A cas9 protein mutant and its application in plant gene editing

Abstract

The application belongs to the technical field of gene editing, and particularly relates to a Cas9 protein mutant and application thereof in plant gene editing. The application discloses a Cas9 protein mutant, an amino acid sequence of which is shown as SEQ ID NO. 1. The Cas9 protein mutant can efficiently screen cutting of various target sites, and is a Cas9 protein not dependent on NGG target site cutting. The application not only widens the targeting range of the Cas9 protein, but also provides the Cas9 protein mutant with higher gene editing efficiency, and has important application value in plant genome editing.

Description

Technical Field

[0001] This invention belongs to the field of gene editing technology, specifically relating to a Cas9 protein mutant and its application in plant gene editing. Background Technology

[0002] The CRISPR / Cas9 system, a revolutionary gene-editing tool, is widely used in plant gene function research, crop improvement, and crop breeding. This system, through the synergistic action of guide RNA (gRNA) and the Cas9 nuclease, can induce double-strand breaks at target DNA sequences, triggering cellular repair mechanisms to achieve genetic operations such as gene knockout, insertion, and replacement. Its high efficiency, precision, and wide applicability make it an important tool in research and application. However, despite the enormous potential of CRISPR / Cas9 technology, its application in plant gene editing still faces challenges. First, the CRISPR / Cas9 system's target recognition depends on specific PAM (protospacer adjacent motif) sequences, with "NGG" being the most commonly used PAM sequence, which limits the range of editable target sites. Second, the editing efficiency of the CRISPR / Cas9 system is uneven across different target sites, which is particularly significant in plant gene editing, increasing the complexity of crop improvement and functional genomics research. Therefore, developing a highly specific, PAM-independent Cas9 protein mutant is of great importance for effectively improving the efficiency and precision of plant gene editing. Summary of the Invention

[0003] To address the aforementioned issues, this invention provides a Cas9 protein mutant and its application in plant gene editing. The Cas9 protein mutant provided by this invention has broad prospects for biotechnological applications, particularly in plant genome editing and functional gene research.

[0004] To achieve the above objectives, the specific technical solution of the present invention is as follows: The first aspect of the present invention provides a Cas9 protein mutant, the amino acid sequence of which is shown in SEQ ID NO.1.

[0005] Furthermore, the Cas9 protein mutant is a Cas9 protein mutant that does not depend on NGG target cleavage.

[0006] A second aspect of the present invention provides a gene editing system comprising sgRNA and the Cas9 protein mutant.

[0007] A third aspect of the present invention provides an expression vector comprising the gene editing system described above.

[0008] The fourth aspect of this invention provides an application of the gene editing system or expression vector described above as an editing tool for editing genomic DNA in related editing of genomic DNA fragments.

[0009] Furthermore, the editing refers to the substitution, insertion, or deletion of bases.

[0010] Furthermore, the genomic DNA fragment is a plant genomic DNA fragment.

[0011] Furthermore, the plant is a birch or a poplar.

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a Cas9 protein mutant and its application in plant gene editing. The amino acid sequence of the Cas9 protein mutant is shown in SEQ ID NO.1. This protein mutant breaks through the dependence of the traditional CRISPR / Cas9 system on the "NGG" PAM sequence, significantly expanding the range of editable plant genome target sites, while improving the specificity and overall efficiency of the editing process. This invention not only provides a more precise and flexible tool for plant gene function research, but also provides efficient and reliable technical support for practical production practices such as crop stress resistance improvement and precision breeding of traits, possessing significant scientific value and broad application prospects. Attached Figure Description

[0013] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0014] Figure 1 To detect Indel mutations using PCR and to evaluate the efficiency of Cas9-M9 gene editing and Cas9-M9-mediated target DNA cleavage using the TCEP (Transient CRISPR / Cas Editing in Plants) method. Figure 1 In the text, 'a' represents the PCR-based Indel mutation detection method, which assesses the Cas9-M9 gene editing efficiency by comparing the amplification efficiency of the target region and the non-target region; 'Reference gene-F1' and 'Bp-Reference gene-R1' represent the primer sets for amplifying the internal reference; 'Target-NNN-F' and 'Target-NNN-F' represent the primer sets for amplifying the target region fragment; and 'Target site' represents the target site. Figure 1b: The TCEP method detects the target cleavage of Cas9-M9. By comparing the amplification of the internal reference and the control sample, it can be determined whether Cas9-M9 cleaves the target and its cleavage efficiency can be calculated. Y1 and Y2 represent the primer sets for amplifying the internal reference; T1 and T2 represent the primer sets for amplifying the target region fragment; Target site represents the target site.

[0015] Figure 2 Statistical analysis of DNA editing efficiency of Cas9-M9 on 64 different PAM sequences.

[0016] Figure 3 Blue-white screening plates were created. Sixteen PAM sites (XNN, where X is any base) were randomly selected from 64 plates, and four plates containing wild-type Cas9-cleavable PAM (NGG) were selected as controls.

[0017] Figure 4 Colony counts on Cas9-M9 transformation plates. Figure 2 In the experimental section, blue and white colonies on the plates were counted. White colonies indicated successful cutting, while blue colonies indicated uncutting.

[0018] Figure 5 To verify the sequencing diagrams of target mutations, the reference sequence is in the top row of each small image, and the actual sequenced sequence is in the bottom row. "-" indicates the location of the mutation, the differential bases are in the red box, the red bases represent PAM site information, and the blue bases represent target sequence information.

[0019] Figure 6 To evaluate the cleavage efficiency of Cas9-M9 against different PAM sequences in birch (a) and poplar (b) using the transient CRISPR / Cas editing system (TCEP), wild-type Cas9 targeting the AGG PAM sequence was used as a control.

[0020] Figure 7 To compare the cleavage efficiency of Cas9-M9-mediated target gene sites in birch (left column) and poplar (right column) with Sanger sequencing analysis results, and with wild-type Cas9 editing results as a control; in each small figure, the reference sequence is in the top row, the actual sequenced sequence is in the bottom row, the differential bases are in the red box, the red bases in the figure represent PAM site information, the blue bases represent target sequence information, and "-" indicates the location of the mutation. Detailed Implementation

[0021] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0022] Example 1: A Cas9 protein mutant and its application in plant gene editing In previous studies, this invention constructed a Cas9 protein mutant library and obtained candidate Cas9 protein mutants capable of efficiently screening and cleaving various target sites, named Cas9-M9. The properties of the Cas9-M9 protein were then investigated through the following experiments.

[0023] The amino acid sequence of Cas9-M9 is shown in SEQ ID NO.1. SEQ ID NO.1:

[0024] I. Experimental Methods 1. Determination of the editing efficiency of Cas9-M9 protein in yeast This invention is based on the reporter gene on the reporter vector. BpEBP1 The CDS design (GenBank accession number: PP081490) targets 64 different PAM (NNN, where N is A, C, G, or T) sites for gRNAs. Each gRNA is then cloned into a gRNA expression vector, and the gRNA expression vector is transformed into a vector containing the reporter vector pGBKT7- BpEBP1 In yeast competent cells expressing the Cas9-M9 vector.

[0025] The method for constructing the reporter vector is as follows: A birch transcription factor with self-activation capability is used. BpEBP1 (GenBank accession number PP081490) was cloned downstream of the GAL4DNA-BD vector pGBKT7 to obtain the reporter vector pGBKT7- BpEBP1 .

[0026] The construction method of gRNA expression vector is as follows: In expression vector pHIS2, using... Sma I and BamH The HIS3 promoter-HIS3 terminator fragment (9bp~1450bp) was excised by restriction endonuclease to obtain a linearized vector, which was then ligated to the pADH1-gRNA expression cassette to obtain the gRNA expression vector pHIS2-gRNA.

[0027] The method for constructing the Cas9-M9 expression vector is as follows: via... Kpn I and Sma The pGADT7-Rec2 vector was digested with enzyme I, its GALDNA-AD domain (1554bp~2037bp) was deleted, and the gene sequence encoding the protein shown in SEQ ID NO.1 was inserted to construct the Cas9-M9 expression vector pGADT7-Cas9-M9.

[0028] Transformed yeast competent cells were cultured to OD. 600 After reaching 1.0, the pGBKT7 plasmid was extracted, and the editing efficiency of Cas9-M9 was evaluated by RT-qPCR.

[0029] To calculate the relative gene editing efficiency, the ΔΔCt value is calculated using the following formula: First, calculate the ΔCt value: ΔCt = Ct(target) - Ct(reference), where Ct(target) represents the threshold cycle (Ct value) of the target gene and Ct(reference) represents the threshold cycle of the reference gene. This value can be used in either the control group or the experimental sample.

[0030] Subsequently, ΔΔCt is calculated using the following formula: ΔΔCt = ΔCt(experimental) - ΔCt(control). The relative gene expression or cleavage efficiency is determined using formula 2. -ΔΔCt Perform the calculation.

[0031] Cutting efficiency is calculated using the following formula: Cutting efficiency = (1 - 2^ -ΔΔCt The cleavage efficiency is defined as 100% (Ct > 35). Primer information is shown in Tables 1-4, and the schematic diagram is shown below. Figure 1 . Figure 1 a: PCR-based Indel mutation detection method. The ternary vector was co-transformed into yeast cells and cultured in SD / -Leu / Trp / URA liquid medium. After collecting yeast cells, the pGBKT7 plasmid was extracted and used as a PCR template. Using the non-target region fragment of BpEBP1 as an internal control, two primer sets were designed to amplify the internal control; the primer sets for amplifying the target region fragment were used as the experimental group (Target-NNN-F / Target-NNN-F, specific primer sequences are shown in Tables 1-4). Mutations at the target site affected the amplification efficiency of the primers in the experimental group, increasing the Ct value. Therefore, the gene editing efficiency of the Cas9-M9 protein could be evaluated by comparing the amplification efficiency of the target and non-target regions.

[0032] Table 1 Primers for Cas9-M9 editing efficiency detection Table 2 is a continuation of Table 1. Table 3 is a continuation of Table 2. Table 4 is a continuation of Table 3. 2. Detection of the editing efficiency of Cas9-M9 in plants See schematic diagram. Figure 1 b: Cas9-M9 was cloned into the pKO119_pEgP237-2A vector to replace SpCas9, transformed using Agrobacterium EHA105, and CRISPR transiently edited plants were cultured using the CPDAT method. Based on the birch... BpEBP1 (NCBI gene accession number PP081490) and Shanxin Yang PdbPFB1 Five target sequences were designed based on the gene sequence (NCBI accession number PV221088) (sequence information is shown in Table 5). The cleavage efficiency was then evaluated using the TCEP (Transient CRISPR / Cas Editing) method. Genomic DNA was extracted two days after transfection, and primer sets were designed to amplify the DNA region containing the target site (T1 and T2 in the schematic diagram). Simultaneously, primer sets were designed to amplify the region without the target site as an internal control (Y1 and Y2 in the schematic diagram). Specific primer sequence information used in the experiment is shown in Tables 6 and 7. Untransformed wild-type plant DNA was used as a control. Cleavage was detected by PCR amplification of the target site: if the target DNA was cleaved by Cas9-M9, the amplification ability of the fragment was weakened, and the Ct value was higher than that of the wild-type control, thus indicating that cleavage had occurred.

[0033] Table 5 Cas9-M9 Cutting Target Sequence Information Table 6. TCEP detection primers for BpEBP1 Table 7. Primers for TCEP detection of PdbBHLH II. Experimental Results 1. Cutting efficiency of Cas9-M9 under different PAM conditions Detection of Indel mutations using PCR ( Figure 1 a) Evaluate the editing efficiency of Cas9-M9 under 64 PAMs ( Figure 2 Of these, 54 PAMs had a Ct > 35, considered as having 100% cutting efficiency; the other 10 PAMs (such as ATC, ACT, AGG, CAG, etc.) had slightly lower efficiency, but still achieved cutting efficiency of 60%~90%. Blue-white screening was performed on 16 representative PAMs from ANN, CNN, GNN, and TNN. Both Cas9-M9 and wild-type Cas9 showed a phenotype primarily consisting of white spots. Figure 3 and Figure 4 ), and sequencing confirmed that the target site had mutated ( Figure 5 ).

[0034] 2. Gene editing of Cas9-M9 in plant cells The editing ability of Cas9-M9 was evaluated in *Birchia spp.* and *Populus tomentosa*. Target PAMs in *Birchia spp.* included GAA, GAG, CGT, CTT, and AAG; target PAMs in *Populus tomentosa* included ACA, ACT, CAC, GTG, and GCT. qPCR analysis using the TCEP method showed that all targets could be cleaved by both Cas9-M9 and wild-type Cas9, but with different efficiencies. Figure 6 Sequencing results further confirmed that all PAM sequences were successfully edited. Figure 7 ).

[0035] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described here to avoid redundancy. Although preferred embodiments of the invention have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this invention.

[0036] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A Cas9 protein mutant, characterized in that, The amino acid sequence of the Cas9 protein mutant is shown in SEQ ID NO.

1.

2. The Cas9 protein mutant according to claim 1, characterized in that, The Cas9 protein mutant is a Cas9 protein mutant that does not depend on NGG target cleavage.

3. A gene editing system, characterized in that, The gene editing system comprises sgRNA and the Cas9 protein mutant as described in claim 1.

4. An expression carrier, characterized in that, The expression vector contains the gene editing system of claim 3.

5. The application of the gene editing system of claim 3 or the expression vector of claim 4 as an editing tool for editing genomic DNA in related editing of genomic DNA fragments.

6. The application according to claim 5, characterized in that, The editing refers to the substitution, insertion, or deletion of bases.

7. The application according to claim 6, characterized in that, The genomic DNA fragment is a plant genomic DNA fragment.

8. The application according to claim 7, characterized in that, The plant in question is either a birch or a poplar.

Images