CN109652422B - Efficient single-base editing system OsSpCas9-eCDA and application thereof - Google Patents

Abstract

The invention relates to the technical field of biotechnology and plant genetic engineering, and discloses an efficient single-base editing system OsSpCas9-eCDA and application thereof. The single base editing system OsSpCas9-eCDA is capable of splicing a plant genome and has: (a) SEQ ID No: 1; or (b) SEQ ID NO: 1 by replacing, deleting or adding one or more nucleotides and can still perform plant genome shearing. The single-base editing system OsSpCas9-eCDA provided by the invention can efficiently carry out single-base editing from C/G to T/A in plants, particularly rice.

Description

Efficient single-base editing system OsSpCas9-eCDA and application thereof

Technical Field

The present invention relates to biotechnology and plant genetic engineering technology. Specifically, the invention relates to a high-efficiency single-base editing system OsSpCas9-eCDA, an expression cassette, an expression vector, a targeting vector, a transgenic cell containing the single-base editing system OsSpCas9-eCDA and application of the high-efficiency single-base editing system OsSpCas 9-eCDA.

Background

The current gene editing technology (ZFN, TALEN, CRISPR/Cas9) relies on the induction of double-strand break at a target site, so that a DNA repair mechanism is activated, and the purpose of gene correction is realized. Therefore, the gene editing technology based on double strand breaks is not only easy to generate DNA fragment insertion and deletion, but also may generate off-target effect, ultimately affecting the function of the target gene. The advent of single base editing techniques has effectively overcome this problem.

Single base gene editing technology (base editors, BEs) refers to gene editing technology that causes a single base change in a genome. The basic principle is the fusion of cytosine deaminase (APOBEC) or adenosine deaminase with existing Cas9n (D10A), a gene editing technique that relies on CRISPR principles to modify a single base at positions 4-7 away from the PAM end of a target. The current single base gene editing comprises two types, one is CBEs (Cytidine base editors) -pyrimidine base conversion technology (C/G to T/A), and the other is ABEs (Adenine base editors) -purine base conversion technology (A/T to G/C).

Based on a CRISPR/Cas9 gene editing system, a new gene editing tool, namely a single-base editing system, is reported on Nature journal by David Liu group of biochemists at Harvard university in 2016. Similar research reports were published in the same year in the Akihiko Kondo group, the university of Japan, and in the Changxing group, the Shanghai transport university in China. The single-base editing system mainly comprises two parts, namely sgRNA and a fusion protein, wherein the fusion protein generally comprises a modified Cas9 protein, cytosine deaminase and a uracil glycosylase inhibitor, and the fusion protein of the family only comprises two parts, namely Cas9 and cytosine deaminase. The sgRNA directs the fusion protein to bind to the target site to function by complementary pairing with the target site. Cytosine deaminase in the fusion protein can deaminate corresponding cytosine C in a non-complementary strand into uracil U, DNA replication further enables the U to be replaced by T, guanine G which is originally complementary to C on the complementary strand can be changed into adenine A, and a uracil glycosylase inhibitor can inhibit U excision, and finally accurate editing of C on the non-complementary strand replaced by T and G on the complementary strand replaced by A is realized.

Transgenic technology has already done a lot of work in improving crop traits, but because of the introduction of exogenous genes and the lack of public science popularization, the popularization and application of transgenic crops have great difficulty. In the long artificial breeding process of crops, human beings directionally breed the gene mutant strains with excellent characters by an artificial selection mode, but the process is time-consuming, labor-consuming and uncontrollable. Using homologous recombination techniques, superior plant strains can be obtained that are site-directed, but the feasibility is not high because the efficiency of homologous recombination is too low. The single base editing technology can efficiently edit the result of a single base in human cells, and the fact that the system can be used for editing the genes with controllable row and fixed point in crops is prompted, and then plants with excellent characters can be obtained quickly and efficiently. In plants, three C/G to T/A editors have been tested, one is the BE3 system using mouse cytosine deaminase (APOBEC), one is the AID system induced by directed activation of sea lamprey cytosine deaminase (PmCDA), and the other is the rBE5 system using a human AID system variant, which are fused with SpCas9 or SacAS9 systems, and have successfully achieved single base editing in rice, maize, wheat, Arabidopsis, tomato and watermelon.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide an efficient single-base editing system OsSpCas9-eCDA and application thereof in plant genome editing.

To achieve the above objects, one aspect of the present invention provides a high efficiency single base editing system ospcas 9-ecca, which is capable of splicing plant genomes and has:

(a) SEQ ID No: 1; or

(b) SEQ ID NO: 1 by replacing, deleting or adding one or more nucleotides and can still perform plant genome shearing.

In a second aspect, the invention provides an expression cassette comprising a single base editing system OsSpCas9-eCDA as described above.

In a third aspect, the invention provides an expression vector, wherein the expression vector is inserted into the single-base editing system OsSpCas9-eCDA or the expression cassette.

In a fourth aspect, the invention provides a targeting vector, wherein the targeting vector is inserted with the single-base editing system OsSpCas9-eCDA or the expression cassette and the target site sequence.

In a fifth aspect, the invention provides a transgenic cell, wherein the transgenic cell is transferred into the single base editing system OsSpCas9-eCDA as described above, the expression cassette as described above, the expression vector as described above or the targeting vector as described above.

In a sixth aspect, the invention provides a use of the single base editing system ospspcas 9-ecca as described above, an expression cassette as described above, an expression vector as described above, a targeting vector as described above or a transgenic cell as described above in plant genome editing, wherein the plant genome editing comprises shearing a plant genome to achieve single base editing from C/G to T/a on the plant genome to obtain a transgenic plant or plant part containing a mutation site.

The single-base editing system OsSpCas9-eCDA provided by the invention can efficiently edit a single base from C/G to T/A in plants, particularly rice.

Drawings

FIG. 1 is a schematic diagram of a specific expression vector pHUN411-eCDA provided by the present invention.

FIG. 2 shows the mutation pattern of rice genome edited with the expression vector pHUN411-eCDA provided by the present invention.

FIG. 3 shows the mutant forms produced by genome editing of rice using the comparative expression vector pHUN 411-CDA.

Detailed Description

The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and these ranges or values should be understood to encompass values close to these ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.

In a first aspect, the present invention provides a single base editing system ospcas 9-eCDA, said single base editing system ospcas 9-eCDA being capable of splicing a plant genome and having:

(a) SEQ ID No: 1; or

(b) The amino acid sequence of SEQ ID NO: 1 by replacing, deleting or adding one or more nucleotides and can still perform plant genome shearing.

Wherein, the term "nucleotide sequence still capable of plant genomic cleavage" refers to a nucleotide sequence that is identical to the nucleotide sequence of SEQ ID No: 1, and compared with the shearing efficiency of the nucleotide sequence shown in SEQ ID NO: 1 by substituting, deleting or adding one or more nucleotides, the shearing efficiency of the nucleotide sequence obtained by the nucleotide sequence shown in SEQ ID NO: 1, e.g., 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%.

It is also to be noted that the term "having" as used herein does not mean any other structure containing a sequence other than SEQ ID No: 1 or a mutant thereof, but refers to a nucleotide sequence that is convenient or advantageous for the nucleotide sequence set forth in SEQ ID No: 1 or a mutant thereof or a nucleotide sequence shown in SEQ ID No: 1 or a mutant thereof, and the like, for example, an enzyme cleavage site, a marker gene, a selection gene, and the like. Thus, the term "having" as used herein means having the sequence of SEQ ID No: 1 or a mutant thereof, but still being capable of effecting the nucleotide sequence set forth in SEQ ID No: 1 or a mutant function sequence thereof.

According to a specific embodiment of the invention, the nucleotide sequence of the single base editing system OsSpCas9-eCDA is shown as SEQ ID No: 1 is shown.

In a second aspect, the invention also provides an expression cassette comprising the single base editing system OsSpCas9-eCDA as described above.

In a third aspect, the invention also provides an expression vector, wherein the expression vector is inserted into the single-base editing system OsSpCas9-eCDA or the expression cassette.

According to the invention, the construction method of the expression vector can be carried out according to the conventional method in the field, for example, the single base editing system OsSpCas9-eCDA and the vector to be inserted are subjected to enzyme digestion by using the same restriction enzyme, and then the single base editing system OsSpCas9-eCDA is connected to the vector by using ligase to obtain the expression vector.

The restriction enzyme can be specifically selected according to the enzyme cutting site introduced into the single base editing system OsSpCas9-eCDA, and for example, the restriction enzyme can be NotI/SacI restriction enzyme.

The ligase may be any one of various ligases conventionally used in the art capable of ligating two nucleic acid fragments, and may be, for example, T4 ligase.

Among them, the vector may be various vectors conventionally used in the art, and preferably, the vector may be PUC57-AMP, pHUN400 or pHUN900, which are commercially available. According to a preferred embodiment of the present invention, the vector is pHUN900, further, the pHUN900 vector and the single base editing system OsSpCas9-eCDA can be cut by NotI/SacI using NotI/SacI cutting sites and recovered, and then the single base editing system OsSpCas9-eCDA is linked to the pHUN900 vector using T4 ligase, so as to obtain the plant expression vector pHUN-OsSpCas9-eCDA, which is named as pHUN 411-eCDA.

In a fourth aspect, the present invention provides a targeting vector, which is inserted with the single base editing system OsSpCas9-eCDA as described above or the expression cassette as described above and the target site sequence.

According to the present invention, the target site sequence may be determined according to a sequence that actually requires genome editing, but the target site sequence requires a PAM sequence including NGG characteristics. According to a specific embodiment of the present invention, the target site sequence is SEQ ID No: 2 (1366-13 of the rice Pi-d2 gene (Os06g 0494100))Nucleotide sequence of 86 th positionCCATTATTATTGTCATTATGCTC, (the underlined part is the reverse complement of the PAM sequence of 5 '-NGG-3' structure, where N is T, A, G or C).

According to the invention, the targeting vector can be obtained by simple annealing and enzyme digestion ligation on the basis of an expression vector.

In a fifth aspect, the invention also provides a transgenic cell, wherein the transgenic cell is transferred into the single base editing system OsSpCas9-eCDA as described above, the expression cassette as described above, the expression vector as described above or the targeting vector as described above.

According to the present invention, the selection of the cell can be determined according to the stage of gene targeting, specifically, for example, when amplification of the single base editing system OsSpCas9-eCDA is to be achieved before targeting vector construction, Escherichia coli can be used as the host cell, when the targeting vector is to be transformed into a plant cell, Agrobacterium tumefaciens (Agrobacterium tumefaciens) can be used as the host cell, and the transgenic cell also includes a plant cell into which the targeting vector is transferred, for example, a callus cell of a plant.

In a sixth aspect, the invention also provides the use of the single base editing system ospspcas 9-ecada as described above, an expression cassette as described above, an expression vector as described above, a targeting vector as described above or a transgenic cell as described above for plant genome editing, wherein the plant genome editing comprises shearing the plant genome to achieve single base editing from C/G to T/a on the plant genome to obtain a transgenic plant or plant part containing a mutation site.

According to the present invention, the plant is preferably a monocotyledon, more preferably rice, still more preferably japonica rice, and yet still more preferably japonica rice nipponica.

According to the invention, the single base editing system OsSpCas9-eCDA is used for identifying a PAM sequence with NGG characteristics, the shearing of a DNA double strand in rice is completed, and a transgenic plant or a plant part with a single base mutation site from C/G to T/A is obtained under the action of a self-repair system.

According to the present invention, the method for introducing a targeting vector into a plant cell can be performed according to a method conventional in the art.

According to a specific embodiment of the present invention, a method for introducing a targeting vector into rice cells comprises the steps of:

(1) removing the hull of the rice seed, sterilizing, separating the embryo, and placing on a callus induction culture medium to generate secondary callus; wherein, the seeds can be mature seeds;

(2) transferring the secondary callus to a new callus induction culture medium for pre-culture;

(3) contacting the callus obtained in the step (2) with agrobacterium carrying a targeting vector of OsSpCas9-eCDA for 15 minutes, and particularly soaking the callus in a suspension of the agrobacterium;

(4) transferring the callus tissue of the step (3) to a culture dish on which three pieces of sterile filter paper (added with 2.5-3.5mL of agrobacterium suspension culture medium) are placed, and culturing for 48 hours at 21-23 ℃;

(5) placing the callus of the step (4) on a pre-screening culture medium for culturing for 5-7 days; wherein the pre-screening medium may be the pre-screening medium listed in table 1;

(6) transferring the callus of the step (5) to a screening culture medium to obtain resistant callus;

(7) transferring the resistant callus to a differentiation regeneration culture medium to differentiate into seedlings;

(8) transferring the seedling in the step (7) to a rooting culture medium for rooting; wherein, the rooting medium can be the rooting medium listed in table 1.

According to the present invention, various media used as above may be media conventionally used in the art, and preferably, the media listed in table 1 may be used, wherein the configuration of the media used may be as follows: yongbo Duan, Chenguang ZHai, Hao Li, Juan Li, Wenqian Mei et al, an effective and high-throughput protocol for Agrobacterium mediated transformation based on phosphorus isomerous amplification selection in Japonica rice (Oryza sativa L.). Plant cell reports,2012,31: 1611-.

TABLE 1

Note: "macroelement N6" means that the macroelement N6 contains [ NO₃ ^-]/[NH₄ ⁺]＝40mM/10mM。

Examples

The present invention will be described in detail below by way of examples.

The operations in the following detailed description are performed by conventional operations commonly used in the art, unless otherwise specifically indicated. The skilled person can easily derive teachings on such routine procedures from the prior art, for example with reference to the textbooks Sambrook and David Russell, molecular μ lar Cloning: A Laboratory Manual,3rd ed., Vols1, 2; charles New Stewart, Alisher Touraev, vitay Citovsky and Tzvi Tzfira, Plant Transformation Technologies, and the like. The raw materials, reagents, materials and the like used in the following examples are all commercially available products unless otherwise specified.

Example 1

This example illustrates splicing of the OsSpCas9-eCDA gene

(1) Performing rice codon optimization on the existing cytosine deaminase (PmCDA) of the sea lamprey to obtain a codon-optimized cytosine deaminase amino acid sequence named as Os-PmCDA. The Os-PmCDA gene was synthesized by KINZHI Biotech, Suzhou, and ligated to PUC57-AMP vector to form PUC57-AMP-PmCDA vector, which was then loaded into E.coli XL-blue strain.

(2) A uracil glycosylase gene inhibitor gene (uracil DNA glycosylase (UDG) inhibitor, UGI) with 3 repetitions is artificially synthesized, named as eUGI, connected to a PUC57-AMP vector to form a PUC57-AMP-eUGI vector, and loaded into an Escherichia coli XL-blue strain.

(3) The SpCas9 gene is obtained from pHUN4c16 vector after rice codon optimization in the laboratory. This vector has been filed for a national invention patent by the present laboratory (a backbone plasmid vector for genetic engineering, patent application No. 201410225911.4).

(4) SpCas9 gene, codon optimized PmCDA gene Os-PmCDA, and 3 repeated uracil glycosylase gene inhibitor gene (eUGI) were ligated together and ligated to PUC57-AMP vector according to Gibson's splicing principle. The specific operation is as follows:

respectively synthesizing primers according to the splicing sequence of the Os-PmCDA gene, the SpCas9 gene and the eUGI gene and the PUC57-AMP vector sequence, wherein the primers are specifically shown in Table 2:

TABLE 2

Using the SpCas9 gene as a template, PCR was performed with primers SpCas9HR FP (SEQ ID No: 2) and SpCas9HR RP (SEQ ID No: 3), and the PCR components were first maintained at 95 ℃ for 5 minutes and then subjected to 35 cycles: PCR products were recovered by extension at 94 ℃ for 45 seconds, 58 ℃ for 45 seconds, 72 ℃ for 4 minutes, and finally at 72 ℃ for 10 minutes.

Using the OsPmCDA gene as a template, PCR amplification was performed with primers PmCDA-SpCas9HR FP (SEQ ID No: 4) and PmCDA-SpCas9HR RP (SEQ ID No: 5), and the PCR components were first kept at 95 ℃ for 5 minutes, followed by 35 cycles: PCR products were recovered by extension at 94 ℃ for 45 seconds, 58 ℃ for 45 seconds, 72 ℃ for 45 seconds, and finally at 72 ℃ for 10 minutes.

PCR amplification was performed with primers Sp-eUGI HR FP (SEQ ID No: 6) and eUGI HR RP (SEQ ID No: 7) using the eUGI gene as template, and the PCR fractions were first maintained at 95 ℃ for 5 minutes and then subjected to 35 cycles: PCR products were recovered by extension at 94 ℃ for 45 seconds, 58 ℃ for 45 seconds, 72 ℃ for 45 seconds, and finally at 72 ℃ for 10 minutes.

The three recovered fragments and the PUC57-AMP vector fragment after EcoRI digestion are combined into a gene which is a SpCas9 gene, OsPmCDA and eUGI gene fused together according to the Gibson splicing principle and the specification of a NEB Golden Gate Assembly Kit, and the gene is named as OsSpCas9-eCDA gene, and has a sequence shown as SEQ ID No: 1, ligated to PUC57-AMP vector and loaded into E.coli XL-blue strain.

Similarly, a gene fused with the SpCas9 gene, OsPmCDA and UGI gene is constructed and named as SpCas9-CDA gene.

Example 2

This example illustrates the construction of a plant targeting vector containing the OsSpCas9-eCDA gene

Plasmid was extracted from E.coli XL-blue containing the OsSpCas9-eCDA vector above using Axygen plasmid extraction kit, digested with NotI/SacI, and the OsSpCas9-eCDA fragment was recovered. At the same time, pHUN900 was linearized with NotI/SacI enzyme to recover pHUN900, and the OsSpCas9-eCDA fragment and pHUN900 fragment were ligated with T4 ligase (available from TaKaRa) to obtain the plant expression vector pHUNSpCas9-eCDA (FIG. 1), which was designated pHUN 411-eCDA.

Selecting the nucleotide sequence of 1366-1386 th site in the gene of rice Pi-d2 (Os06g0494100)CCATTATTATTGTCATTATGCTC, (the underlined part is the PAM sequence reverse complement of the 5 'NGG-3' structure) as the targeting site. The target site sequence was fused with pHUN411-eCDA to form pHUN411-eCDA-Pi-d 2. The plant expression vector was transferred to Agrobacterium tumefaciens (Agrobacterium tumefaciens) EHA105 strain (stored by Rice research institute of agricultural sciences, Anhui) by freeze-thawing for genetic transformation.

Similarly, a pHUN41-CDA-Pi-d2 expression vector is constructed and transferred into an agrobacterium tumefaciens EHA105 strain for genetic transformation.

Example 3

This example illustrates the genetic transformation of rice using pHUN411-eCDA-Pi-d2 as a targeting vector and the acquisition of mutants.

1. Induction and preculture of mature embryo calli

Removing shells of mature seeds of Nipponbare, selecting seeds with normal appearance, cleanness and no mildew stains, shaking for 90sec with 70% alcohol, and pouring off the alcohol; then 50% sodium hypochlorite solution containing Tween20 (the effective chlorine concentration of the stock solution is more than 4%, 1 drop of Tween20 is added per 100 ml) is used for cleaning the seeds, and the seeds are shaken on a shaking table for 45min (180 r/min). Pouring out sodium hypochlorite, washing with sterile water for 5-10 times until no smell of sodium hypochlorite exists, adding sterile water, and soaking at 30 deg.C overnight. Embryos were separated along the aleurone layer with scalpel blade, scutellum up placed on induction medium (see table 1 for ingredients), 12 grains/dish, dark cultured at 30 ℃ to induce callus.

After two weeks spherical, rough and yellowish secondary callus appeared, which was pre-cultured by transferring the secondary callus to a new callus induction medium and pre-culturing for 5 days at 30 ℃ in the dark. After the pre-culture is finished, collecting the small particles with good state and vigorous division into a 50mL sterile centrifuge tube by using a spoon for agrobacterium infection.

2. Culture and suspension preparation of Agrobacterium strains

The Agrobacterium strain EHA105 containing the pHUN411-eCDA-Pi-d2 vector was streaked on LB solid medium containing 50mg/L kanamycin (see Table 1 for components), dark-cultured at 28 ℃ for 24 hours, and then the activated Agrobacterium was inoculated with a sterile inoculating loop onto a fresh LB plate containing 50mg/L kanamycin for a second activation, and dark-cultured at 28 ℃ overnight. 20-30mL of Agrobacterium suspension medium (see Table 1 for ingredients) was added to a 50mL sterile centrifuge tube, the Agrobacterium activated 2 times was scraped off with an inoculating loop, OD660 was adjusted to about 0.10-0.25, and the tube was allowed to stand at room temperature for more than 30 min.

3. Infection and Co-cultivation

To the prepared callus (see step 1), the Agrobacterium suspension was added and soaked for 15min with occasional gentle shaking. After soaking, pouring off the liquid (dripping the liquid as far as possible), sucking the redundant agrobacterium liquid on the surface of the callus by using sterile filter paper, and drying the callus by using sterile wind in a super clean bench. Three sterile filter papers were placed on a 100X 25mm disposable sterile petri dish pad, 2.5mL of Agrobacterium suspension medium (see Table 1 for components) was added, and the blotted calli were uniformly spread on the filter papers and cultured in the dark at 23 ℃ for 48 hours.

4. Pre-screening and screening cultures

After the end of the co-culture, the co-cultured calli were spread evenly on the pre-selection medium (see Table 1 for composition) and cultured in the dark at 30 ℃ for 5 days. After the pre-screening culture is finished, transferring the callus onto a screening culture medium (the components are shown in table 1), inoculating 25 calli into each culture dish, culturing in the dark at the temperature of 30 ℃, and after 2-3 weeks, obviously growing the resistant callus and performing differentiation regeneration operation.

5. Differentiation and regeneration

2-3 fresh small particles with good growth state were selected from each independent transformant and transferred to differentiation regeneration medium (see table 1 for composition). Each culture dish was inoculated with 5 independent transformants. Culturing at 28 ℃ under illumination, wherein the illumination period is 16h, the illumination period is 8h, and the light intensity is 3000-6000 lx.

6. Rooting and transplanting

When the bud differentiated from the resistant callus grows to about 2cm, only one well-grown seedling is taken from each independent transformant and is transferred to a rooting medium (the components are shown in the table 1), the seedling is cultured under the light of 28 ℃, the light period is 16h, the light is 8h and the dark, and the light intensity is 3000-. After two weeks, seedlings with developed root systems are selected, washed with water to remove the culture medium, and transplanted into soil.

7. Molecular identification

Before transplanting, a rice leaf sample is taken, and DNA is subjected to DNA miniextraction by a CTAB method. The resulting genomic DNA samples were used for PCR analysis. PCR primers 5'-ACTAACTTCTTCCCTCCTGCGG-3' (SEQ ID NO: 8) and 5'-AGATCCAAACCCTCCCTGACCA-3' (SEQ ID NO: 9) were designed to amplify sequences around 150bp near the Pi-d2 target. The PCR components were first kept at 95 ℃ for 5 minutes and then subjected to 32 cycles: 94 ℃ for 45 seconds, 56 ℃ for 45 seconds, 72 ℃ for 45 seconds, and finally extension at 72 ℃ for 10 minutes. The PCR product was sequenced. The results were aligned with the wild type sequence (FIGS. 2 and 3).

The results show that: among the plants obtained from pHUN411-eCDA-Pi-d2, 24 mutations were detected in 28 plants, all of which were G-to-A at different positions in the target sequence, with a single base mutation efficiency of 85.7%, and no unwanted mutation, such as G-to-C (FIG. 2). Similarly, in the plants obtained from pHUN411-CDA-Pi-d2, 22 target sequences in 40 tested plants showed single base variation, and the mutation rate reached 55%. However, the mutation of G into A, namely the clean mutant plant, is only 5, the mutation rate is only 8 percent, and the rest of the mutation forms are that G is mutated into C, or unnecessary bases are inserted (figure 3). Thus, pHUN411-eCDA can not only obtain higher single base mutation rate, but also obtain higher ideal clean single base mutation rate.

The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.

SEQUENCE LISTING

<110> institute of Rice research of agricultural science institute of Anhui province

<120> efficient single-base editing system OsSpCas9-eCDA and application thereof

<130> HFI00863-NYSD

<160> 9

<170> PatentIn version 3.3

<210> 1

<211> 6047

<212> DNA

<213> Single base editing System OsSpCas9-eCDA

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gcggccgcat ggacaagaag tactccatcg gcctcgctat cggcaccaat tctgttggct 60

gggccgtgat caccgacgag tacaaggtgc cgtccaagaa gttcaaggtc ctcggcaaca 120

ccgaccgcca ctccatcaag aagaatctca tcggcgccct gctgttcgac tctggcgaga 180

cagccgaggc tacaaggctc aagaggaccg ctagacgcag gtacaccagg cgcaagaacc 240

gcatctgcta cctccaagag atcttctcca acgagatggc caaggtggac gacagcttct 300

tccacaggct cgaggagagc ttcctcgtcg aggaggacaa gaagcacgag cgccatccga 360

tcttcggcaa catcgtggat gaggtggcct accacgagaa gtacccgacc atctaccacc 420

tccgcaagaa gctcgtcgac tccaccgata aggccgacct caggctcatc tacctcgccc 480

tcgcccacat gatcaagttc aggggccact tcctcatcga gggcgacctc aacccggaca 540

actccgatgt ggacaagctg ttcatccagc tcgtgcagac ctacaaccag ctgttcgagg 600

agaacccgat caacgcctct ggcgttgacg ccaaggctat tctctctgcc aggctctcta 660

agtcccgcag gctcgagaat ctgatcgccc aacttccggg cgagaagaag aatggcctct 720

tcggcaacct gatcgccctc tctcttggcc tcaccccgaa cttcaagtcc aacttcgacc 780

tcgccgagga cgccaagctc cagctttcca aggacaccta cgacgacgac ctcgacaatc 840

tcctcgccca gattggcgat cagtacgccg atctgttcct cgccgccaag aatctctccg 900

acgccatcct cctcagcgac atcctcaggg tgaacaccga gatcaccaag gccccactct 960

ccgcctccat gatcaagagg tacgacgagc accaccagga cctcacactc ctcaaggccc 1020

tcgtgagaca gcagctccca gagaagtaca aggagatctt cttcgaccag tccaagaacg 1080

gctacgccgg ctacatcgat ggcggcgctt ctcaagagga gttctacaag ttcatcaagc 1140

cgatcctcga gaagatggac ggcaccgagg agctgctcgt gaagctcaat agagaggacc 1200

tcctccgcaa gcagcgcacc ttcgataatg gctccatccc gcaccagatc cacctcggcg 1260

agcttcatgc tatcctccgc aggcaagagg acttctaccc gttcctcaag gacaaccgcg 1320

agaagattga gaagatcctc accttccgca tcccgtacta cgtgggcccg ctcgccaggg 1380

gcaactccag gttcgcctgg atgaccagaa agtccgagga gacaatcacc ccctggaact 1440

tcgaggaggt ggtggataag ggcgcctctg cccagtcttt catcgagcgc atgaccaact 1500

tcgacaagaa cctcccgaac gagaaggtgc tcccgaagca ctcactcctc tacgagtact 1560

tcaccgtgta caacgagctg accaaggtga agtacgtgac cgaggggatg aggaagccag 1620

ctttccttag cggcgagcaa aagaaggcca tcgtcgacct gctgttcaag accaaccgca 1680

aggtgaccgt gaagcagctc aaggaggact acttcaagaa aatcgagtgc ttcgactccg 1740

tcgagatctc cggcgtcgag gataggttca atgcctccct cgggacctac cacgacctcc 1800

tcaagattat caaggacaag gacttcctcg acaacgagga gaacgaggac atcctcgagg 1860

acatcgtgct caccctcacc ctcttcgagg accgcgagat gatcgaggag cgcctcaaga 1920

catacgccca cctcttcgac gacaaggtga tgaagcagct gaagcgcagg cgctataccg 1980

gctggggcag gctctctagg aagctcatca acggcatccg cgacaagcag tccggcaaga 2040

cgatcctcga cttcctcaag tccgacggct tcgccaaccg caacttcatg cagctcatcc 2100

acgacgactc cctcaccttc aaggaggaca tccaaaaggc ccaggtgtcc ggccaaggcg 2160

attccctcca tgagcatatc gccaatctcg ccggctcccc ggctatcaag aagggcattc 2220

tccagaccgt gaaggtggtg gacgagctgg tgaaggtgat gggcaggcac aagccagaga 2280

acatcgtgat cgagatggcc cgcgagaacc agaccacaca gaagggccaa aagaactccc 2340

gcgagcgcat gaagaggatc gaggagggca ttaaggagct gggctcccag atcctcaagg 2400

agcacccagt cgagaacacc cagctccaga acgagaagct ctacctctac tacctccaga 2460

acggccgcga catgtacgtg gaccaagagc tggacatcaa ccgcctctcc gactacgacg 2520

tggaccatat tgtgccgcag tccttcctga aggacgactc catcgacaac aaggtgctca 2580

cccgctccga caagaacagg ggcaagtccg ataacgtgcc gtccgaagag gtcgtcaaga 2640

agatgaagaa ctactggcgc cagctcctca acgccaagct catcacccag aggaagttcg 2700

acaacctcac caaggccgag agaggcggcc tttccgagct tgataaggcc ggcttcatca 2760

agcgccagct cgtcgagaca cgccagatca caaagcacgt ggcccagatc ctcgactccc 2820

gcatgaacac caagtacgac gagaacgaca agctcatccg cgaggtgaag gtcatcaccc 2880

tcaagtccaa gctcgtgtcc gacttccgca aggacttcca gttctacaag gtgcgcgaga 2940

tcaacaacta ccaccacgcc cacgacgcct acctcaatgc cgtggtgggc acagccctca 3000

tcaagaagta cccaaagctc gagtccgagt tcgtgtacgg cgactacaag gtgtacgacg 3060

tgcgcaagat gatcgccaag tccgagcaag agatcggcaa ggcgaccgcc aagtacttct 3120

tctactccaa catcatgaat ttcttcaaga ccgagatcac gctcgccaac ggcgagatta 3180

ggaagaggcc gctcatcgag acaaacggcg agacaggcga gatcgtgtgg gacaagggca 3240

gggatttcgc cacagtgcgc aaggtgctct ccatgccgca agtgaacatc gtgaagaaga 3300

ccgaggttca gaccggcggc ttctccaagg agtccatcct cccaaagcgc aactccgaca 3360

agctgatcgc ccgcaagaag gactgggacc cgaagaagta tggcggcttc gattctccga 3420

ccgtggccta ctctgtgctc gtggttgcca aggtcgagaa gggcaagagc aagaagctca 3480

agtccgtcaa ggagctgctg ggcatcacga tcatggagcg cagcagcttc gagaagaacc 3540

caatcgactt cctcgaggcc aagggctaca aggaggtgaa gaaggacctc atcatcaagc 3600

tcccgaagta cagcctcttc gagcttgaga acggccgcaa gagaatgctc gcctctgctg 3660

gcgagcttca gaagggcaac gagcttgctc tcccgtccaa gtacgtgaac ttcctctacc 3720

tcgcctccca ctacgagaag ctcaagggct ccccagagga caacgagcaa aagcagctgt 3780

tcgtcgagca gcacaagcac tacctcgacg agatcatcga gcagatctcc gagttctcca 3840

agcgcgtgat cctcgccgat gccaacctcg ataaggtgct cagcgcctac aacaagcacc 3900

gcgataagcc aattcgcgag caggccgaga acatcatcca cctcttcacc ctcaccaacc 3960

tcggcgctcc agccgccttc aagtacttcg acaccaccat cgaccgcaag cgctacacct 4020

ctaccaagga ggttctcgac gccaccctca tccaccagtc tatcacaggc ctctacgaga 4080

cacgcatcga cctctcacaa ctcggcggcg atccaaagaa gaaacgcaag gtggggggcg 4140

gccccggtgc cgagtacgtg agggccctct tcgatttcaa cggcaacgac gaggaggacc 4200

tcccgttcaa gaagggcgac atcctccgca ttcgtgacaa gccggaggag cagtggtgga 4260

acgcggagga ctccgaaggg aagcgcggga tgatcctcgt gccatacgtg gaaaagtact 4320

ccggcgatta caaggaccac gatggcgact ataaggatca cgacatcgac tacaaggatg 4380

acgacgacaa gagcggcatg accgatgcgg agtacgtgcg catccacgag aagctcgaca 4440

tttacacctt caagaagcag ttcttcaaca acaaaaagag cgtctcccac cgctgctacg 4500

tgctcttcga gctgaaacgc cgcggcgaaa ggagggcttg tttctggggc tacgcggtga 4560

acaagccgca gagcggcaca gaaaggggca tccacgcgga gatcttcagc attcgcaagg 4620

tggaggagta tctccgcgac aacccgggcc agttcaccat caactggtac agctcttggt 4680

ccccgtgcgc ggactgcgcc gagaagatcc tcgagtggta caaccaagaa ttacgtggca 4740

atggccacac cctcaaaatc tgggcttgta agctctatta cgagaagaac gcccgcaacc 4800

aaatcggcct ctggaacctc cgcgataatg gcgtgggcct caatgtgatg gtgtccgagc 4860

actaccagtg ctgccgcaag atcttcatcc agtcctccca caaccagctc aacgagaatc 4920

gttggctcga gaagaccctc aagagggccg agaagaggag gagcgaatta tccatcatga 4980

tccaagttaa gatcctccat accaccaagt ccccggcggt gggcccgaag aagaagagga 5040

aggtggggcc ggagggccgc ggctctttac tcacttgtgg cgacgtggag gaaaatccgg 5100

gcccgaccaa tctgtccgac atcatcgaga aagaaaccgg caaacaactc gtcatccaag 5160

agagcatcct catgctgccg gaagaagtgg aggaagtgat cggcaataag ccggagagcg 5220

atatcctcgt gcataccgcg tacgatgagt ccaccgatga gaacgtgatg ctcctcacca 5280

gcgacgcccc ggaatataaa ccgtgggccc tcgtcatcca agacagcaat ggcgaaaata 5340

agatcaaaat gctcccgaaa aaaaagagga aggtcgcgac caacttctct ttactcaagc 5400

aagccggcga tgtggaagaa aaccccggtc cgacaaacct ctccgacatc attgagaagg 5460

aaactggcaa gcaactcgtg atccaagaat ccattttaat gctcccagag gaagtggagg 5520

aggtgatcgg caacaagcca gagtccgaca tcctcgtgca cacagcgtac gacgaaagca 5580

ccgacgagaa cgtcatgtta ttaacatccg atgccccgga gtataagccg tgggcgctcg 5640

tcatccaaga tagcaatggc gaaaacaaga tcaaaatgct gccgaagaag aagaggaaag 5700

tcgaggggcg cggctccctc ctcacatgcg gggatgtgga ggagaacccc ggtccaacca 5760

atttatccga catcatcgaa aaggaaactg gtaagcagct ggtgatccaa gaaagcattc 5820

tcatgctccc agaagaggtg gaagaggtca tcggcaataa gccggaatcc gatatcctcg 5880

tccacaccgc ctatgacgag agcaccgatg aaaatgtcat gttattaacc agcgatgcgc 5940

cagagtacaa gccgtgggct ttagtgatcc aagatagcaa tggcgagaac aaaatcaaaa 6000

tgctcagcgg gggcagcccg aagaagaaaa ggaaggtgtg agagctc 6047

<210> 2

<211> 49

<212> DNA

<213> SpCas9 HR FP

<400> 2

ttacgccaag ctgcccttgg cggccgcatc ggcaccaatt ctgttggct 49

<210> 3

<211> 22

<212> DNA

<213> SpCas9 HR RP

<400> 3

atcgccgccg agttgtgaga gg 22

<210> 4

<211> 44

<212> DNA

<213> PmCDA-SpCas9 HR FP

<400> 4

cctctcacaa ctcggcggcg atccaaagaa gaaacgcaag gtgg 44

<210> 5

<211> 19

<212> DNA

<213> PmCDA-SpCas9 HR RP

<400> 5

cggccccacc ttcctcttc 19

<210> 6

<211> 43

<212> DNA

<213> Sp-eUGI HR FP

<400> 6

gaagaggaag gtggggccgg agggccgcgg ctctttactc act 43

<210> 7

<211> 48

<212> DNA

<213> eUGI HR RP

<400> 7

gcgaattgaa gctgcccttg gagctctcac accttccttt tcttcttc 48

<210> 8

<211> 22

<212> DNA

<213> molecular identification of forward primer

<400> 8

actaacttct tccctcctgc gg 22

<210> 9

<211> 22

<212> DNA

<213> molecular identification reverse primer

<400> 9

agatccaaac cctccctgac ca 22

Claims (11)

1. A high-efficiency single-base editing system OsSpCas9-eCDA, which is characterized in that the single-base editing system OsSpCas9-eCDA can cut plant genome and has a nucleotide sequence shown in SEQ ID No: 1 is shown.

2. An expression cassette comprising the single base editing system OsSpCas9-eCDA of claim 1.

3. An expression vector, wherein the expression vector is inserted with the single base editing system OsSpCas9-eCDA of claim 1 or the expression cassette of claim 2.

4. The expression vector according to claim 3, wherein the vector is PUC57-AMP, pHUN400 or pHUN 900.

5. A targeting vector, wherein the single base editing system OsSpCas9-eCDA of claim 1 or the expression cassette of claim 2 and a target site sequence are inserted into the targeting vector.

6. A transgenic cell transformed with the single base editing system ospspcas 9-eCDA of claim 1, the expression cassette of claim 2, the expression vector of claim 3 or 4, or the targeting vector of claim 5;

wherein the transgenic cell is escherichia coli or agrobacterium tumefaciens (A)Agrobacterium tumefaciens)。

7. Use of the single base editing system OsSpCas9-eCDA of claim 1, the expression cassette of claim 2, the expression vector of claim 3 or 4, the targeting vector of claim 5, or the transgenic cell of claim 6 in plant genome editing, wherein the plant genome editing comprises shearing a plant genome to achieve single base editing from C/G to T/A on the plant genome to obtain a transgenic plant or plant part containing a mutation site.

8. Use according to claim 7, wherein the plant is a monocotyledonous plant.

9. The use of claim 8, wherein the plant is rice.

10. Use according to claim 9, wherein the plant is japonica rice.

11. The use of claim 10, wherein the plant is japonica rice nipponica.

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