CN110964741B

CN110964741B - Nuclear localization signal FNB and application thereof in improving base editing efficiency

Info

Publication number: CN110964741B
Application number: CN201911323189.7A
Authority: CN
Inventors: 王飞鹏; 杨进孝; 宋伟; 李璐; 袁爽
Original assignee: Beijing Academy of Agriculture and Forestry Sciences
Current assignee: Beijing Academy of Agriculture and Forestry Sciences
Priority date: 2019-12-20
Filing date: 2019-12-20
Publication date: 2022-03-01
Anticipated expiration: 2039-12-20
Also published as: CN110964741A

Abstract

The invention discloses a nuclear localization signal FNB and application thereof in improving base editing efficiency. The nuclear localization signal FNB consists of a nuclear localization signal A and a nuclear localization signal B, and the nuclear localization signal A comprises 3 Flag1 tag protein and NLS1 protein; the nuclear localization signal b comprises a bpNLS protein; the amino acid sequence of the 3 Flag1 tag protein is 1 st to 22 nd of the sequence 8; the amino acid sequence of the NLS1 protein is 25 th to 31 th of a sequence 8; the amino acid sequence of the bpNLS protein is sequence 7. Experiments prove that: the nuclear localization signal FNB can improve the base editing efficiency and has good application prospect in the field of biological genome editing.

Description

Nuclear localization signal FNB and application thereof in improving base editing efficiency

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a nuclear localization signal FNB and application thereof in improving base editing efficiency.

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.

In order to improve the working efficiency and reduce the working cost, the improvement of the base substitution efficiency has been the research direction of the base editing system of the animal and plant genome. The existing base editing system still has the condition that the base editing efficiency is not high, or a target point which can not be edited by the base editing, or the editing efficiency of a certain target base in the target point is low or the target base can not be edited.

Disclosure of Invention

The object of the present invention is to improve the efficiency of base editing in a base editing system.

In order to achieve the above object, the present invention firstly provides a kit comprising a sgRNA or a biological material related to the sgRNA, a Cas9 nuclease or a biological material related to the Cas9 nuclease, a deaminase or a biological material related to the deaminase, a nuclear localization signal a or a biological material related to the nuclear localization signal a, a nuclear localization signal b or a biological material related to the nuclear localization signal b;

the nuclear localization signal A comprises 3 Flag1 tag protein and NLS1 protein;

the nuclear localization signal b comprises a bpNLS protein;

the amino acid sequence of the 3 Flag1 tag protein is 1 st to 22 nd of the sequence 8;

the amino acid sequence of the NLS1 protein is 25 th to 31 th of a sequence 8;

the amino acid sequence of the bpNLS protein is sequence 7.

In the kit, in the nuclear localization signal a, the number of the 3 Flag 1-tagged proteins may be 1 or 2 or more, and the number of the NLS1 proteins may also be 1 or 2 or more. In a specific embodiment of the invention, said nuclear localization signal a comprises 1 of said 3 Flag1 tag protein and 1 of said NLS1 protein.

In the nuclear localization signal B, the number of the bpNLS protein can be 1 or 2 or more. In a specific embodiment of the invention, said nuclear localization signal b comprises 1 of said bpNLS proteins.

Further, the amino acid sequence of the nuclear localization signal A is A1) or A2):

A1) the amino acid sequence is a protein shown in a sequence 8;

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 8 in the sequence table and has the same function;

the biological material related to the nuclear localization signal A is any one of B1) to B5):

B1) a nucleic acid molecule encoding the nuclear localization signal A;

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 amino acid sequence of the nuclear localization signal B is C1) or C2):

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;

the biological material related to the nuclear localization signal B is any one of D1) to D5):

D1) a nucleic acid molecule encoding the nuclear localization signal b;

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 D2) the expression cassette.

In a still further aspect of the present invention,

B1) the nucleic acid molecule is b1) or b2) or b 3):

b1) a cDNA molecule or DNA molecule shown in 1 st to 93 th sites of a sequence 5 in a sequence table;

b2) a cDNA or DNA molecule having 75% or more identity with the nucleotide sequence defined in b1) and encoding the nuclear localization signal A;

b3) a cDNA molecule or a DNA molecule which hybridizes with the nucleotide sequence defined by b1) or b2) under strict conditions and codes the nuclear localization signal A;

D1) the nucleic acid molecule is d1) or d2) or d 3):

d1) a cDNA molecule or DNA molecule shown in the 8647-8697 th site of the sequence 1 in the sequence table;

d2) a cDNA or DNA molecule having 75% or more identity to the nucleotide sequence defined in d1) and encoding the nuclear localization signal B;

d3) a cDNA molecule or a DNA molecule which hybridizes with the nucleotide sequence defined by d1) or d2) under stringent conditions and codes for the nuclear localization signal B.

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 variants (such as xCas9, Cas9-NG, Cas9-VQR, Cas9-VRER and the like) recognizing different PAMs, Cas9 high fidelity enzyme variants (such as HypaCas9, eSpCas9(1.1), Cas9-HF1 and the like) and the like.

The deaminase may be a cytosine deaminase or an adenine deaminase.

The cytosine deaminase can be human APOBEC3A, human AID, PmCDA1 or rAPOBEC1 and other proteins.

The adenine deaminase may be derived from different sources, such as adenine deaminase derived from Escherichia coli (e.g., protein such as ecTadA and ecTadA), and adenine deaminase derived from plant (e.g., protein such as OsTadA derived from rice or AtTadA derived from Arabidopsis).

In one embodiment of the present invention,

the Cas9 nuclease is a Cas9n protein;

the deaminase is cytosine deaminase; the cytosine deaminase is rAPOBEC1 protein;

the Cas9n protein is J1) or J2):

J1) the amino acid sequence is a protein shown in a sequence 3;

J2) 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;

the biological material related to the Cas9n protein is any one of K1) to K5):

K1) a nucleic acid molecule encoding the Cas9n protein;

K2) an expression cassette comprising the nucleic acid molecule of K1);

K3) a recombinant vector containing K1) the nucleic acid molecule or a recombinant vector containing K2) the expression cassette;

K4) a recombinant microorganism containing K1) the nucleic acid molecule, or a recombinant microorganism containing K2) the expression cassette, or a recombinant microorganism containing K3) the recombinant vector;

K5) a transgenic cell line containing K1) the nucleic acid molecule or a transgenic cell line containing K2) the expression cassette;

the rAPOBEC1 protein is L1) or L2):

l1) the amino acid sequence is a protein shown in the sequence 2;

l2) the amino acid sequence shown in the sequence 2 in the sequence table is substituted and/or deleted and/or added by one or more amino acid residues and has the same function;

the biological material related to the rAPOBEC1 protein is any one of M1) to M5):

m1) a nucleic acid molecule encoding said rAPOBEC1 protein;

m2) an expression cassette containing the nucleic acid molecule of M1);

m3) a recombinant vector containing the nucleic acid molecule of M1) or a recombinant vector containing the expression cassette of M2);

m4) a recombinant microorganism containing M1) the nucleic acid molecule, or a recombinant microorganism containing M2) the expression cassette, or a recombinant microorganism containing M3) the recombinant vector;

m5) a transgenic cell line containing M1) the nucleic acid molecule or a transgenic cell line containing M2) the expression cassette;

the sgRNA targets a target sequence;

the sgRNA is tRNA-sgRNA;

the tRNA-sgRNA structure is as follows: tRNA-RNA transcribed from the target sequence-esgRNA backbone;

the tRNA is N1) or N2) or N3):

n1) replacing T in the 474-550 th position of the sequence 1 with U to obtain an RNA molecule;

n2) carrying out substitution and/or deletion and/or addition of one or more nucleotides on the RNA molecule shown in N1) and having the same function;

n3) and N1) or N2) and has the same function;

the esgRNA backbone is P1) or P2) or P3):

p1) replacing T in the 571-656 position of the sequence 1 with U to obtain an RNA molecule;

p2) carrying out substitution and/or deletion and/or addition of one or more nucleotides on the RNA molecule shown in P1) and having the same function;

p3) and P1) or P2) and has the same function.

In the above kit, when the deaminase is a cytosine deaminase, the kit may further comprise UGI protein or biological material related to the UGI protein.

The UGI protein is Q1) or Q2):

q1) the amino acid sequence is the protein shown in the sequence 4;

q2) is protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in the sequence 4 in the sequence table and has the same function;

the biological material related to the UGI protein is any one of R1) to R5):

r1) a nucleic acid molecule encoding said UGI protein;

r2) an expression cassette containing the nucleic acid molecule according to R1);

r3) a recombinant vector containing the nucleic acid molecule according to R1) or a recombinant vector containing the expression cassette according to R2);

r4) a recombinant microorganism containing the nucleic acid molecule of R1), or a recombinant microorganism containing R2) the expression cassette, or a recombinant microorganism containing R3) the recombinant vector;

r5) a transgenic cell line which contains the nucleic acid molecule described in R1) or a transgenic cell line which contains the expression cassette described in R2).

The protein of A2), C2), J2), L2) and Q2) is a protein having 75% or more identity to the amino acid sequence of the protein represented by SEQ ID NO. 8, SEQ ID NO. 7, SEQ ID NO. 3, SEQ ID NO. 2 or SEQ ID NO. 4 and having the same function. The identity of 75% or more than 75% is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity.

The protein of A2), C2), J2), L2) and Q2) can be artificially synthesized, or can be obtained by synthesizing the coding gene and then performing biological expression.

The coding genes of the proteins A2), C2), J2), L2) and Q2) can be obtained by carrying out missense mutation of one or more base pairs and/or codons which are deleted one or more amino acid residues in the DNA sequence shown by the positions 1 to 93 (the protein shown by a coding sequence 8) of the sequence 5, the positions 8647 and 8697 (the protein shown by a coding sequence 7) of the sequence 1, the positions 4012 and 8112 (the protein shown by a coding sequence 3) of the sequence 1, the positions 3280 and 3963 (the protein shown by a coding sequence 2) of the sequence 1, and the positions 8386 and 8634 (the protein shown by a coding sequence 4) of the sequence 1.

Further, K1) the nucleic acid molecule is K1) or K2) or K3):

k1) a cDNA molecule or DNA molecule shown in 4012-8112 site of the sequence 1 in the sequence table;

k2) a cDNA molecule or DNA molecule having 75% or more identity to the nucleotide sequence defined by k1) and encoding the Cas9 n;

k3) a cDNA or DNA molecule hybridizing under stringent conditions to a nucleotide sequence defined by k1) or k2) and encoding the Cas9 n;

m1) the nucleic acid molecule is M1) or M2) or M3):

m1) the cDNA molecule or DNA molecule shown in the 3280-3963 site of the sequence 1 in the sequence table;

m2) has 75% or more than 75% identity with the nucleotide sequence defined by m1) and encodes the cDNA molecule or DNA molecule of rAPOBEC 1;

m3) hybridizes under stringent conditions with the nucleotide sequence defined in m1) or m2) and encodes a cDNA molecule or a DNA molecule of the rAPOBEC 1;

r1) the nucleic acid molecule is R1) or R2) or R3):

r1) shown in the sequence 1 at the 8386-8634 site in the sequence list;

r2) has 75% or more than 75% identity with the nucleotide sequence defined by r1) and encodes the cDNA or DNA molecule of the UGI;

r3) hybridizes under stringent conditions with the nucleotide sequence defined in r1) or r2) and encodes a cDNA molecule or a DNA molecule of said UGI.

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 of the present invention encoding the nuclear localization signal a or the nuclear localization signal b or the Cas9n or the rAPOBEC1 or the UGI 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 nuclear localization signal A or the nuclear localization signal B or the Cas9n 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 nuclear localization signal A or the nuclear localization signal B or the Cas9n 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 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 sequences

8, 7, 3, 2, 4 of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

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

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

B2) The expression cassette containing a nucleic acid molecule encoding a nuclear localization signal A (nuclear localization signal A expression cassette) refers to a DNA capable of expressing the nuclear localization signal A in a host cell, and the DNA may include not only a promoter for initiating transcription of the nuclear localization signal A but also a terminator for terminating transcription of the nuclear localization signal A. Further, the expression cassette may also include an enhancer sequence. The recombinant vector containing the nuclear localization signal A expression cassette can be constructed by using the existing expression vector.

D2) The expression cassette containing a nucleic acid molecule encoding a nuclear localization signal B (a nuclear localization signal B expression cassette) refers to a DNA capable of expressing the nuclear localization signal B in a host cell, and the DNA may include not only a promoter for initiating transcription of the nuclear localization signal B but also a terminator for terminating transcription of the nuclear localization signal B. Further, the expression cassette may also include an enhancer sequence. The recombinant vector containing the nuclear localization signal B expression cassette can be constructed by using the existing expression vector.

K2) The expression cassette containing a nucleic acid molecule encoding a Cas9n protein (Cas9n gene expression cassette) refers to a DNA capable of expressing a Cas9n protein in a host cell, and the DNA can comprise a promoter for starting the transcription of a Cas9n gene and a terminator for stopping the transcription of the Cas9n gene. Further, the expression cassette may also include an enhancer sequence. Existing expression vectors can be used to construct recombinant vectors containing the Cas9n gene expression cassette.

M2) the expression cassette containing a nucleic acid molecule encoding rAPOBEC1 protein (rAPOBEC1 gene expression cassette) refers to DNA capable of expressing rAPOBEC1 protein in a host cell, which DNA may include not only a promoter that initiates transcription of the rAPOBEC1 gene, but also a terminator that terminates transcription of the 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.

R2) the expression cassette containing a nucleic acid molecule encoding UGI protein (UGI gene expression cassette) refers to a DNA capable of expressing UGI protein in a host cell, which may include not only a promoter for initiating transcription of UGI gene but also a terminator for terminating transcription of 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 FNB-sCBE-1 recombinant expression vector.

The FNB-sCBE-1 recombinant expression vector is a sequence obtained by replacing the bpNLS nucleotide sequence shown in the 3229-3279 th site of the sequence 1 in the sequence of the sCBE-1 recombinant expression vector with a sequence 5 and keeping other sequences unchanged.

The microorganism may be a yeast, bacterium, algae or fungus. Wherein the bacterium can be an Agrobacterium, such as Agrobacterium EHA 105. In a particular embodiment of the invention, said recombinant microorganism is in particular Agrobacterium EHA105 containing said recombinant expression vector FNB-sCBE-1.

The transgenic cell line does not include propagation material.

The use of the above kit is as follows:

s1) editing of a genomic target sequence of an organism or cell of an organism;

s2) preparing an edited product of a genomic target sequence of the organism or cell of the organism;

s3) improving the efficiency of editing a genomic target sequence of an organism or a cell of an organism;

s4) to produce a product that increases the efficiency of editing a target sequence in the genome of an organism or cell of an organism.

The nuclear localization signal A or the biological material related to the nuclear localization signal A and/or the nuclear localization signal B or the biological material related to the nuclear localization signal B also belong to the protection scope of the invention.

In order to achieve the above object, the present invention also provides a new use of the above kit and/or the above nuclear localization signal a or a biological material related to the nuclear localization signal a and/or the above nuclear localization signal b or a biological material related to the nuclear localization signal b.

The invention provides the use of the kit as described above and/or of the nuclear localization signal A as described above or of a biological material associated with the nuclear localization signal A as described above and/or of the nuclear localization signal B as described above or of a biological material associated with the nuclear localization signal B as described above in any of S1) -S4):

s1) editing of a genomic target sequence of an organism or cell of an organism;

In order to achieve the above object, the present invention also provides the method of T1) or T2):

t1) or a method for increasing the efficiency of editing a genomic target sequence of an organism or cell comprising the steps of: allowing an organism or a biological cell to express the nuclear localization signal A, the nuclear localization signal B, the sgRNA, the Cas9 nuclease, and the deaminase; the sgRNA targets the target sequence;

t2) biological mutant, comprising the following steps: editing the genome of the organism according to the method described in T1) to obtain a biological mutant.

In the above method, when the deaminase is a cytosine deaminase, T1) wherein the sgRNA is tRNA-esgRNA, the tRNA-esgRNA obtained by transcription of the DNA molecule of the tRNA-esgRNA is an immature RNA precursor, and the tRNA in the RNA precursor is cleaved by two enzymes (RNase P and RNase Z) to obtain mature RNA. And (b) obtaining a plurality of independent mature RNAs by the number of targets in a recombinant expression vector, wherein each mature RNA consists of RNA transcribed by the target sequence and the esgRNA skeleton in sequence or consists of RNA transcribed by the target sequence, the esgRNA skeleton and residual individual bases of the tRNA in sequence.

In the above method, when the deaminase is a cytosine deaminase, the T1) may further comprise a step of introducing the UGI protein into an organism or an organism cell, and the number of UGIs may be 1 or 2 or more. In a specific embodiment of the present invention, the number of the UGIs is specifically 2.

Further, the method for expressing the nuclear localization signal a, the nuclear localization signal b, the sgRNA, the Cas9 nuclease and the deaminase in an organism or a biological cell is to introduce a gene encoding the nuclear localization signal a, a gene encoding the nuclear localization signal b, a DNA molecule for transcribing the sgRNA, a gene encoding the Cas9 nuclease, a gene encoding the cytosine deaminase and a gene encoding UGI into the organism or the biological cell.

Furthermore, the coding gene of the nuclear localization signal A, the coding gene of the nuclear localization signal B, the DNA molecule for transcribing the sgRNA, the coding gene of the Cas9 nuclease, the coding gene of the cytosine deaminase and the coding gene of the UGI are introduced into an organism or an organism cell through a recombinant expression vector. The coding gene of the nuclear localization signal A, the coding gene of the nuclear localization signal B, the DNA molecule for transcribing the sgRNA, the coding gene of the Cas9 nuclease, the coding gene of the cytosine deaminase and the coding gene of the UGI can be introduced into organisms or biological cells through the same recombinant expression vector, or can be introduced into the organisms or biological cells through two or more recombinant expression vectors.

In a specific embodiment of the invention, the recombinant expression vector comprises an expression cassette consisting of a promoter, a coding gene of a nuclear localization signal A, a coding gene of cytosine deaminase rAPOBEC1, a coding gene of Cas9n nuclease, a coding gene of UGI, a coding gene of a nuclear localization signal B and a terminator in sequence. The recombinant expression vector can be the FNB-sCBE-1 recombinant expression vector.

In the kit or use or method, the number of target sequences may be 1 or 2 or more.

In the above kit or use or method, the editing of the target sequence may be a mutation of base a to base G or a mutation of base C to base T. The base A can be a base A at any position on the target sequence, and the base C can be a base C at any position on the target sequence.

In the above-described kit of parts or use or method,

the organism is X1) or X2) or X3) or X4):

x1) plant or animal;

x2) a monocot or dicot;

x3) gramineous plants;

x4) rice;

the biological cell is Y1) or Y2) or Y3) or Y4):

y1) plant cells or animal cells;

y2) a monocotyledonous or dicotyledonous plant cell;

y3) a graminaceous plant cell;

y4) rice cells.

The invention adds 3 Flag1&1 NLS1 nuclear localization signal (nuclear localization signal A) in front of rAPOBEC1 element in rAPOBEC1& Cas9n & UGI base editing system, and finds that after adding bpNLS nuclear localization signal (nuclear localization signal B) behind UGI element: the base editing system rAPOBEC1& Cas9n & UGI has obviously improved C.T base replacement efficiency (up to 85.7 percent).

Drawings

FIG. 1 is a schematic diagram of a structure of a recombinant expression vector of a cytosine base editor.

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'-GCGAATGGCCACAGGG-3' and primer T1-R: 5'-TCTGATCATCATGGATTCCTTC-3', and is used for amplifying target points T1 and T2.

Primer pair T3 was composed of primer T3-F: 5'-CTCATCCACGACACATCCATAC-3' and primer T3-R: 5'-ATACTTCTGGCTTATGCTTGCG-3', and is used for amplifying target T3.

Primer pair T4 was composed of primer T4-F: 5'-GCCATCAACTAAACACAGCC-3' and primer T4-R: 5'-CATGAGCGTGAGAATTCTGATC-3', and is used for amplifying target T4.

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 was equal to the number of positive T0 seedlings with C · T base substitution/total positive T0 seedlings analyzed × 100%.

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.

Example 1 application of Nuclear localization Signal FNB to improvement of C.T base substitution efficiency

Design and construction of recombinant expression vector

1. Design of recombinant expression vectors

A nuclear localization signal is added in an rAPOBEC1& Cas9n & UGI base editing system, the added nuclear localization signal is divided into four design types according to different added nuclear localization signals, and the structural schematic diagram of the recombinant expression vector containing the four different design types is shown in figure 1. The design modes of four different design types are as follows:

sCBE system: the bpNLS nuclear localization signal was added before the rAPOBEC1 element in the rAPOBEC1& Cas9n & UGI base editing system and after the UGI element. The amino acid sequence of the bpNLS nuclear localization signal is as follows: KRTADGSEFEPKKKRKV (SEQ ID NO: 7). This type of design is designated bpNLS-bpNLS.

FNLS-sCBE system: at rAPOBEC1&Cas9n&Addition of 3 Flag1 to rAPOBEC1 element in UGI base editing System&1 × NLS1 nuclear localization signal, and 1 × NLS1 nuclear localization signal was added after UGI element. 3 Flag1&The 1 × NLS1 nuclear localization signal sequentially comprises 13 × Flag1 tag protein and 1 NLS1 protein, and 3 × Flag1&1-NLS 1 amino acid sequenceThe following were used:

(sequence 8), wherein the amino acid sequence of the 3 Flag1 tag protein is shown by underlining, and the amino acid sequence of the NLS1 protein is shown by wavy lines. The 1 × NLS1 nuclear localization signal includes 1 NLS1 protein, and the amino acid sequence of 1 × NLS1 is as follows: PKKKRKV. This design type is denoted as 3 Flag1&1*NLS1-1*NLS1。

F4NLS-sCBE System: at rAPOBEC1&Cas9n&Addition of 3 Flag2 to rAPOBEC1 element in UGI base editing System&4 × NLS2 nuclear localization signal, and 4 × NLS2 nuclear localization signal was added after UGI element. 3 Flag2&The 4 × NLS2 nuclear localization signal sequentially comprises 13 × Flag2 tag protein and 4NLS 2 protein, 3 × Flag2&4NLS 2 the amino acid sequence of the nuclear localization signal is as follows:

the amino acid sequence of the 3 Flag2 tag protein is shown by underlining, and the amino acid sequence of the NLS2 protein is shown by wavy lines. The 4 × NLS2 nuclear localization signal includes 4NLS 2 proteins, and the amino acid sequence of the 4 × NLS2 nuclear localization signal is as follows: PKKKRKVGGSPKKKRKVGGSPKKKRKVGGSPKKKRKV are provided. This design type is denoted as 3 Flag2&4*NLS2-4*NLS2。

FNB-sCBE system: the 3 Flag1&1 NLS1 nuclear localization signal was added before the rAPOBEC1 element in the rAPOBEC1& Cas9n & UGI base editing system and the bpNLS nuclear localization signal was added after the UGI element. 3 Flag1&1 NLS1 nuclear localization signal amino acid sequence of 8, bpNLS nuclear localization signal amino acid sequence of 7. This design type was designated 3 Flag1&1 NLS1-bpnls (fnb).

2. Construction of recombinant expression vectors

The following recombinant expression vectors were artificially synthesized, each of which was a circular plasmid:

a recombinant expression vector comprising bpNLS-bpNLS: sCBE-1;

a recombinant expression vector containing 3 Flag1&1 NLS1-1 NLS 1: FNLS-sCBE-1;

a recombinant expression vector containing 3 Flag2& 4NLS 2-4 NLS 2: f4 NLS-sCBE-1;

a recombinant expression vector containing 3 Flag1&1 NLS 1-bpNLS: FNB-sCBE-1.

The nucleotide sequence of the sCBE-1 recombinant expression vector is sequence 1 in a sequence table. Wherein, the 131-467 site of the sequence 1 is a nucleotide sequence of OsU3 promoter, the 474-550 site, 657-733 site, 840-916 site and 1023-1099 site are nucleotide sequences of tRNA, the 551-656 site, 734-839 site, 917-1022 site and 1100-1205 site are nucleotide sequences of esgRNA, the 551-570 site, 734-753 site, 917-936 site and 1100-1119 site are nucleotide sequences of T1, T2, T3 and T4 targets, respectively, the 571-656 site, 754-839 site, 937-1022 site and 1120-1119 site are nucleotide sequences of esgRNA framework, and the 1206-1496 site is a nucleotide sequence of OsU3 terminator; the 1503-3216 site of the sequence 1 is a nucleotide sequence of an OsUbq3 promoter, the 3229-3279 site is a bpNLS nucleotide sequence, the 3280-3963 site is a coding sequence (without a stop codon) of an rAPOBEC1 protein, and the coding sequence 2 shows the rAPOBEC1 protein; the 4012-8112 site of the sequence 1 is a coding sequence (without a stop codon) of the Cas9n protein, and the coding sequence 3 shows the Cas9n protein; the 8125-8373 and 8386-8634 of the sequence 1 are both UGI protein coding sequences (without stop codons), and the UGI protein shown in the coding sequence 4; the 8647-8697 th site of the sequence 1 is a bpNLS nucleotide sequence; the 9040-position 9292 of the sequence 1 is a Nos terminator sequence; the 9333-11325 site of the sequence 1 is the nucleotide sequence of ZmUbi1 promoter, the 11332-12357 site is the coding sequence of hygromycin phosphotransferase, and the 12384-12599 site is the nucleotide sequence of CaMV35S polyA. The four targets of the sCBE-1 recombinant expression vector are T1, T2, T3 and T4 respectively, and the sequences are shown in Table 1.

The nucleotide sequence of the FNLS-sCBE-1 recombinant expression vector is a sequence obtained by replacing the bpNLS nucleotide sequence shown in the 3229-3279 th site of the sequence 1 in the sCBE-1 recombinant expression vector sequence with a sequence 5, replacing the bpNLS nucleotide sequence shown in the 8647-8697 th site of the sequence 5 with a nucleotide sequence shown in the 73-93 th site of the sequence 5 and keeping other sequences unchanged. Wherein, the 1 st to 66 th positions of the sequence 5 are 3 Flag1 nucleotide sequences, the 73 th to 93 th positions are NLS1 nucleotide sequences, and the sequence 5 totally contains 1 NLS1 nucleotide sequences.

The nucleotide sequence of the F4NLS-sCBE-1 recombinant expression vector is a sequence obtained by replacing the bpNLS nucleotide sequence shown in 3229-3279 th site of the sequence 1 in the sCBE-1 recombinant expression vector sequence with a sequence 6, replacing the bpNLS nucleotide sequence shown in 8647-8697 th site with a nucleotide sequence shown in 55-201 th site of the sequence 6 and keeping other sequences unchanged. Wherein, the 1 st to 66 th positions of the sequence 6 are 3 Flag2 nucleotide sequences, the 73 rd to 93 th positions, the 103 rd and 123 th positions, the 133 th and 153 th positions and the 163 th and 183 th positions are NLS2 nucleotide sequences, and the sequence 6 totally contains 4NLS 2 nucleotide sequences.

The FNB-sCBE-1 recombinant expression vector is a sequence obtained by replacing the nucleotide sequence of bpNLS shown in the 3229-3279 th site of the sequence 1 in the sequence of the sCBE-1 recombinant expression vector with the sequence 5 and keeping other sequences unchanged.

The target nucleotide sequence and the corresponding PAM sequence of each vector are shown in table 1.

TABLE 1

Second, obtaining the Positive T0 Rice seedlings

Respectively operating the sCBE-1 vector, the FNLS-sCBE-1 vector, the F4NLS-sCBE-1 vector and the FNB-sCBE-1 vector obtained in the step one according to the following steps 1-9:

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. And (3) putting the rice resistant callus obtained in the step (7) on a differentiation culture medium, performing illumination culture at 25 ℃ for about 1 month, transplanting the differentiated plantlets on a rooting culture medium, and performing illumination culture at 25 ℃ for 2 weeks to obtain rice T0 seedlings.

9. Extracting genome DNA of rice T0 seedling, using the genome DNA as a template, and performing PCR amplification by using a primer pair consisting of a primer F (5'-CCGAGGAGACTATCACCCCT-3') and a primer R (5'-CGACCCATAACCTTGACAAGC-3') to obtain a PCR amplification product; the PCR amplification product was subjected to agarose gel electrophoresis, followed by judgment as follows: if the PCR amplification product contains a DNA fragment of about 853bp, the corresponding rice T0 seedling is a rice positive T0 seedling; if the PCR amplification product does not contain the DNA fragment of about 853bp, the corresponding rice T0 seedling is not the rice positive T0 seedling.

Third, result analysis

1. Taking the genomic DNA of the rice positive T0 seedling obtained in the step two as a template for each vector, and carrying out PCR amplification on T1 by adopting primers for T1 and T2 targets to obtain PCR amplification products; for the T3 target, carrying out PCR amplification on T3 by adopting a primer pair to obtain a PCR amplification product; for the T4 target, PCR amplification is carried out by adopting a primer pair T4 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 positive T0 seedlings with C.T base substitution at T1, T2, T3 and T4 is counted respectively, and the C.T base substitution efficiency is calculated, and the result is shown in Table 2.

The nuclear localization signal used by the sCBE system is bpNLS-bpNLS; the nuclear localization signal used by the FNLS-sccbe system was 3 Flag1&1 NLS1-1 NLS 1; the nuclear localization signal used by the F4 NLS-sccbe system was 3 Flag2& 4NLS 2-4 NLS 2; the nuclear localization signal used by the FNB-sccbe system was 3 Flag1&1 NLS 1-bpNLS.

The base editing results of different systems on the T1, T2, T3 and T4 targets show that: compared with the sCBE and FNLS-sCBE systems, the replacement efficiency of C.T basic groups of all four target points, namely the FNB-sCBE system, is greatly improved. Compared with the F4NLS-sCBE system, the FNB-sCBE system has the advantages that the C.T base substitution efficiency of the FNB-sCBE system is greatly improved compared with that of the F4NLS-sCBE system except for the T2 target point. Particularly for the T3 target point, the other three systems can not realize editing, and the FNB-sCBE system can realize editing. It can be seen that FNB-sCBE using the nuclear localization signals 3 Flag &1 NLS1 and bpNLS in combination can significantly improve the efficiency of C · T base substitution.

TABLE 2

Sequence listing

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

<120> nuclear localization signal FNB and application thereof in improving base editing efficiency

<160>8

<170>PatentIn version 3.5

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<211>19005

<212>DNA

<213>Artificial Sequence

<400>1

ggtggcagga tatattgtgg tgtaaacatg gcactagcct caccgtcttc gcagacgagg 60

ccgctaagtc gcagctacgc tctcaacggc actgactagg tagtttaaac gtgcacttaa 120

ttaaggtacc gaagcaactt aaagttatca ggcatgcatg gatcttggag gaatcagatg 180

tgcagtcagg gaccatagca caagacaggc gtcttctact ggtgctacca gcaaatgctg 240

gaagccggga acactgggta cgttggaaac cacgtgatgt gaagaagtaa gataaactgt 300

aggagaaaag catttcgtag tgggccatga agcctttcag gacatgtatt gcagtatggg 360

ccggcccatt acgcaattgg acgacaacaa agactagtat tagtaccacc tcggctatcc 420

acatagatca aagctgattt aaaagagttg tgcagatgat ccgtggcgga tccaacaaag 480

caccagtggt ctagtggtag aatagtaccc tgccacggta cagacccggg ttcgattccc 540

ggctggtgca ttgtaatcaa ctccagtgtc gtttcagagc tatgctggaa acagcatagc 600

aagttgaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgcaaca 660

aagcaccagt ggtctagtgg tagaatagta ccctgccacg gtacagaccc gggttcgatt 720

cccggctggt gcaccttctc caggaatgac ggagtttcag agctatgctg gaaacagcat 780

agcaagttga aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgca 840

acaaagcacc agtggtctag tggtagaata gtaccctgcc acggtacaga cccgggttcg 900

attcccggct ggtgcagacc agccagcgtc tggcgcgttt cagagctatg ctggaaacag 960

catagcaagt tgaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt 1020

gcaacaaagc accagtggtc tagtggtaga atagtaccct gccacggtac agacccgggt 1080

tcgattcccg gctggtgcaa atcctgatga tgctgcagtg tttcagagct atgctggaaa 1140

cagcatagca agttgaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc 1200

ggtgcttttt tttttcgttt tgcattgagt tttctccgtc gcatgtttgc agttttattt 1260

tccgttttgc attgaaattt ctccgtctca tgtttgcagc gtgttcaaaa agtacgcagc 1320

tgtatttcac ttatttacgg cgccacattt tcatgccgtt tgtgccaact atcccgagct 1380

agtgaataca gcttggcttc acacaacact ggtgacccgc tgacctgctc gtacctcgta 1440

ccgtcgtacg gcacagcatt tggaattaaa gggtgtgatc gatactgctt gctgctaagc 1500

ttacaaattc gggtcaaggc ggaagccagc gcgccacccc acgtcagcaa atacggaggc 1560

gcggggttga cggcgtcacc cggtcctaac ggcgaccaac aaaccagcca gaagaaatta 1620

cagtaaaaaa aaagtaaatt gcactttgat ccacctttta ttacctaagt ctcaatttgg 1680

atcaccctta aacctatctt ttcaatttgg gccgggttgt ggtttggact accatgaaca 1740

acttttcgtc atgtctaact tccctttcag caaacatatg aaccatatat agaggagatc 1800

ggccgtatac tagagctgat gtgtttaagg tcgttgattg cacgagaaaa aaaaatccaa 1860

atcgcaacaa tagcaaattt atctggttca aagtgaaaag atatgtttaa aggtagtcca 1920

aagtaaaact tatagataat aaaatgtggt ccaaagcgta attcactcaa aaaaaatcaa 1980

cgagacgtgt accaaacgga gacaaacggc atcttctcga aatttcccaa ccgctcgctc 2040

gcccgcctcg tcttcccgga aaccgcggtg gtttcagcgt ggcggattct ccaagcagac 2100

ggagacgtca cggcacggga ctcctcccac cacccaaccg ccataaatac cagccccctc 2160

atctcctctc ctcgcatcag ctccaccccc gaaaaatttc tccccaatct cgcgaggctc 2220

tcgtcgtcga atcgaatcct ctcgcgtcct caaggtacgc tgcttctcct ctcctcgctt 2280

cgtttcgatt cgatttcgga cgggtgaggt tgttttgttg ctagatccga ttggtggtta 2340

gggttgtcga tgtgattatc gtgagatgtt taggggttgt agatctgatg gttgtgattt 2400

gggcacggtt ggttcgatag gtggaatcgt ggttaggttt tgggattgga tgttggttct 2460

gatgattggg gggaattttt acggttagat gaattgttgg atgattcgat tggggaaatc 2520

ggtgtagatc tgttggggaa ttgtggaact agtcatgcct gagtgattgg tgcgatttgt 2580

agcgtgttcc atcttgtagg ccttgttgcg agcatgttca gatctactgt tccgctcttg 2640

attgagttat tggtgccatg ggttggtgca aacacaggct ttaatatgtt atatctgttt 2700

tgtgtttgat gtagatctgt agggtagttc ttcttagaca tggttcaatt atgtagcttg 2760

tgcgtttcga tttgatttca tatgttcaca gattagataa tgatgaactc ttttaattaa 2820

ttgtcaatgg taaataggaa gtcttgtcgc tatatctgtc ataatgatct catgttacta 2880

tctgccagta atttatgcta agaactatat tagaatatca tgttacaatc tgtagtaata 2940

tcatgttaca atctgtagtt catctatata atctattgtg gtaatttctt tttactatct 3000

gtgtgaagat tattgccact agttcattct acttatttct gaagttcagg atacgtgtgc 3060

tgttactacc tatctgaata catgtgtgat gtgcctgtta ctatcttttt gaatacatgt 3120

atgttctgtt ggaatatgtt tgctgtttga tccgttgttg tgtccttaat cttgtgctag 3180

ttcttaccct atctgtttgg tgattatttc ttgcagtacg taagcatgaa gaggaccgcc 3240

gacggcagcg agttcgagcc gaagaagaag aggaaggtgt ccagcgagac aggaccagtg 3300

gcagtcgacc caacactgcg caggcggatc gagccacacg agttcgaggt gttcttcgat 3360

ccgagggagc tccggaagga gacatgcctc ctgtacgaga tcaactgggg cggccgccac 3420

tctatctgga ggcatacctc acagaacaca aataagcatg tggaggtcaa cttcatcgag 3480

aagttcacca cagagcggta cttctgcccg aatacgcgct gctccatcac ctggttcctg 3540

tcgtggtccc catgcggaga gtgctcgagg gcaatcacgg agttcctctc ccgctacccg 3600

cacgtcaccc tgttcatcta catcgcacgg ctctaccacc atgcggaccc gcggaatagg 3660

cagggcctcc gcgatctgat ctcttcaggc gtgacaatcc agatcatgac ggagcaggag 3720

tcaggctact gctggaggaa cttcgtcaat tacagcccat ctaacgaggc acactggccg 3780

cgctacccgc atctctgggt gcgcctctac gtgctcgagc tgtactgcat catcctcggc 3840

ctgccgccat gcctcaatat cctgcgcagg aagcagccgc agctgacgtt cttcaccatc 3900

gccctccaga gctgccacta ccagcggctc cctccgcata tcctgtgggc gacaggcctc 3960

aagtcaggct cggagacacc tggcacgtcc gagagcgcca ccccggagtc tgacaagaag 4020

tactccatcg gcctcgccat cggcaccaac agcgtcggct gggcggtgat caccgacgag 4080

tacaaggtcc cgtccaagaa gttcaaggtc ctgggcaaca ccgaccgcca ctccatcaag 4140

aagaacctca tcggcgccct cctcttcgac tccggcgaga cggcggaggc gacccgcctc 4200

aagcgcaccg cccgccgccg ctacacccgc cgcaagaacc gcatctgcta cctccaggag 4260

atcttctcca acgagatggc gaaggtcgac gactccttct tccaccgcct cgaggagtcc 4320

ttcctcgtgg aggaggacaa gaagcacgag cgccacccca tcttcggcaa catcgtcgac 4380

gaggtcgcct accacgagaa gtaccccact atctaccacc ttcgtaagaa gcttgttgac 4440

tctactgata aggctgatct tcgtctcatc taccttgctc tcgctcacat gatcaagttc 4500

cgtggtcact tccttatcga gggtgacctt aaccctgata actccgacgt ggacaagctc 4560

ttcatccagc tcgtccagac ctacaaccag ctcttcgagg agaaccctat caacgcttcc 4620

ggtgtcgacg ctaaggcgat cctttccgct aggctctcca agtccaggcg tctcgagaac 4680

ctcatcgccc agctccctgg tgagaagaag aacggtcttt tcggtaacct catcgctctc 4740

tccctcggtc tgacccctaa cttcaagtcc aacttcgacc tcgctgagga cgctaagctt 4800

cagctctcca aggataccta cgacgatgat ctcgacaacc tcctcgctca gattggagat 4860

cagtacgctg atctcttcct tgctgctaag aacctctccg atgctatcct cctttcggat 4920

atccttaggg ttaacactga gatcactaag gctcctcttt ctgcttccat gatcaagcgc 4980

tacgacgagc accaccagga cctcaccctc ctcaaggctc ttgttcgtca gcagctcccc 5040

gagaagtaca aggagatctt cttcgaccag tccaagaacg gctacgccgg ttacattgac 5100

ggtggagcta gccaggagga gttctacaag ttcatcaagc caatccttga gaagatggat 5160

ggtactgagg agcttctcgt taagcttaac cgtgaggacc tccttaggaa gcagaggact 5220

ttcgataacg gctctatccc tcaccagatc caccttggtg agcttcacgc catccttcgt 5280

aggcaggagg acttctaccc tttcctcaag gacaaccgtg agaagatcga gaagatcctt 5340

actttccgta ttccttacta cgttggtcct cttgctcgtg gtaactcccg tttcgcttgg 5400

atgactagga agtccgagga gactatcacc ccttggaact tcgaggaggt tgttgacaag 5460

ggtgcttccg cccagtcctt catcgagcgc atgaccaact tcgacaagaa cctccccaac 5520

gagaaggtcc tccccaagca ctccctcctc tacgagtact tcacggtcta caacgagctc 5580

accaaggtca agtacgtcac cgagggtatg cgcaagcctg ccttcctctc cggcgagcag 5640

aagaaggcta tcgttgacct cctcttcaag accaaccgca aggtcaccgt caagcagctc 5700

aaggaggact acttcaagaa gatcgagtgc ttcgactccg tcgagatcag cggcgttgag 5760

gaccgtttca acgcttctct cggtacctac cacgatctcc tcaagatcat caaggacaag 5820

gacttcctcg acaacgagga gaacgaggac atcctcgagg acatcgtcct cactcttact 5880

ctcttcgagg atagggagat gatcgaggag aggctcaaga cttacgctca tctcttcgat 5940

gacaaggtta tgaagcagct caagcgtcgc cgttacaccg gttggggtag gctctcccgc 6000

aagctcatca acggtatcag ggataagcag agcggcaaga ctatcctcga cttcctcaag 6060

tctgatggtt tcgctaacag gaacttcatg cagctcatcc acgatgactc tcttaccttc 6120

aaggaggata ttcagaaggc tcaggtgtcc ggtcagggcg actctctcca cgagcacatt 6180

gctaaccttg ctggttcccc tgctatcaag aagggcatcc ttcagactgt taaggttgtc 6240

gatgagcttg tcaaggttat gggtcgtcac aagcctgaga acatcgtcat cgagatggct 6300

cgtgagaacc agactaccca gaagggtcag aagaactcga gggagcgcat gaagaggatt 6360

gaggagggta tcaaggagct tggttctcag atccttaagg agcaccctgt cgagaacacc 6420

cagctccaga acgagaagct ctacctctac tacctccaga acggtaggga tatgtacgtt 6480

gaccaggagc tcgacatcaa caggctttct gactacgacg tcgaccacat tgttcctcag 6540

tctttcctta aggatgactc catcgacaac aaggtcctca cgaggtccga caagaacagg 6600

ggtaagtcgg acaacgtccc ttccgaggag gttgtcaaga agatgaagaa ctactggagg 6660

cagcttctca acgctaagct cattacccag aggaagttcg acaacctcac gaaggctgag 6720

aggggtggcc tttccgagct tgacaaggct ggtttcatca agaggcagct tgttgagacg 6780

aggcagatta ccaagcacgt tgctcagatc ctcgattcta ggatgaacac caagtacgac 6840

gagaacgaca agctcatccg cgaggtcaag gtgatcaccc tcaagtccaa gctcgtctcc 6900

gacttccgca aggacttcca gttctacaag gtccgcgaga tcaacaacta ccaccacgct 6960

cacgatgctt accttaacgc tgtcgttggt accgctctta tcaagaagta ccctaagctt 7020

gagtccgagt tcgtctacgg tgactacaag gtctacgacg ttcgtaagat gatcgccaag 7080

tccgagcagg agatcggcaa ggccaccgcc aagtacttct tctactccaa catcatgaac 7140

ttcttcaaga ccgagatcac cctcgccaac ggcgagatcc gcaagcgccc tcttatcgag 7200

acgaacggtg agactggtga gatcgtttgg gacaagggtc gcgacttcgc tactgttcgc 7260

aaggtccttt ctatgcctca ggttaacatc gtcaagaaga ccgaggtcca gaccggtggc 7320

ttctccaagg agtctatcct tccaaagaga aactcggaca agctcatcgc taggaagaag 7380

gattgggacc ctaagaagta cggtggtttc gactccccta ctgtcgccta ctccgtcctc 7440

gtggtcgcca aggtggagaa gggtaagtcg aagaagctca agtccgtcaa ggagctcctc 7500

ggcatcacca tcatggagcg ctcctccttc gagaagaacc cgatcgactt cctcgaggcc 7560

aagggctaca aggaggtcaa gaaggacctc atcatcaagc tccccaagta ctctcttttc 7620

gagctcgaga acggtcgtaa gaggatgctg gcttccgctg gtgagctcca gaagggtaac 7680

gagcttgctc ttccttccaa gtacgtgaac ttcctctacc tcgcctccca ctacgagaag 7740

ctcaagggtt cccctgagga taacgagcag aagcagctct tcgtggagca gcacaagcac 7800

tacctcgacg agatcatcga gcagatctcc gagttctcca agcgcgtcat cctcgctgac 7860

gctaacctcg acaaggtcct ctccgcctac aacaagcacc gcgacaagcc catccgcgag 7920

caggccgaga acatcatcca cctcttcacg ctcacgaacc tcggcgcccc tgctgctttc 7980

aagtacttcg acaccaccat cgacaggaag cgttacacgt ccaccaagga ggttctcgac 8040

gctactctca tccaccagtc catcaccggt ctttacgaga ctcgtatcga cctttcccag 8100

cttggtggtg attccggcgg cagcaccaac ctctccgaca tcatcgagaa ggagacaggc 8160

aagcagctcg tgatccagga gagcatcctc atgctcccgg aggaggtgga ggaggtcatc 8220

ggcaacaagc cggagtccga catcctcgtg cacaccgcct acgacgagtc caccgacgag 8280

aacgtgatgc tcctcacctc agatgcacca gagtacaagc catgggcact cgtgatccag 8340

gacagcaacg gcgagaacaa gatcaagatg ctctccggcg gctccaccaa cctctccgac 8400

atcatcgaga aggagacagg caagcagctc gtgatccagg agagcatcct catgctcccg 8460

gaggaggtgg aggaggtcat cggcaacaag ccggagtccg acatcctcgt gcacaccgcc 8520

tacgacgagt ccaccgacga gaacgtgatg ctcctcacct cagatgcacc agagtacaag 8580

ccatgggcac tcgtgatcca ggacagcaac ggcgagaaca agatcaagat gctctccggc 8640

ggctccaaga ggaccgccga cggcagcgag ttcgagccga agaagaagag gaaggtgtag 8700

actagttcag ccagtttggt ggagctgccg atgtgcctgg tcgtcccgag cctctgttcg 8760

tcaagtattt gtggtgctga tgtctacttg tgtctggttt aatggaccat cgagtccgta 8820

tgatatgtta gttttatgaa acagtttcct gtgggacagc agtatgcttt atgaataagt 8880

tggatttgaa cctaaatatg tgctcaattt gctcatttgc atctcattcc tgttgatgtt 8940

ttatctgagt tgcaagtttg aaaatgctgc atattcttat taaatcgtca tttactttta 9000

tcttaatgag ctttgcaatg gcctatggga tataaaagag atcgttcaaa catttggcaa 9060

taaagtttct taagattgaa tcctgttgcc ggtcttgcga tgattatcat ataatttctg 9120

ttgaattacg ttaagcatgt aataattaac atgtaatgca tgacgttatt tatgagatgg 9180

gtttttatga ttagagtccc gcaattatac atttaatacg cgatagaaaa caaaatatag 9240

cgcgcaaact aggataaatt atcgcgcgcg gtgtcatcta tgttactaga tcggcgcctg 9300

tccgggcgcg cctggtggat cgtccgccta ggctgcagtg cagcgtgacc cggtcgtgcc 9360

cctctctaga gataatgagc attgcatgtc taagttataa aaaattacca catatttttt 9420

ttgtcacact tgtttgaagt gcagtttatc tatctttata catatattta aactttactc 9480

tacgaataat ataatctata gtactacaat aatatcagtg ttttagagaa tcatataaat 9540

gaacagttag acatggtcta aaggacaatt gagtattttg acaacaggac tctacagttt 9600

tatcttttta gtgtgcatgt gttctccttt ttttttgcaa atagcttcac ctatataata 9660

cttcatccat tttattagta catccattta gggtttaggg ttaatggttt ttatagacta 9720

atttttttag tacatctatt ttattctatt ttagcctcta aattaagaaa actaaaactc 9780

tattttagtt tttttattta ataatttaga tataaaatag aataaaataa agtgactaaa 9840

aattaaacaa atacccttta agaaattaaa aaaactaagg aaacattttt cttgtttcga 9900

gtagataatg ccagcctgtt aaacgccgtc gacgagtcta acggacacca accagcgaac 9960

cagcagcgtc gcgtcgggcc aagcgaagca gacggcacgg catctctgtc gctgcctctg 10020

gacccctctc gagagttccg ctccaccgtt ggacttgctc cgctgtcggc atccagaaat 10080

tgcgtggcgg agcggcagac gtgagccggc acggcaggcg gcctcctcct cctctcacgg 10140

caccggcagc tacgggggat tcctttccca ccgctccttc gctttccctt cctcgcccgc 10200

cgtaataaat agacaccccc tccacaccct ctttccccaa cctcgtgttg ttcggagcgc 10260

acacacacac aaccagatct cccccaaatc cacccgtcgg cacctccgct tcaaggtacg 10320

ccgctcgtcc tccccccccc cccctctcta ccttctctag atcggcgttc cggtccatgg 10380

ttagggcccg gtagttctac ttctgttcat gtttgtgtta gatccgtgtt tgtgttagat 10440

ccgtgctgct agcgttcgta cacggatgcg acctgtacgt cagacacgtt ctgattgcta 10500

acttgccagt gtttctcttt ggggaatcct gggatggctc tagccgttcc gcagacggga 10560

tcgatttcat gatttttttt gtttcgttgc atagggtttg gtttgccctt ttcctttatt 10620

tcaatatatg ccgtgcactt gtttgtcggg tcatcttttc atgctttttt ttgtcttggt 10680

tgtgatgatg tggtctggtt gggcggtcgt tctagatcgg agtagaattc tgtttcaaac 10740

tacctggtgg atttattaat tttggatctg tatgtgtgtg ccatacatat tcatagttac 10800

gaattgaaga tgatggatgg aaatatcgat ctaggatagg tatacatgtt gatgcgggtt 10860

ttactgatgc atatacagag atgctttttg ttcgcttggt tgtgatgatg tggtgtggtt 10920

gggcggtcgt tcattcgttc tagatcggag tagaatactg tttcaaacta cctggtgtat 10980

ttattaattt tggaactgta tgtgtgtgtc atacatcttc atagttacga gtttaagatg 11040

gatggaaata tcgatctagg ataggtatac atgttgatgt gggttttact gatgcatata 11100

catgatggca tatgcagcat ctattcatat gctctaacct tgagtaccta tctattataa 11160

taaacaagta tgttttataa ttattttgat cttgatatac ttggatgatg gcatatgcag 11220

cagctatatg tggatttttt tagccctgcc ttcatacgct atttatttgc ttggtactgt 11280

ttcttttgtc gatgctcacc ctgttgtttg gtgttacttc tgcaggagct catgaaaaag 11340

cctgaactca ccgcgacgtc tgtcgagaag tttctgatcg aaaagttcga cagcgtctcc 11400

gacctgatgc agctctcgga gggcgaagaa tctcgtgctt tcagcttcga tgtaggaggg 11460

cgtggatatg tcctgcgggt aaatagctgc gccgatggtt tctacaaaga tcgttatgtt 11520

tatcggcact ttgcatcggc cgcgctcccg attccggaag tgcttgacat tggggagttt 11580

agcgagagcc tgacctattg catctcccgc cgttcacagg gtgtcacgtt gcaagacctg 11640

cctgaaaccg aactgcccgc tgttctacaa ccggtcgcgg aggctatgga tgcgatcgct 11700

gcggccgatc ttagccagac gagcgggttc ggcccattcg gaccgcaagg aatcggtcaa 11760

tacactacat ggcgtgattt catatgcgcg attgctgatc cccatgtgta tcactggcaa 11820

actgtgatgg acgacaccgt cagtgcgtcc gtcgcgcagg ctctcgatga gctgatgctt 11880

tgggccgagg actgccccga agtccggcac ctcgtgcacg cggatttcgg ctccaacaat 11940

gtcctgacgg acaatggccg cataacagcg gtcattgact ggagcgaggc gatgttcggg 12000

gattcccaat acgaggtcgc caacatcttc ttctggaggc cgtggttggc ttgtatggag 12060

cagcagacgc gctacttcga gcggaggcat ccggagcttg caggatcgcc acgactccgg 12120

gcgtatatgc tccgcattgg tcttgaccaa ctctatcaga gcttggttga cggcaatttc 12180

gatgatgcag cttgggcgca gggtcgatgc gacgcaatcg tccgatccgg agccgggact 12240

gtcgggcgta cacaaatcgc ccgcagaagc gcggccgtct ggaccgatgg ctgtgtagaa 12300

gtactcgccg atagtggaaa ccgacgcccc agcactcgtc cgagggcaaa gaaatagagt 12360

agatgccgac cgggatctgt cgatcgacaa gctcgagttt ctccataata atgtgtgagt 12420

agttcccaga taagggaatt agggttccta tagggtttcg ctcatgtgtt gagcatataa 12480

gaaaccctta gtatgtattt gtatttgtaa aatacttcta tcaataaaat ttctaattcc 12540

taaaaccaaa atccagtact aaaatccaga tcccccgaat taattcggcg ttaattcagc 12600

ctgcaggacg cgtttaatta agtgcacgcg gccgcctact tagtcaagag cctcgcacgc 12660

gactgtcacg cggccaggat cgcctcgtga gcctcgcaat ctgtacctag tgtttaaact 12720

atcagtgttt gacaggatat attggcgggt aaacctaaga gaaaagagcg tttattagaa 12780

taacggatat ttaaaagggc gtgaaaaggt ttatccgttc gtccatttgt atgtgcatgc 12840

caaccacagg gttcccctcg ggatcaaagt actttgatcc aacccctccg ctgctatagt 12900

gcagtcggct tctgacgttc agtgcagccg tcttctgaaa acgacatgtc gcacaagtcc 12960

taagttacgc gacaggctgc cgccctgccc ttttcctggc gttttcttgt cgcgtgtttt 13020

agtcgcataa agtagaatac ttgcgactag aaccggagac attacgccat gaacaagagc 13080

gccgccgctg gcctgctggg ctatgcccgc gtcagcaccg acgaccagga cttgaccaac 13140

caacgggccg aactgcacgc ggccggctgc accaagctgt tttccgagaa gatcaccggc 13200

accaggcgcg accgcccgga gctggccagg atgcttgacc acctacgccc tggcgacgtt 13260

gtgacagtga ccaggctaga ccgcctggcc cgcagcaccc gcgacctact ggacattgcc 13320

gagcgcatcc aggaggccgg cgcgggcctg cgtagcctgg cagagccgtg ggccgacacc 13380

accacgccgg ccggccgcat ggtgttgacc gtgttcgccg gcattgccga gttcgagcgt 13440

tccctaatca tcgaccgcac ccggagcggg cgcgaggccg ccaaggcccg aggcgtgaag 13500

tttggccccc gccctaccct caccccggca cagatcgcgc acgcccgcga gctgatcgac 13560

caggaaggcc gcaccgtgaa agaggcggct gcactgcttg gcgtgcatcg ctcgaccctg 13620

taccgcgcac ttgagcgcag cgaggaagtg acgcccaccg aggccaggcg gcgcggtgcc 13680

ttccgtgagg acgcattgac cgaggccgac gccctggcgg ccgccgagaa tgaacgccaa 13740

gaggaacaag catgaaaccg caccaggacg gccaggacga accgtttttc attaccgaag 13800

agatcgaggc ggagatgatc gcggccgggt acgtgttcga gccgcccgcg cacgtctcaa 13860

ccgtgcggct gcatgaaatc ctggccggtt tgtctgatgc caagctggcg gcctggccgg 13920

ccagcttggc cgctgaagaa accgagcgcc gccgtctaaa aaggtgatgt gtatttgagt 13980

aaaacagctt gcgtcatgcg gtcgctgcgt atatgatgcg atgagtaaat aaacaaatac 14040

gcaaggggaa cgcatgaagg ttatcgctgt acttaaccag aaaggcgggt caggcaagac 14100

gaccatcgca acccatctag cccgcgccct gcaactcgcc ggggccgatg ttctgttagt 14160

cgattccgat ccccagggca gtgcccgcga ttgggcggcc gtgcgggaag atcaaccgct 14220

aaccgttgtc ggcatcgacc gcccgacgat tgaccgcgac gtgaaggcca tcggccggcg 14280

cgacttcgta gtgatcgacg gagcgcccca ggcggcggac ttggctgtgt ccgcgatcaa 14340

ggcagccgac ttcgtgctga ttccggtgca gccaagccct tacgacatat gggccaccgc 14400

cgacctggtg gagctggtta agcagcgcat tgaggtcacg gatggaaggc tacaagcggc 14460

ctttgtcgtg tcgcgggcga tcaaaggcac gcgcatcggc ggtgaggttg ccgaggcgct 14520

ggccgggtac gagctgccca ttcttgagtc ccgtatcacg cagcgcgtga gctacccagg 14580

cactgccgcc gccggcacaa ccgttcttga atcagaaccc gagggcgacg ctgcccgcga 14640

ggtccaggcg ctggccgctg aaattaaatc aaaactcatt tgagttaatg aggtaaagag 14700

aaaatgagca aaagcacaaa cacgctaagt gccggccgtc cgagcgcacg cagcagcaag 14760

gctgcaacgt tggccagcct ggcagacacg ccagccatga agcgggtcaa ctttcagttg 14820

ccggcggagg atcacaccaa gctgaagatg tacgcggtac gccaaggcaa gaccattacc 14880

gagctgctat ctgaatacat cgcgcagcta ccagagtaaa tgagcaaatg aataaatgag 14940

tagatgaatt ttagcggcta aaggaggcgg catggaaaat caagaacaac caggcaccga 15000

cgccgtggaa tgccccatgt gtggaggaac gggcggttgg ccaggcgtaa gcggctgggt 15060

tgtctgccgg ccctgcaatg gcactggaac ccccaagccc gaggaatcgg cgtgacggtc 15120

gcaaaccatc cggcccggta caaatcggcg cggcgctggg tgatgacctg gtggagaagt 15180

tgaaggccgc gcaggccgcc cagcggcaac gcatcgaggc agaagcacgc cccggtgaat 15240

cgtggcaagc ggccgctgat cgaatccgca aagaatcccg gcaaccgccg gcagccggtg 15300

cgccgtcgat taggaagccg cccaagggcg acgagcaacc agattttttc gttccgatgc 15360

tctatgacgt gggcacccgc gatagtcgca gcatcatgga cgtggccgtt ttccgtctgt 15420

cgaagcgtga ccgacgagct ggcgaggtga tccgctacga gcttccagac gggcacgtag 15480

aggtttccgc agggccggcc ggcatggcca gtgtgtggga ttacgacctg gtactgatgg 15540

cggtttccca tctaaccgaa tccatgaacc gataccggga agggaaggga gacaagcccg 15600

gccgcgtgtt ccgtccacac gttgcggacg tactcaagtt ctgccggcga gccgatggcg 15660

gaaagcagaa agacgacctg gtagaaacct gcattcggtt aaacaccacg cacgttgcca 15720

tgcagcgtac gaagaaggcc aagaacggcc gcctggtgac ggtatccgag ggtgaagcct 15780

tgattagccg ctacaagatc gtaaagagcg aaaccgggcg gccggagtac atcgagatcg 15840

agctagctga ttggatgtac cgcgagatca cagaaggcaa gaacccggac gtgctgacgg 15900

ttcaccccga ttactttttg atcgatcccg gcatcggccg ttttctctac cgcctggcac 15960

gccgcgccgc aggcaaggca gaagccagat ggttgttcaa gacgatctac gaacgcagtg 16020

gcagcgccgg agagttcaag aagttctgtt tcaccgtgcg caagctgatc gggtcaaatg 16080

acctgccgga gtacgatttg aaggaggagg cggggcaggc tggcccgatc ctagtcatgc 16140

gctaccgcaa cctgatcgag ggcgaagcat ccgccggttc ctaatgtacg gagcagatgc 16200

tagggcaaat tgccctagca ggggaaaaag gtcgaaaagg tctctttcct gtggatagca 16260

cgtacattgg gaacccaaag ccgtacattg ggaaccggaa cccgtacatt gggaacccaa 16320

agccgtacat tgggaaccgg tcacacatgt aagtgactga tataaaagag aaaaaaggcg 16380

atttttccgc ctaaaactct ttaaaactta ttaaaactct taaaacccgc ctggcctgtg 16440

cataactgtc tggccagcgc acagccgaag agctgcaaaa agcgcctacc cttcggtcgc 16500

tgcgctccct acgccccgcc gcttcgcgtc ggcctatcgc ggccgctggc cgctcaaaaa 16560

tggctggcct acggccaggc aatctaccag ggcgcggaca agccgcgccg tcgccactcg 16620

accgccggcg cccacatcaa ggcaccctgc ctcgcgcgtt tcggtgatga cggtgaaaac 16680

ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc 16740

agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc 16800

cagtcacgta gcgatagcgg agtgtatact ggcttaacta tgcggcatca gagcagattg 16860

tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc 16920

gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc 16980

ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata 17040

acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg 17100

cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct 17160

caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa 17220

gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc 17280

tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt 17340

aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg 17400

ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg 17460

cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct 17520

tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc 17580

tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg 17640

ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc 17700

aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt 17760

aagggatttt ggtcatgcat tctaggtact aaaacaattc atccagtaaa atataatatt 17820

ttattttctc ccaatcaggc ttgatcccca gtaagtcaaa aaatagctcg acatactgtt 17880

cttccccgat atcctccctg atcgaccgga cgcagaaggc aatgtcatac cacttgtccg 17940

ccctgccgct tctcccaaga tcaataaagc cacttacttt gccatctttc acaaagatgt 18000

tgctgtctcc caggtcgccg tgggaaaaga caagttcctc ttcgggcttt tccgtcttta 18060

aaaaatcata cagctcgcgc ggatctttaa atggagtgtc ttcttcccag ttttcgcaat 18120

ccacatcggc cagatcgtta ttcagtaagt aatccaattc ggctaagcgg ctgtctaagc 18180

tattcgtata gggacaatcc gatatgtcga tggagtgaaa gagcctgatg cactccgcat 18240

acagctcgat aatcttttca gggctttgtt catcttcata ctcttccgag caaaggacgc 18300

catcggcctc actcatgagc agattgctcc agccatcatg ccgttcaaag tgcaggacct 18360

ttggaacagg cagctttcct tccagccata gcatcatgtc cttttcccgt tccacatcat 18420

aggtggtccc tttataccgg ctgtccgtca tttttaaata taggttttca ttttctccca 18480

ccagcttata taccttagca ggagacattc cttccgtatc ttttacgcag cggtattttt 18540

cgatcagttt tttcaattcc ggtgatattc tcattttagc catttattat ttccttcctc 18600

ttttctacag tatttaaaga taccccaaga agctaattat aacaagacga actccaattc 18660

actgttcctt gcattctaaa accttaaata ccagaaaaca gctttttcaa agttgttttc 18720

aaagttggcg tataacatag tatcgacgga gccgattttg aaaccgcggt gatcacaggc 18780

agcaacgctc tgtcatcgtt acaatcaaca tgctaccctc cgcgagatca tccgtgtttc 18840

aaacccggca gcttagttgc cgttcttccg aatagcatcg gtaacatgag caaagtctgc 18900

cgccttacaa cggctctccc gctgacgccg tcccggactg atgggctgcc tgtatcgagt 18960

ggtgattttg tgccgagctg ccggtcgggg agctgttggc tggct 19005

<210>2

<211>228

<212>PRT

<213>Artificial Sequence

<400>2

Ser Ser Glu Thr Gly Pro Val Ala Val Asp Pro Thr Leu Arg Arg Arg

1 5 10 15

Ile Glu Pro His Glu Phe Glu Val Phe Phe Asp Pro Arg Glu Leu Arg

20 25 30

Lys Glu Thr Cys Leu Leu Tyr Glu Ile Asn Trp Gly Gly Arg His Ser

35 40 45

Ile Trp Arg His Thr Ser Gln Asn Thr Asn Lys His Val Glu Val Asn

50 55 60

Phe Ile Glu Lys Phe Thr Thr Glu Arg Tyr Phe Cys Pro Asn Thr Arg

65 70 75 80

Cys Ser Ile Thr Trp Phe Leu Ser Trp Ser Pro Cys Gly Glu Cys Ser

85 90 95

Arg Ala Ile Thr Glu Phe Leu Ser Arg Tyr Pro His Val Thr Leu Phe

100 105 110

Ile Tyr Ile Ala Arg Leu Tyr His His Ala Asp Pro Arg Asn Arg Gln

115 120 125

Gly Leu Arg Asp Leu Ile Ser Ser Gly Val Thr Ile Gln Ile Met Thr

130 135 140

Glu Gln Glu Ser Gly Tyr Cys Trp Arg Asn Phe Val Asn Tyr Ser Pro

145 150 155 160

Ser Asn Glu Ala His Trp Pro Arg Tyr Pro His Leu Trp Val Arg Leu

165 170 175

Tyr Val Leu Glu Leu Tyr Cys Ile Ile Leu Gly Leu Pro Pro Cys Leu

180 185 190

Asn Ile Leu Arg Arg Lys Gln Pro Gln Leu Thr Phe Phe Thr Ile Ala

195 200 205

Leu Gln Ser Cys His Tyr Gln Arg Leu Pro Pro His Ile Leu Trp Ala

210 215 220

Thr Gly Leu Lys

225

<210>3

<211>1367

<212>PRT

<213>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>83

<212>PRT

<213>Artificial Sequence

<400>4

Thr Asn Leu Ser Asp Ile Ile Glu Lys Glu Thr Gly Lys Gln Leu Val

1 5 10 15

Ile Gln Glu Ser Ile Leu Met Leu Pro Glu Glu Val Glu Glu Val Ile

20 25 30

Gly Asn Lys Pro Glu Ser Asp Ile Leu Val His Thr Ala Tyr Asp Glu

35 40 45

Ser Thr Asp Glu Asn Val Met Leu Leu Thr Ser Asp Ala Pro Glu Tyr

50 55 60

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

65 70 75 80

Lys Met Leu

<210>5

<211>117

<212>DNA

<213>Artificial Sequence

<400>5

gactacaagg accacgacgg cgactacaag gatcatgaca tcgactacaa ggacgacgac 60

gacaagatgg ccccgaagaa gaagaggaaa gtgggcatcc acggcgtgcc ggccgcc 117

<210>6

<211>207

<212>DNA

<213>Artificial Sequence

<400>6

gactacaagg accacgacgg ggattacaaa gaccacgaca tagactacaa ggatgacgat 60

gacaaaatgg caccgaagaa aaaaaggaag gtcggcggct ccccgaagaa aaaaaggaag 120

gtcggcggct ccccgaagaa aaaaaggaag gtcggcggct ccccgaagaa aaaaaggaag 180

gtcggaatcc atggcgttcc agctgcc 207

<210>7

<211>17

<212>PRT

<213>Artificial Sequence

<400>7

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

1 5 10 15

Val

<210>8

<211>31

<212>PRT

<213>Artificial Sequence

<400>8

Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp Ile Asp Tyr

1 5 10 15

Lys Asp Asp Asp Asp Lys Met Ala Pro Lys Lys Lys Arg Lys Val

20 25 30

Claims

1. A kit comprising sgRNA, Cas9 nuclease, deaminase, nuclear localization signal a, nuclear localization signal b, UGI protein;

the Cas9 nuclease is a Cas9n protein;

the deaminase is rAPOBEC1 protein;

the kit is obtained by expressing a recombinant expression vector, wherein the recombinant expression vector comprises an expression cassette which consists of a promoter, a coding gene of a nuclear localization signal A, a coding gene of cytosine deaminase rAPOBEC1, a coding gene of Cas9n nuclease, a coding gene of UGI, a coding gene of a nuclear localization signal B and a terminator in sequence;

the nuclear localization signal A comprises 13 Flag1 tag protein and 1 NLS1 protein, and the amino acid sequence of the nuclear localization signal A is shown as a sequence 8;

the nuclear localization signal B comprises 1 bpNLS protein, and the amino acid sequence of the nuclear localization signal B is shown as a sequence 7.

2. The kit of claim 1, wherein:

the nucleic acid molecule for coding the nuclear localization signal A is a DNA molecule shown in 1 st to 93 th sites of a sequence 5 in a sequence table;

the nucleic acid molecule for coding the nuclear localization signal B is a DNA molecule shown in 8647-8697 th site of the sequence 1 in the sequence table.

3. The kit of claim 1 or 2, wherein:

the amino acid sequence of the Cas9n protein is shown as sequence 3;

the amino acid sequence of the rAPOBEC1 protein is shown as a sequence 2;

the sgRNA targets a target sequence;

the sgRNA is tRNA-sgRNA;

the tRNA is an RNA molecule obtained by replacing T in the 474-550 th position of the sequence 1 with U;

the esgRNA framework is an RNA molecule obtained by replacing T in the 571-656 position of the sequence 1 with U.

4. The kit of claim 3, wherein: the amino acid sequence of the UGI protein is shown as a sequence 4.

5. Use of the kit of any one of claims 1 to 4 in any one of S1) -S4):

s1) editing of a genomic target sequence of an organism or cell of an organism;

s4) preparing a product for improving the editing efficiency of the genome target sequence of the organism or the organism cell;

the organism is a plant; the biological cell is a plant cell.

6. Use according to claim 5, characterized in that: the target sequence is edited by mutating a base C into a base T.

7. Use according to claim 5 or 6, characterized in that: the plant is a monocotyledon or a dicotyledon;

the plant cell is a monocotyledon cell or a dicotyledon cell.

8. Use according to claim 7, characterized in that: the monocotyledon is a gramineous plant; the monocotyledon cell is a gramineae plant cell.

9. Use according to claim 8, characterized in that: the gramineous plant is rice; the gramineous plant cell is a rice cell.

10, T1) or T2):

t1) or a method for increasing the efficiency of editing a genomic target sequence of an organism or cell comprising the steps of: causing an organism or biological cell to express a nuclear localization signal a as described in claims 1-5, a nuclear localization signal b as described in claims 1-5, a sgRNA as described in claims 1-5, a Cas9 nuclease as described in claims 1-5, a deaminase as described in claims 1-5; the sgRNA targets the target sequence;

the organism or the biological cell expresses a nuclear localization signal A as described in claims 1 to 5, a nuclear localization signal B as described in claims 1 to 5, a sgRNA as described in claims 1 to 5, a Cas9 nuclease as described in claims 1 to 5, a deaminase as described in claims 1 to 5 by introducing a gene encoding the nuclear localization signal A, a gene encoding the nuclear localization signal B, a DNA molecule transcribing the sgRNA, a gene encoding the Cas9 nuclease, a gene encoding the cytosine deaminase, and a gene encoding UGI into the organism or the biological cell;

the coding gene of the nuclear localization signal A, the coding gene of the nuclear localization signal B, a DNA molecule for transcribing the sgRNA, the coding gene of the Cas9 nuclease, the coding gene of the cytosine deaminase and the coding gene of the UGI are introduced into an organism or an organism cell through a recombinant expression vector;

the recombinant expression vector comprises an expression cassette which consists of a promoter, a coding gene of a nuclear localization signal A, a coding gene of cytosine deaminase rAPOBEC1, a coding gene of Cas9n nuclease, a coding gene of UGI, a coding gene of a nuclear localization signal B and a terminator in sequence;

t2) biological mutant, comprising the following steps: editing the genome of the organism according to the method of T1) to obtain a biological mutant;

the organism is a plant; the biological cell is a plant cell.

11. The method of claim 10, wherein: the target sequence is edited by mutating a base C into a base T.

12. The method according to claim 10 or 11, characterized in that: the plant is a monocotyledon or a dicotyledon;

the plant cell is a monocotyledon cell or a dicotyledon cell.

13. The method of claim 12, wherein: the monocotyledon is a gramineous plant; the monocotyledon cell is a gramineae plant cell.

14. The method of claim 13, wherein: the gramineous plant is rice; the gramineous plant cell is a rice cell.