screening agent resistance gene, further realizing the enrichment of cells with C.T base substitution of the target sequence of the target gene of the genome of an organism or an organism cell or improving the C.T 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 in which a C.T base substitution has occurred for a target sequence in the genome of an organism or a cell of an organism or a method for increasing the efficiency of a C.T base substitution for a target sequence in the genome of an organism or a cell of an organism, comprising the steps of: introducing into an organism or cell of an organism a gene encoding a Cas9 nuclease, a DNA molecule transcribing a sgRNA targeted to a target gene target sequence, a DNA molecule transcribing a sgRNA targeted to the loss-of-function screener resistance gene target sequence, a gene encoding a cytosine deaminase, and a loss-of-function screener resistance gene of any one of claims 1-5, such that the Cas9 nuclease, the sgRNA, the cytosine deaminase are all expressed; under the guidance of sgRNA of the target sequence of the screening agent resistance gene with the targeted function loss, the Cas9 nuclease and the cytosine deaminase can restore the function of the screening agent resistance gene with the function loss by carrying out C.T base substitution on the target sequence of the screening agent resistance gene with the function loss, and further enrich the cells with the C.T base substitution of the screening agent resistance gene, thereby realizing the enrichment of the cells with the C.T base substitution of the target sequence of the target gene of the genome of the organism or the organism cell or improving the C.T base substitution efficiency of the target sequence of the target gene of the genome of the organism or the organism cell;
n3) A method for enriching cells in which a C.T base substitution has occurred for a target sequence in the genome of an organism or a cell of an organism or a method for increasing the efficiency of a C.T base substitution for a target sequence in the genome of an organism or a cell of an organism, comprising the steps of: 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, cytosine deaminase, UGI, and a loss-of-function screener resistance gene into an organism or biological cell; under the guidance of sgRNA of the target sequence of the screening agent resistance gene with targeted function loss, the Cas9 nuclease, the cytosine deaminase and the UGI can restore the function of the screening agent resistance gene with function loss by carrying out C.T base substitution on the target sequence of the screening agent resistance gene with function loss, thereby realizing the enrichment of cells with C.T base substitution of the screening agent resistance gene, further realizing the enrichment of cells with C.T base substitution of the target sequence of the target gene of the genome of an organism or an organism cell or improving the C.T base substitution efficiency of the target sequence of the target gene of the genome of the organism or the organism cell;
n4) A method for enriching cells in which a C.T base substitution has occurred for a target sequence in the genome of an organism or a cell of an organism or a method for increasing the efficiency of a C.T base substitution for a target sequence in the genome of an organism or a cell of an organism, comprising the steps of: 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, cytosine 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 function loss, the Cas9 nuclease and the cytosine deaminase can restore the function of the screening agent resistance gene with the function loss by carrying out C.T base substitution on the target sequence of the screening agent resistance gene with the function loss, and further enrich the cells with the C.T base substitution of the screening agent resistance gene, thereby realizing the enrichment of the cells with the C.T base substitution of the target sequence of the target gene of the genome of the organism or the organism cell or improving the C.T base substitution efficiency of the target sequence of the target gene of the genome of the organism or the organism cell;
n5) biological mutant, comprising the following steps: editing the genome of the organism according to the method of N1) or N2) or N3) or N4) to obtain an organism mutant; the biological mutant is an organism in which C.T 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 C.T base is replaced by a base C and mutated into a base T.
10. The kit of any one of claims 1 to 5 or the use of claim 7 or the method of claim 8, wherein: the organism is P1) or P2) or P3) or P4):
p1) plants or animals;
p2) monocotyledonous or dicotyledonous plants;
p3) gramineous plants;
p4) rice;
and/or, the biological cell is Q1) or Q2) or Q3) or Q4):
q1) plant cells or animal cells;
q2) a monocotyledonous or dicotyledonous plant cell;
q3) a graminaceous plant cell;
q4) Rice cells.
Technical Field
The invention relates to the field of biotechnology, in particular to a cell enrichment technology of C.T 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 2016, two laboratories such as David Liu and Akihiko Kondo independently report two different types of Cytosine Base Editors (CBEs), respectively, and use two different types of cytidine deaminases rAPOBEC1(rat APOBEC1) and PmCDA1(activation-induced Cytosine deaminase (AID) orthogonal template), which are based on the principle that the base editing of a single Cytosine (C) base is directly realized by using the cytidine deaminase, but not by generating DSB and initiating HDR repair, so that the base editing efficiency of C to be replaced by Thymine (Thymine, T) is greatly improved. Specifically, dead Cas9(dCas9) or the Cas9 nickase (Cas9n) are positioned to a target point through sgRNA together with rAPOBEC1 or PmCDA1, rAPOBEC1 or PmCDA1 catalyzes cytosine deamination reaction of C on unpaired single-stranded DNA to Uracil (U), and the U is paired with Adenine (Adenine, a) through DNA repair and finally paired with a through DNA replication, thereby realizing C-to-T conversion. The mean mutation rate of SpCas9n (D10A) & rAPOBEC1/PmCDA1& UGI base editing system (which contains uracil DNA glycosylase inhibitor, UGI)) was higher in the editor tested for two reasons: firstly, UGI can inhibit Uracil DNA Glycosylase (UDG) from catalyzing and removing U in DNA, and secondly, SpCas9n (D10A) generates a nick on a non-editing chain, and induces a eukaryotic mismatch repair mechanism or a long-patch BER (base-extension repair) repair mechanism to promote more preferential repair of U: G mismatch into U: A.
At present, research on enrichment of C.T base-substituted cells in plants by reporter gene-mediated cell enrichment technology is very limited, and no report is available on enrichment of C.T base-substituted cells at the cellular level and improvement of C.T base substitution efficiency by using a selection marker used in the transformation process.
Disclosure of Invention
The invention aims to provide a cell enrichment technology of C.T base substitution by taking an inactivated screening agent resistance gene as a report system, which can realize the enrichment of C.T base substituted cells on the cell level and further improve the C.T 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, a c.t 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 c.t base substitution system comprises Cas9 nuclease or a biological material associated with the Cas9 nuclease and a cytosine deaminase or a biological material associated with the cytosine deaminase;
the C.T base replacement system can restore the function of the screening agent resistance gene with the loss of function by carrying out C.T base replacement 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 targeted loss of function;
the sgRNA backbone is S1) or S2) or S3):
s1) replacing T in the 571-646 th site of the sequence 1 with U to obtain an RNA molecule;
s2) carrying out substitution and/or deletion and/or addition of one or more nucleotides on the RNA molecule shown in S1) and having the same function;
s3) and S1) or S2) and has the same function.
In the kit, the sgRNA may be specifically tRNA-sgRNA; the tRNA-sgRNA consists of tRNA-sgRNA targeting a target gene target sequence and tRNA-sgRNA targeting the loss-of-function screening agent resistance gene target sequence;
the tRNA-sgRNA structure is as follows: tRNA-RNA transcribed from the target sequence-sgRNA backbone;
the tRNA is R1) or R2) or R3):
r1) is replaced by U in the 474-550 th position of the sequence 1;
r2) the RNA molecule shown in R1) is substituted and/or deleted and/or added by one or more nucleotides and has the same function;
r3) and R1) or R2) have more than 75 percent of identity or 75 percent of identity and have 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 C.T base substitution is carried out on a 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 C.T base substitution, 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: and C.T base substitution is carried out on the surrogate target sequence through a C.T base substitution system, so that the function of the selection agent resistance gene with the lost function can be recovered. 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 present invention, the surrogate target sequence is 11305 th-11327 th of SEQ ID NO. 1. The target sequence of the screening agent resistance gene with the loss of function is 11305 th-11324 th site of the sequence 1. The C.T base substitution system can perform C.T base substitution on the proxy target sequence under the guidance of tRNA-sgRNA of the target sequence of the screening agent resistance gene with the target of the loss of function, so that the 5 th base C of the proxy target sequence is mutated into the base T 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.
In another embodiment of the invention, the surrogate target sequence is sequence 10. The target sequence of the screening agent resistance gene with the loss of function is 1 st-20 th site of the sequence 10. The C.T base substitution system can perform C.T base substitution on the proxy target sequence under the guidance of tRNA-sgRNA of the target sequence of the screening agent resistance gene with the target of the loss of function, so that the 7 th base C of the proxy target sequence is mutated into the base T to form ATG, and further the function of the screening marker gene is recovered.
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 kit, the c.t base substitution system further comprises UGI or biological material related to the UGI.
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 cytosine deaminase can be an hAPOBE3A protein, a human AID protein, a PmCDA1 protein, or an rAPOBEC1 protein. In a specific embodiment of the invention, the cytosine deaminase is PmCDA1 protein. In another specific embodiment of the invention, the cytosine deaminase is an rAPOBEC1 protein.
Further, the SpCas9n protein is a1) or a2) or A3):
A1) the amino acid sequence is a protein shown in a sequence 2;
A2) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 2 in the sequence table and has the same function;
A3) a fusion protein obtained by connecting a label to the N terminal or/and the C terminal of A1) or A2);
the 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 HypaCas9n protein is C1) or C2) or C3):
C1) the amino acid sequence is a protein shown in a sequence 7;
C2) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 7 in the sequence table and has the same function;
C3) a fusion protein obtained by connecting a label to the N terminal or/and the C terminal of C1) or C2);
the biological material related to the HypaCas9n is any one of D1) to D5):
D1) a nucleic acid molecule encoding the HypaCas9 n;
D2) an expression cassette comprising the nucleic acid molecule of D1);
D3) a recombinant vector containing the nucleic acid molecule of D1) or a recombinant vector containing the expression cassette of D2);
D4) a recombinant microorganism containing D1) the nucleic acid molecule, or a recombinant microorganism containing D2) the expression cassette, or a recombinant microorganism containing D3) the recombinant vector;
D5) a transgenic cell line comprising D1) the nucleic acid molecule or a transgenic cell line comprising the expression cassette of D2);
the PmCDA1 protein is E1) or E2) or E3):
E1) the amino acid sequence is a protein shown in a sequence 3;
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 3 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 the PmCDA1 protein is any one of F1) to F5):
F1) a nucleic acid molecule encoding the PmCDA1 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 rAPOBEC1 protein is G1) or G2) or G3):
G1) the amino acid sequence is a protein shown in a sequence 12;
G2) 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 as the sequence 12 in the sequence table and has the same function;
G3) a fusion protein obtained by connecting a tag to the N-terminus or/and the C-terminus of G1) or G2);
the biological material related to the rAPOBEC1 protein is any one of H1) to H5):
H1) a nucleic acid molecule encoding said rAPOBEC1 protein;
H2) an expression cassette comprising the nucleic acid molecule of H1);
H3) a recombinant vector containing H1) the nucleic acid molecule or a recombinant vector containing H2) the expression cassette;
H4) a recombinant microorganism containing H1) the nucleic acid molecule, or a recombinant microorganism containing H2) the expression cassette, or a recombinant microorganism containing H3) the recombinant vector;
H5) a transgenic cell line comprising H1) the nucleic acid molecule or a transgenic cell line comprising H2) the expression cassette;
the UGI protein is I1) or I2) or I3):
I1) the amino acid sequence is a protein shown in a sequence 4;
I2) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 4 in the sequence table and has the same function;
I3) a fusion protein obtained by connecting labels at the N terminal or/and the C terminal of I1) or I2);
the biological material related to the UGI protein is any one of J1) to J5):
J1) a nucleic acid molecule encoding the UGI protein;
J2) an expression cassette comprising the nucleic acid molecule of J1);
J3) a recombinant vector comprising J1) said nucleic acid molecule, or a recombinant vector comprising J2) said expression cassette;
J4) a recombinant microorganism containing J1) the nucleic acid molecule, or a recombinant microorganism containing J2) the expression cassette, or a recombinant microorganism containing J3) the recombinant vector;
J5) a transgenic cell line comprising J1) the nucleic acid molecule or a transgenic cell line comprising J2) the expression cassette;
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 protein of A1), C1), E1), G1), I1), the amino terminal or carboxyl terminal of the protein consisting of the amino acid sequence shown in the sequence 2 or 3 or 4 or 7 or 12 in the sequence table is attached 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), C2), E2), G2) and I2) is a protein having 75% or more or 75% or more identity to the amino acid sequence of the protein shown in SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 7 or SEQ ID NO. 12 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), C2), E2), G2) and I2) can be artificially synthesized, or can be obtained by synthesizing the coding gene and then performing biological expression.
The coding gene for the proteins A2), C2), E2), G2), I2) described above can be obtained by deleting one or several amino acid residues from the DNA sequence shown at positions 3529-7797 of the sequence 1 (protein shown by the coding sequence 2), 8089-8712 of the sequence 1 (protein shown by the coding sequence 3), 8734-9030 of the sequence 1 (protein shown by the coding sequence 4), 6 (protein shown by the coding sequence 7) or 1-687 of the sequence 9 (protein shown by the coding sequence 12), and/or by carrying out missense mutation of one or several base pairs, and/or by attaching a coding sequence to the 5 'end and/or 3' end of which a tag shown in the above table is attached.
Further, B1) the nucleic acid molecule is B1) or B2) or B3):
b1) a cDNA molecule or DNA molecule shown in 3529-7797 site of a sequence 1 in a sequence table;
b2) a cDNA or DNA molecule having 75% or more identity to the nucleotide sequence defined in b1) and encoding said 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;
D1) the nucleic acid molecule is d1) or d2) or d 3):
d1) a cDNA molecule or DNA molecule shown in a sequence 6 in a sequence table;
d2) a cDNA or DNA molecule having 75% or more identity to the nucleotide sequence defined by d1) and encoding the HypaCas9 n;
d3) a cDNA or DNA molecule hybridizing under stringent conditions to the nucleotide sequence defined by d1) or d2) and encoding said HypaCas9 n;
F1) the nucleic acid molecule is f1) or f2) or f 3):
f1) a cDNA molecule or DNA molecule shown in the 8089-8712 site of the sequence 1 in the sequence table;
f2) a cDNA molecule or DNA molecule having 75% or more identity with the nucleotide sequence defined by f1) and encoding the PmCDA 1;
f3) hybridizing with the nucleotide sequence defined by f1) or f2) under strict conditions, and encoding the cDNA molecule or DNA molecule of the PmCDA 1;
H1) the nucleic acid molecule is h1) or h2) or h 3):
h1) a cDNA molecule or DNA molecule shown in 1 st to 687 th sites of a sequence 9 in a sequence table;
h2) a cDNA or DNA molecule having 75% or more identity with the nucleotide sequence defined by h1) and encoding said rAPOBEC 1;
h3) hybridizing under stringent conditions with a nucleotide sequence defined by h1) or h2) and encoding a cDNA molecule or a DNA molecule of the rAPOBEC 1;
J1) the nucleic acid molecule is j1) or j2) or j 3):
j1) a cDNA molecule or DNA molecule shown in 8734-9030 site of a sequence 1 in a sequence table;
j2) a cDNA molecule or DNA molecule having 75% or more identity to the nucleotide sequence defined in j1) and encoding said UGI;
j3) a cDNA molecule or DNA molecule which hybridizes with the nucleotide sequence defined by j1) or j2) under strict conditions and codes the UGI;
K1) the resistance gene of the screening agent with the loss of function is a DNA molecule shown in 11305-12351 position of the sequence 1 or a sequence obtained by replacing 11305-11328 in the DNA molecule shown in 11305-12351 position of the sequence 1 with the sequence 10 and keeping other sequences unchanged.
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 HypaCas9n or the PmCDA1 or the rAPOBEC1 or the UGI of the present invention can be easily mutated by a person of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which are artificially modified to have 75% or more identity to the nucleotide sequence of the SpCas9n or the HypaCas9n or the PmCDA1 or the rAPOBEC1 or the UGI of the present invention are derived from the nucleotide sequence of the present invention and are identical to the sequence of the present invention as long as they encode the SpCas9n or the HypaCas9n or the PmCDA1 or the rAPOBEC1 or the UGI and have the same function.
The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes nucleotide sequences that are 75% or greater, or 85% or greater, or 90% or greater, or 95% or greater, identical to the nucleotide sequence of a protein consisting of the amino acid sequence set forth in coding sequence 2, 3, 4, 7, or 12 of the 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.
D2) The expression cassette containing the nucleic acid molecule encoding the HypaCas9n protein (HypaCas9n gene expression cassette) refers to a DNA capable of expressing the HypaCas9n protein in a host cell, and the DNA may include not only a promoter for initiating transcription of the HypaCas9n gene, but also a terminator for terminating transcription of the HypaCas9n 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 HypaCas9n gene expression cassette.
F2) The expression cassette containing the nucleic acid molecule encoding the PmCDA1 protein (PmCDA1 gene expression cassette) refers to a DNA capable of expressing the PmCDA1 protein in a host cell, and the DNA may include not only a promoter for initiating transcription of the PmCDA1 gene, but also a terminator for terminating transcription of the PmCDA1 gene. Further, the expression cassette may also include an enhancer sequence. The recombinant vector containing the PmCDA1 gene expression cassette can be constructed by using the existing expression vector.
H2) The expression cassette containing a nucleic acid molecule encoding rAPOBEC1 protein (rAPOBEC1 gene expression cassette) refers to DNA capable of expressing rAPOBEC1 protein in host cells, and the DNA can not only comprise a promoter for starting transcription of rAPOBEC1 gene, but also comprise a terminator for stopping transcription of rAPOBEC1 gene. Further, the expression cassette may also include an enhancer sequence. The recombinant vector containing the rAPOBEC1 gene expression cassette can be constructed by using the existing expression vector.
J2) The expression cassette containing a nucleic acid molecule encoding the UGI protein (UGI gene expression cassette) refers to a DNA capable of expressing the UGI protein in a host cell, and the DNA may include not only a promoter for initiating transcription of the UGI gene but also a terminator for terminating transcription of the UGI gene. Further, the expression cassette may also include an enhancer sequence. The recombinant vector containing the UGI 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 and sgRNA-ATG-Hyg-ATG/Hypacas9n-sgRNA-GT-1 recombinant expression vector, sgRNA-ATG-Hyg-ATG/Hypacas9n-sgRNA-GT-2 recombinant expression vector or sgRNA-ATG-Hyg-ATGa/r-sgRNA-GT 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 six 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 five target sequences in the sequence 1, namely ALS-T1, CDC48-T1, NRT1.1B-T1, Waxy and ALS-T2 are sequentially and respectively replaced by the following five target sequences: ALS-T3, CDC48-T2, NRT1.1B-T3, NRT1.1B-T2 and DEP1, 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/HypaCas 9n-sgRNA-GT-1 recombinant expression vector is obtained by replacing the coding sequence of the SpCas9n protein shown in 3529-7797 site of the sequence 1 with the coding sequence of the HypaCas9n protein shown in the sequence 6 (the HypaCas9n protein shown in the coding sequence 7) and keeping other sequences unchanged.
The sgRNA-ATG-Hyg-ATGThe sequence of the/HypaCas 9n-sgRNA-GT-2 recombinant expression vector is obtained by replacing the coding sequence of the SpCas9n protein shown in the 3529-7797 site of the sequence 1 with the coding sequence of the HypaCas9n protein shown in the sequence 6, replacing the 131-1802 site with the sequence 8 and keeping other sequences unchanged.
The sgRNA-ATG-Hyg-ATGThe sequence of the/r-sgRNA-GT recombinant expression vector is that the 3529-th and 8712-th positions of the sequence 1 are replaced by the sequence 9(rAPOBEC1 fuses with SpCas9n sequence), the 11305-th and 11328-th positions of the sequence 1 are replaced by the sequence 10(rAPOBEC1 surrogate target point target sequence), and the first five target point sequences of the first expression cassette of the sequence 1 are sequentially and respectively replaced by the following five target point sequences: ALS-T1, wax, NRT1.1B-T1, wax-T2 and rAPOBEC1-sug, and the sixth target sequence shown in the 1339 th-1511 th position of the sequence 1, the nucleotide sequence of tRNA thereof and the nucleotide sequence of sgRNA are deleted, and other sequences are kept unchanged. The corresponding target sequences are shown in Table 3.
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-ATG/Hypacas9n-sgRNA-GT-1 recombinant expression vector or sgRNA-ATG-Hyg-ATG/Hypacas9n-sgRNA-GT-2 recombinant expression vector or sgRNA-ATG-Hyg-ATGThe agrobacterium EHA105 of the/r-sgRNA-GT recombinant expression vector.
The transgenic cell line does not include propagation material.
The kit has the following uses:
m1) enriching the cells with C.T base substitution of the genome target sequence of the organism or the organism cells;
m2) preparing products for enriching the cells with the C.T base substitution of the genome target sequences of organisms or organism cells;
m3) improving the efficiency of C.T base substitution of the genome target sequence of an organism or an organism cell;
m4) preparing a product for improving the replacement efficiency of the C.T base of the genome target sequence of the organism or the organism cell;
m5) a c.t base substitution in a target sequence of a genome of an organism or a cell of an organism;
m6) preparation of products for C.T base substitution in target sequences of organisms or biological cells.
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 C.T base substitution of the genome target sequence of the organism or the organism cells;
m2) preparing products for enriching the cells with the C.T base substitution of the genome target sequences of organisms or organism cells;
m3) improving the efficiency of C.T base substitution of the genome target sequence of an organism or an organism cell;
m4) preparing a product for improving the replacement efficiency of the C.T base of the genome target sequence of the organism or the organism cell;
m5) a c.t base substitution in a target sequence of a genome of an organism or a cell of an organism;
m6) preparation of products for C.T base substitution in target sequences of organisms or biological cells.
In order to achieve the above object, the present invention also provides the method described in N1) or N2) or N3) or N4) or N5):
n1) A method for enriching cells in which a C.T base substitution has occurred for a target sequence in the genome of an organism or a cell of an organism or a method for increasing the efficiency of a C.T base substitution for a target sequence in the genome of an organism or a cell of an organism, comprising the steps of: introducing the coding gene of the Cas9 nuclease, the DNA molecule of the sgRNA transcribed and targeted to the target gene target sequence, the DNA molecule of the sgRNA transcribed and targeted to the target sequence of the loss-of-function screening agent resistance gene, the coding gene of cytosine deaminase, the coding gene of UGI 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, the cytosine deaminase and the UGI; under the guidance of sgRNA of the target sequence of the screening agent resistance gene with targeted function loss, the Cas9 nuclease, the cytosine deaminase and the UGI can restore the function of the screening agent resistance gene with function loss by carrying out C.T base substitution on the target sequence of the screening agent resistance gene with function loss, thereby realizing the enrichment of cells with C.T base substitution of the screening agent resistance gene, further realizing the enrichment of cells with C.T base substitution of the target sequence of the target gene of the genome of an organism or an organism cell or improving the C.T 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 in which a C.T base substitution has occurred for a target sequence in the genome of an organism or a cell of an organism or a method for increasing the efficiency of a C.T base substitution for a target sequence in the genome of an organism or a cell of an organism, comprising the steps of: 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 cytosine deaminase and the loss-of-function screening agent resistance gene into an organism or biological cells so as to express the Cas9 nuclease, the sgRNA and the cytosine deaminase; under the guidance of sgRNA of the target sequence of the screening agent resistance gene with the targeted function loss, the Cas9 nuclease and the cytosine deaminase can restore the function of the screening agent resistance gene with the function loss by carrying out C.T base substitution on the target sequence of the screening agent resistance gene with the function loss, and further enrich the cells with the C.T base substitution of the screening agent resistance gene, thereby realizing the enrichment of the cells with the C.T base substitution of the target sequence of the target gene of the genome of the organism or the organism cell or improving the C.T base substitution efficiency of the target sequence of the target gene of the genome of the organism or the organism cell;
n3) A method for enriching cells in which a C.T base substitution has occurred for a target sequence in the genome of an organism or a cell of an organism or a method for increasing the efficiency of a C.T base substitution for a target sequence in the genome of an organism or a cell of an organism, comprising the steps of: introducing the Cas9 nuclease, sgRNA targeting a target gene target sequence, sgRNA targeting the loss-of-function screening agent resistance gene target sequence, cytosine deaminase, UGI and the 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 targeted function loss, the Cas9 nuclease, the cytosine deaminase and the UGI can restore the function of the screening agent resistance gene with function loss by carrying out C.T base substitution on the target sequence of the screening agent resistance gene with function loss, thereby realizing the enrichment of cells with C.T base substitution of the screening agent resistance gene, further realizing the enrichment of cells with C.T base substitution of the target sequence of the target gene of the genome of an organism or an organism cell or improving the C.T base substitution efficiency of the target sequence of the target gene of the genome of the organism or the organism cell;
n4) A method for enriching cells in which a C.T base substitution has occurred for a target sequence in the genome of an organism or a cell of an organism or a method for increasing the efficiency of a C.T base substitution for a target sequence in the genome of an organism or a cell of an organism, comprising the steps of: introducing the Cas9 nuclease, the sgRNA targeting the target gene target sequence, the sgRNA targeting the loss-of-function screening agent resistance gene target sequence, cytosine deaminase and the 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 function loss, the Cas9 nuclease and the cytosine deaminase can restore the function of the screening agent resistance gene with the function loss by carrying out C.T base substitution on the target sequence of the screening agent resistance gene with the function loss, and further enrich the cells with the C.T base substitution of the screening agent resistance gene, thereby realizing the enrichment of the cells with the C.T base substitution of the target sequence of the target gene of the genome of the organism or the organism cell or improving the C.T base substitution efficiency of the target sequence of the target gene of the genome of the organism or the organism cell;
n5) biological mutant, comprising the following steps: editing the genome of the organism according to the method of N1) or N2) or N3) or N4) to obtain an organism mutant; the biological mutant is an organism in which C.T base substitution occurs.
In the method, the sgRNA of the target gene target sequence is tRNA-sgRNA of the target gene target sequence, and the sgRNA of the screening agent resistance gene target sequence with targeted loss of function is tRNA-sgRNA of the screening agent resistance gene target sequence with targeted loss of function. Further, the tRNA-sgRNA obtained by transcribing the DNA molecule of the tRNA-sgRNA that is transcribed to target a target sequence of the target gene or the DNA molecule of the tRNA-sgRNA that is transcribed to target a target sequence of the selection agent resistance gene that is lost of function is an immature RNA precursor, and the tRNA in the RNA precursor is cut off by two enzymes (RNase P and RNase Z) to obtain mature RNA. And obtaining independent mature RNAs according to the number of targets in a recombinant expression vector, wherein each mature RNA consists of RNA transcribed by the target sequence and the sgRNA framework in sequence or consists of individual base remained by the tRNA, RNA transcribed by the target sequence and the sgRNA framework in sequence.
In the above method, the number of the UGIs may be one or two or more in the N1) or N3). In a specific embodiment of the present invention, the number of the UGIs is specifically one.
In the above method, in N1), the gene encoding Cas9 nuclease, the DNA molecule of sgRNA transcription-targeted to the target gene sequence, the DNA molecule of sgRNA transcription-targeted to the loss-of-function screening agent-resistant gene target sequence, the gene encoding cytosine deaminase, and the gene encoding UGI are introduced into an organism or an organism cell via a recombinant vector containing an expression cassette of the gene encoding Cas9 nuclease, an expression cassette of the DNA molecule transcription-targeted to the sgRNA transcription-targeted to the target gene sequence, an expression cassette of the DNA molecule transcription-targeted to the loss-of-function screening agent-resistant gene target sequence, an expression cassette of the gene encoding cytosine deaminase, and an expression cassette of the gene encoding UGI. Each of the above-mentioned expression cassettes may be introduced into an organism or a biological cell by the same recombinant expression vector, or may be introduced into an organism or a biological cell by two or more recombinant expression vectors together.
In a specific embodiment of the present invention, each of the expression cassettes is introduced into an organism or a biological cell through the same recombinant expression vector, 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-ATG/Hypacas9n-sgRNA-GT-1 recombinant expression vector or sgRNA thereof-ATG-Hyg-ATG/Hypacas9n-sgRNA-GT-2 recombinant expression vector or sgRNA thereof-ATG-Hyg-ATGa/r-sgRNA-GT recombinant expression vector.
In the kit of parts or the use or the method, the base C is mutated to the base T instead of the base C. The base C 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 inactivated resistance gene of the screening agent as a reporter gene for C.T base substitution is established, so that cells with C.T base substitution on the reporter gene can grow in a medium containing the screening agent, and cells without C.T base substitution can not grow in a medium containing the screening agent. On the basis of the reporter gene, if C.T 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 C.T base replacement of the endogenous target gene target spot, so that enrichment of the cells with the C.T base replacement of the endogenous target gene target spot is realized, and the C.T 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 C.T base replacement. 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 C.T base replacement cells on the cellular level for different deaminase mediated base editors or different Cas9 enzyme mediated base editors, and greatly improves the C.T base replacement efficiency.
Drawings
FIG. 1 is a schematic structural diagram of a non-cell enrichment technology vector sgRNA-GT.
FIG. 2 shows a cell enrichment technology vector sgRNA-ATG-Hyg-ATGSchematic structural diagram of/sgRNA-GT.
FIG. 3 is a schematic diagram of the operation principle of the cell enrichment technique.
FIG. 4 shows a cell enrichment technology vector sgRNA-ATG-Hyg-ATGComparing the efficiency of C.T base replacement of the target in resistance healing of the/sgRNA-GT and the non-cell enrichment technology vector sgRNA-GT.
FIG. 5 shows a cell enrichment technology vector sgRNA-ATG-Hyg-ATGThe efficiencies of C.T base replacement of target spots in T0 seedlings by the sgRNA-GT and the non-cell enrichment technology vector sgRNA-GT are compared.
FIG. 6 shows a cell enrichment technology vector sgRNA-ATG-Hyg-ATGComparing the efficiencies of the sgRNA-GT and the non-cell enrichment technology vector sgRNA-GT in the T0 vaccine for the homozygous replacement of the target C.T base substitution.
FIG. 7 is Hypacas9n&PmCDA1&UGI-mediated cell enrichment technology vector sgRNA-ATG-Hyg-ATGStructural schematic diagrams of/HypaCas 9n-sgRNA-GT and acellular enrichment technology vector HypaCas9 n-sgRNA-GT.
FIG. 8 shows a cell enrichment technology vector sgRNA-ATG-Hyg-ATGComparison of C.T base replacement efficiency of the/HypaCas 9n-sgRNA-GT and the acellular enrichment technology vector HypaCas9n-sgRNA-GT on the target spot in the T0 vaccine.
FIG. 9 shows rAPOBEC1&Cas9n&UGI-mediated cell enrichment technology vector sgRNA-ATG-Hyg-ATGThe structure schematic diagram of/r-sgRNA-GT and non-cell enrichment technology vector r-sgRNA-GT.
FIG. 10 shows a cell enrichment technology vector sgRNA-ATG-Hyg-ATGAnd comparing the C.T base replacement efficiency of the/r-sgRNA-GT with that of the r-sgRNA-GT in a non-cell enrichment technology vector to a target point in a T0 seedling.
FIG. 11 shows a cell enrichment technology vector sgRNA-ATG-Hyg-ATGSchematic structural diagrams of/sgRNA-GT, Dissugs and acellular enrichment technology vectors sgRNA-GT.
FIG. 12 shows a cell enrichment technology vector sgRNA-ATG-Hyg-ATGC.T base replacement efficiency of the target point in T0 seedlings by/sgRNA-GT and Dissugs is compared.
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'-gtaagaaccaccagcgacac-3' and primer T1-R: 5'-gtaattgtgcttggtgatgga-3', and is used for amplifying target ALS-T1.
Primer pair T2 was composed of primer T2-F: 5'-aatatgccattcaggtgctgg-3' and primer T2-R: 5'-atcataggcagcacatgctcc-3', and is used for amplifying target ALS-T2.
Primer pair T3 was composed of primer T3-F: 5'-atggctacgaccgccgcgg-3' and primer T3-R: 5'-gcctcaattttccctgtcacacgatc-3', and is used for amplifying target ALS-T3.
Primer pair T4 was composed of primer T4-F: 5'-attgtggctcgtgctctacc-3' and primer T4-R: 5'-agacacacccacaggaacatt-3', and is used for amplifying the target DEP 1.
Primer pair T5 was composed of primer T5-F: 5'-cttcaaattctaatccccaatcc-3' and primer T5-R: 5'-ggttgttgttgaggtttaggatc-3', for amplifying the target spot wax.
Primer pair T6 was composed of primer T6-F: 5'-ttacgaactttataactttgtcgg-3' and primer T6-R: 5'-atggaggcgatgaggaagac-3', and is used for amplifying target NRT1.1B-T1.
Primer pair T7 was composed of primer T7-F: 5'-ctaatcctaccaattaacgagtcg-3' and primer T7-R: 5'-accagttgaagaagcgcatc-3', and is used for amplifying target NRT1.1B-T2.
Primer pair T8 was composed of primer T8-F: 5'-cctccatcctcctcaccg-3' and primer T8-R: 5'-tgaccttgtggacgatggtg-3', and is used for amplifying target NRT1.1B-T3.
Primer pair T9 was composed of primer T9-F: 5'-acatcgagatggagaagcgg-3' and primer T9-R: 5'-ccatgctccaatcgatgaatac-3', and is used for amplifying target CDC 48-T1.
Primer pair T10 was composed of primer T10-F: 5'-agacaccatctgcattgttct-3' and primer T10-R: 5'-ggatgtaagaaggcgacactag-3', and is used for amplifying target CDC 48-T2.
Primer pair T11 was composed of primer T11-F: 5'-gtagcttcaaattctaatcc-3' and primer T11-R: 5'-ggaggccaccgaggacgtc-3', and is used for amplifying the target point wax-T2.
In the following examples, C.T base substitutions refer to mutations from C to T at any position in the target sequence.
The efficiency of C · T base substitution is equal to the number of positive resistant calli (or positive T0 seedlings) in which C · T base substitution occurred/the number of total positive resistant calli analyzed (or total positive T0 seedlings) × 100%.
The homozygous replacement efficiency of c.t base replacement was equal to the number of homozygous mutated T0 seedlings/total number of T0 seedlings with c.t base replacement × 100%. Homozygous mutated T0 seedlings were defined as T0 seedlings in which all sites where c.t base substitutions occurred were homozygous mutations.
Japanese fine rice: reference documents: the effects of sodium nitroprusside and its photolysis products on the growth of Nippon rice seedlings and the expression of 5 hormone marker genes [ J ]. proceedings of university of Master Henan (Nature edition), 2017(2): 48-52.; the public is available from the agroforestry academy of sciences of Beijing.
Recovering the culture medium: n6 solid medium containing 200mg/L timentin.
Screening a culture medium: n6 solid medium containing 50mg/L hygromycin.
Differentiation medium: n6 solid culture medium containing 2mg/L KT, 0.2mg/L NAA, 0.5g/L glutamic acid and 0.5g/L proline.
Rooting culture medium: n6 solid medium containing 0.2mg/L NAA, 0.5g/L glutamic acid, 0.5g/L proline.