HTR2 promoter, CRISPR / Cas9 gene editing vector and application of HTR2 promoter and CRISPR / Cas9 gene editing vector in plant gene editing

Abstract

The invention discloses an HTR2 promoter, a CRISPR / Cas9 gene editing vector and application of the CRISPR / Cas9 gene editing vector in plant gene editing, and belongs to the technical field of biology. According to the present invention, the HTR2 promoter having the nucleotide sequence represented by SEQ ID NO: 1 can efficiently drive the transcription of the Cas9 gene in the CRISPR / Cas9 gene editing vector, can edit the target gene, can significantly improve the gene editing efficiency, and can reduce the off-target effect and the chimera generation; the homozygous mutation rate of the CRISPR / Cas9 gene editing vector of the Cas9 gene driven by the HTR2 promoter is obviously higher than that of common promoters such as a CaMV35S promoter, and the CRISPR / Cas9 gene editing vector has a better application prospect in the field of plant gene editing.

Description

Technical Field

[0001] The present invention relates to the field of gene editing technology, and in particular to an HTR2 promoter, a CRISPR / Cas9 gene editing vector and their application in plant gene editing. Background Art

[0002] As a revolutionary gene-editing technology, the CRISPR / Cas9 system has been widely used in fields such as biomedicine and agricultural science due to its high efficiency, precision, and ease of use. In plants, CRISPR / Cas9 technology has been successfully applied to gene function research, crop trait improvement, and stress tolerance breeding.

[0003] The efficiency and specificity of gene editing are affected by many factors. Among them, the promoter is the core element of gene expression regulation, which determines the spatiotemporal specificity and expression level of Cas9 expression, thereby affecting the efficiency of gene editing and potential off-target effects. Therefore, screening highly active promoters is the key to improving the efficiency of precise gene editing.

[0004] The S phase of the cell cycle is the active period of DNA replication. During this period, the chromosome structure is loose, and gene editing of DNA during this period may be more efficient.

[0005] HTR2 (Histone H3K4 Methyltransferase 2, AT1G09200) encodes a histone H3K4 methyltransferase involved in chromatin regulation. Its expression is cell cycle-dependent and peaks in the S phase.

[0006] BRI1 (BRASSINOSTEROID INSENSITIVE 1) is a receptor for the plant sterol hormone brassinosteroid (BR) and plays an important role in plant growth and development. When the BRI1 (AT4G39400) gene is knocked out in Arabidopsis thaliana, homozygous mutants exhibit visible phenotypes such as dwarfism, dark green leaves, and curled leaves. Heterozygous mutants exhibit mild growth and developmental defects in plant height, fertility, leaf color, and morphology, but these defects are not as severe as those in homozygous mutants and are intermediate between those in homozygous and wild type, demonstrating a dosage effect.

[0007] These phenotypes are easily observable and quantifiable, making BRI1 an ideal target gene for evaluating gene editing efficiency.

[0008] Current plant gene editing technologies mostly utilize the CaMV 35S (hereafter referred to as 35S) promoter to drive Cas9. However, sustained high expression of Cas9 driven by the 35S promoter can lead to the following problems: formation of early embryonic mosaics, reducing the proportion of homozygous mutants; increased probability of cleavage at non-target sites, resulting in off-target effects; and failure to fully utilize the repair mechanisms during DNA replication to improve editing efficiency (Zhang, Y., et al. (2016). Applications of CRISPR-Cas9 based genome editing in plant biology. Acta Pharm. Sin. B 6, 535-539.). Summary of the Invention

[0009] In view of this, the purpose of the present invention is to provide an HTR2 promoter, a CRISPR / Cas9 gene editing vector and its application in plant gene editing, which can significantly improve the plant gene editing efficiency and the proportion of homozygous mutants while reducing off-target effects and chimera production.

[0010] In order to achieve the above object, the present invention adopts the following technical solutions: The present invention provides an HTR2 promoter, wherein the HTR2 promoter has a nucleotide sequence shown in SEQ ID NO: 1.

[0011] In the present invention, the aforementioned HTR2 promoter is the Arabidopsis thaliana (Latin name, Arabidopsis thaliana) AtHTR2 promoter.

[0012] The present invention also provides an expression cassette, wherein the expression cassette comprises the HTR2 promoter.

[0013] In the present invention, the expression cassette comprises the HTR2 promoter and the Cas gene, and the HTR2 promoter drives the transcription of the Cas gene.

[0014] In the present invention, the aforementioned Cas gene is the Cas9 gene.

[0015] Common Cas genes include Cas9 gene, Cas12 gene, Cas13 gene and Cas3 gene. In the present invention, as a widely used gene editing tool, Cas9 gene is preferred.

[0016] The present invention also provides a gene editing vector, comprising: the aforementioned HTR2 promoter or the aforementioned expression cassette.

[0017] In the present invention, the aforementioned gene editing vector is a CRISPR / Cas gene editing vector, and the aforementioned HTR2 promoter drives the transcription of the aforementioned Cas gene.

[0018] In the present invention, the aforementioned Cas gene is the Cas9 gene.

[0019] In the present invention, the aforementioned CRISPR / Cas gene editing vector further comprises an expression cassette of a gRNA targeting the editing site.

[0020] Preferably, the CRISPR / Cas gene editing vector of the present invention is the plasmid pHZM184B-AtHTR2_Pro.

[0021] Preferably, the expression cassette of the gRNA targeting the editing site contained in the CRISPR / Cas gene editing vector of the present invention is a gRNA designed according to the exon of the target gene AtBRI1, and its nucleotide sequence is 5'-GCATTTGAAGGATCACACCG-3'.

[0022] The present invention also provides a host cell comprising: the aforementioned HTR2 promoter, the aforementioned expression cassette or the aforementioned gene editing vector.

[0023] In the present invention, host cells include microbial cells and plant cells. Among them, plant cells do not have the ability to develop into complete plant individuals. In addition, in the present invention, the host cell is Agrobacterium GV3101.

[0024] The present invention also provides a plant gene editing method based on CRISPR / Cas, which comprises the following steps: 1) Assemble the aforementioned CRISPR / Cas gene editing vector; 2) Transforming the aforementioned CRISPR / Cas gene editing vector into plants; 3) Positive transformed plants were obtained through screening.

[0025] Preferably, the CRISPR / Cas-based plant gene editing method of the present invention uses AtBRI1 as the target gene, and the primers AtBRI1-gRNA.F and AtBRI1-gRNA.R can be used to construct an editing vector for targeted knockout of the AtBRI1 gene.

[0026] The beneficial effects of the present invention include at least: the HTR2 gene promoter-driven CRISPR / Cas9 system significantly outperforms commonly used promoters such as the CaMV 35S promoter in terms of the proportion of homozygous mutants produced. Furthermore, cell cycle-specific expression of the HTR2 gene promoter improves gene editing efficiency. In particular, the HTR2 gene promoter-driven CRISPR / Cas9 system achieved a homozygous mutation rate of 50.98% in Arabidopsis thaliana, significantly higher than the 5.56% of the 35S promoter. Phenotypic analysis showed that BRI1 knockout plants exhibited typical phenotypes such as dwarfing and leaf curling, further verifying the high efficiency of gene editing. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the present invention or the prior art, a brief introduction is given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0028] Figure 1 This is the phenotypic analysis result of the AtBRI1 gene knockout plants in Example 4 of the present invention.

[0029] Figure 2 This is the comparison result of the gene editing efficiency driven by the AtHTR2 promoter in Example 5 of the present invention and common promoters such as the 35S promoter. DETAILED DESCRIPTION

[0030] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0031] Unless otherwise specified, the methods used in the following examples are conventional methods. For specific steps, please refer to: Molecular Cloning: A Laboratory Manual (Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor).

[0032] The methods for obtaining the various biological materials described in the examples merely provide experimental methods for achieving the disclosed objectives and should not be construed as limiting the sources of the biological materials used in this invention. In fact, the sources of the biological materials used are diverse; any legally and ethically accessible biological material can be substituted for it as indicated in the examples. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0033] The nucleotides involved in the following examples can all be synthesized using existing techniques.

[0034] Example 1: Construction of gene editing vector.

[0035] The CRISPR / Cas9 gene editing vector pHZM184B used in this experiment was constructed using pHZM172m as its backbone. Constructed and preserved in this experiment, pHZM172m is a 35S-driven vector. The nucleotide sequence of pHZM172m consists of SEQ ID NO: 3 and SEQ ID NO: 4, with bp 1-7287 of pHZM172m being SEQ ID NO: 3 and bp 7288-14547 of pHZM172m being SEQ ID NO: 4.

[0036] Specifically, the steps for constructing a CRISPR / Cas9 gene editing vector that drives Cas9 gene transcription with 35S are as follows: First, the Cas9 gene was amplified using CasF and CasR (Table 1) (provided by Researcher Xie Qi, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Li, J., Zhang, H., Si, X., Tian, ​​Y., Chen, K., Liu, J., Chen,Q., and Xie, Q. (2015). Generation of thermotolerant tomato plants byexpressing Arabidopsis thaliana heat shock transcription factor HsfA1a withIRES. Mol. Plant 8, 1820–1823.) and inserted into pHZM172m between the XhoI and PacI restriction sites using in-fusion technology. Secondly, U26.F and U26.R (Table 1) were used to amplify the gRNA expression cassette (provided by Professor Qijun Chen of China Agricultural University, 12.Xing, S., Chen, K., Zhu, H., and Chen, Q. (2014). Fine-tuning sugar content in Arabidopsis seeds by CRISPR-Cas9-directed mutagenesisof the SWEET11 and SWEET12 sucrose transporters. BMC Plant Biol. 14, 327.) and inserted into the EcoRI and StuI restriction sites of pHZM172m by in-fusion technology. The final constructed CRISPR / Cas9 gene editing vector was named pHZM184B, whose nucleotide sequence was composed of SEQ ID NO: 5 and SEQ ID NO: 6, with 1-8998 bp of SEQ ID NO: 5 and 8999-17542 bp of SEQ ID NO: 6.

[0037] Example 2: Construction of a specific promoter-driven CRISPR / Cas9 gene editing vector.

[0038] 1) Construction of CRISPR / Cas9 gene editing vector specifically driven by the AtHTR2 promoter.

[0039] Promoter acquisition: Using 184-HTR2.F and 184-HTR2.R (Table 1) as primers, KOD high-fidelity enzyme and Arabidopsis thaliana Col-0 DNA as a template, the AtHTR2 promoter was obtained, the nucleotide sequence of which is SEQ ID NO: 1.

[0040] Assembly of gene editing vectors driven by specific promoters: Based on pHZM184B, the amplified AtHTR2 promoter fragment was ligated between StuI and XhoI by the In-Fusion method, replacing 35S in pHZM184B, and finally a gene editing vector was constructed in which the AtHTR2 promoter specifically drives Cas9 gene transcription: pHZM184B-AtHTR2_Pro.

[0041] 2) Construction of CRISPR / Cas9 gene editing vector specifically driven by the DR5v2 promoter.

[0042] Promoter acquisition: Using primers 184I.F and 184I.R (Table 1) and plasmid pDR5v2 (Liao, C.-Y., Smet, W., Brunoud, G., Yoshida, S., Vernoux, T., and Weijers, D. (2015). Reporters for sensitive and quantitative measurement of auxin response. Nat. Methods 12, 207–210.) as a template, the DR5v2 promoter was obtained, the nucleotide sequence of which is shown in SEQ ID NO: 2.

[0043] Assembly of gene editing vectors driven by specific promoters: Based on pHZM184B, the amplified DR5v2 promoter fragment was ligated between StuI and XhoI by the In-Fusion method, replacing 35S in pHZM184B, and finally a gene editing vector pHZM184B-I was constructed in which the DR5v2 promoter specifically drives Cas9 gene transcription.

[0044] Example 3: Construction of gene editing vector for targeted knockout of AtBRI1.

[0045] gRNA design: The CRISPR-P2.0 online tool (http: / / crispr.hzau.edu.cn / CRISPR2 / ) was used to design gRNA based on the exons of the AtBRI1 gene. The nucleotide sequence was 5'-GCATTTGAAGGATCACACCG-3' and used as a primer to synthesize AtBRI1-gRNA.F and AtBRI1-gRNA.R (Table 1) to construct an editing vector for targeted knockout of the AtBRI1 gene.

[0046] These vectors were digested with BsaI, and the AtBRI1 gRNA expression cassette was subsequently recombined into the pHZM184B, pHZM184B-AtHTR2_Pro, and pHZM184B-I vectors using T4 ligase. These plasmids were named 184-35S_Pro, 184-AtHTR2_Pro, and 184I, respectively. After sequencing verification, these plasmids were transformed into GV3101 Agrobacterium tumefaciens for later use.

[0047] Table 1: Primer list

[0048] Example 4: Arabidopsis genetic transformation.

[0049] Plant material: Arabidopsis Col-0 wild-type was used as the transformation recipient. Agrobacterium-mediated transformation: Agrobacterium containing the gene-editing vector was transformed into Arabidopsis thaliana using the Agrobacterium-mediated floral dip method (Clough, SJ, & Bent, AF (1998). Floral dip: A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735-743). At least 30 Arabidopsis plants were transformed with each vector. Screening for positively transformed plants: T0 generation seeds were sown in nutrient soil (peat soil: vermiculite = 1:1) and screened. Bri1 homozygous mutants with extremely dwarfed plants and wrinkled leaves were observed ( Figure 1 ). Figure 1Phenotypic analysis of AtBRI1 knockout plants. WT denotes wild-type Arabidopsis Col-0, Homozygote denotes a homozygous mutant of CRISPR / Cas9-mediated AtBRI1 knockout, and Hterozygote denotes a heterozygous mutant of CRISPR / Cas9-mediated AtBRI1 knockout. Transformation success was confirmed by spraying surviving T0 plants with 3‰ Basta. Basta-resistance screening and PCR detection yielded the following number of T0-generation positively transformed plants: 184-35S_Pro: 36 plants, 184-AtHTR2_Pro: 51 plants, and 18 plants of 184I.

[0050] Example 5: Detection of gene editing efficiency.

[0051] 1) Extraction of genomic DNA The genomic DNA of T0 generation positive transformed plants was extracted using the CTAB method.

[0052] 2) PCR amplification Primers (AtBRI1-Test.F and AtBRI1-Test.R (Table 1)) were designed to amplify the target region of the AtBRI1 gene, and the primers contained sequencing adapter sequences.

[0053] 3) High-throughput sequencing The PCR products were sequenced by Illumina MiSeq high-throughput sequencing.

[0054] 4) Data Analysis The sequencing data were analyzed using CRISPResso2 software (Clement, K., et al. (2019). CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat. Biotechnol. 37, 224–226.). The read numbers and ratios of different genotypes (homozygous mutations, heterozygous mutations, and wild type) were calculated to determine the gene editing efficiency.

[0055] 5) Genotype determination criteria Homozygous mutations: insertion-deletion polymorphisms (Indels) cause frameshifts and the mutation reads account for ≥90%; heterozygous mutations: mutation reads account for 20%-80%; wild-type: mutation reads account for <5%. The number of positive lines, homozygous mutants, and heterozygous mutants obtained by transforming with different vectors was counted.

[0056] The homozygous mutation rate (homozygous mutation rate = number of homozygous mutant strains / positive strain coefficient) and heterozygous mutation rate (heterozygous mutation rate = number of heterozygous mutant strains / positive strain coefficient) were calculated. One-way analysis of variance (ANOVA) and Tukey's multiple comparison test were performed using GraphPad Prism 8 software to compare the differences in gene editing efficiency between different promoters. High-throughput sequencing results showed that there were significant differences in gene editing efficiency between CRISPR / Cas9 systems driven specifically by different promoters (Table 2, Figure 2 ).exist Figure 2 In the data, 184-35S_Pro, 184-AtHTR2_Pro, and 184I represent the knockout efficiency of the BRI1 gene driven by Cas9 using the CaMV35S promoter, the AtHTR2 promoter, and the DR5v2 promoter, respectively. In particular, the homozygous mutation rate of 184-AtHTR2_Pro was significantly higher than that of commonly used promoters (P<0.05). The results showed that the AtHTR2 promoter-driven CRISPR / Cas9 system exhibited the highest gene editing efficiency. The homozygous mutation rate driven by the AtHTR2 promoter reached 50.98%, significantly higher than the 5.56% of the 35S promoter. No homozygous mutations were detected using the 184I vector.

[0057] Table 2: Gene editing efficiency driven by promoters such as AtHTR2

[0058] Note: The values ​​in parentheses are the numbers of transgenic lines.

[0059] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. An HTR2 promoter, characterized in that The HTR2 promoter has a nucleotide sequence shown in SEQ ID NO:

1.

2. An expression cassette, characterized in that The expression cassette comprises the HTR2 promoter of claim 1.

3. The expression cassette according to claim 2, characterized in that The expression cassette comprises the HTR2 promoter and the Cas gene of claim 1, and the HTR2 promoter drives the transcription of the Cas gene.

4. The expression cassette according to claim 3, characterized in that The Cas gene is the Cas9 gene.

5. A gene editing vector, characterized in that: Comprising: the HTR2 promoter according to claim 1 or the expression cassette according to any one of claims 2 to 4.

6. The gene editing vector according to claim 5, characterized in that The gene editing vector is a CRISPR / Cas gene editing vector, and the HTR2 promoter drives the transcription of the Cas gene.

7. The gene editing vector according to claim 5 or 6, characterized in that The Cas gene is the Cas9 gene.

8. The gene editing vector according to any one of claims 5 to 7, characterized in that The CRISPR / Cas gene editing vector also contains an expression cassette for a gRNA targeting the editing site.

9. A host cell, characterized in that Comprising: the HTR2 promoter according to claim 1, the expression cassette according to any one of claims 2 to 4, or the gene editing vector according to any one of claims 5 to 8.

10. A plant gene editing method based on CRISPR / Cas, characterized in that: The following steps are involved: 1) Assembling the CRISPR / Cas gene editing vector according to any one of claims 5 to 8; 2) Transforming the CRISPR / Cas gene editing vector into plants; 3) Positive transformed plants were obtained through screening.

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