Method for improving plant genome editing efficiency based on transcription factor AtSOG1 and utilization thereof

Abstract

The invention provides a method for improving plant genome editing efficiency based on a transcription factor AtSOG1 and application of the method, and belongs to the technical field of gene editing. The invention finds that the AtSOG1 transcription factor has multiple influences on gene editing, a target plant is co-transformed by using an expression vector constructed by the AtSOG1 transcription factor and a gene editing vector, or an expression element of the AtSOG1 transcription factor is constructed into the gene editing vector, and then the target plant is transformed, so that the plant genome editing efficiency can be improved. The method provided by the invention not only can promote targeted gene knock-in based on a PE editor and HDR mediation, but also can improve the knockout efficiency of long fragments. Meanwhile, in the embodiment of the invention, an experiment is further designed, it is found that the AtSOG1 can promote a T-DNA unconformity knock-in event, and a T-DNA free knock-in plant is directly obtained in a T0 generation.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of gene editing, and particularly relates to a method for improving plant genome editing efficiency based on a transcription factor AtSOG1 and utilization thereof. BACKGROUND

[0002] Gene editing is a technology capable of precisely modifying the genome of an organism, and the core thereof is to recognize a specific sequence in the genome through a specific tool, modify a DNA double strand or a single strand, and then utilize the DNA repair mechanism of the organism itself to complete gene modification, complete "cutting", "insertion" or "replacement" of a target gene, and thus realize precise regulation of a trait.

[0003] At present, the most widely used gene editing technologies include CRISPR-Cas9, TALEN (transcription activator-like effector nuclease), ZFN (zinc finger nuclease), single-base editor (BE) and prime editor (PE), and although gene editing has been widely applied in plants, there are still many technical and application challenges, such as significant differences in "responsiveness" of different plants to gene editing tools, large differences in editing efficiency, low off-target rate, and T-DNA free knock-in plants obtained only in T1 generation or even T2 generation. Therefore, it is urgent to improve the gene editing efficiency, especially to seek a method for obtaining T-DNA free knock-in plants in T0 generation. SUMMARY

[0004] The application provides a method for improving plant genome editing efficiency based on a transcription factor AtSOG1 and utilization thereof, and the transcription factor AtSOG1 can significantly improve the plant genome editing efficiency and promote T-DNA non-integrated knock-in events, so that T-DNA free knock-in plants are directly obtained in T0 generation.

[0005] The application provides application of Arabidopsis thaliana transcription factor AtSOG1 in improving plant genome editing efficiency, and the nucleotide sequence of the Arabidopsis thaliana transcription factor AtSOG1 is shown in SEQ ID No. 1.

[0006] In a preferred mode of the application, the plant genome editing includes PE editor-based gene editing and / or HDR-mediated gene editing.

[0007] In a preferred mode of the application, the genome editing includes gene knock-in and / or knock-out.

[0008] The present invention also provides a method for constructing an expression vector of Arabidopsis transcription factor AtSOG1, comprising the following steps: using the 35S::SOG1-GFP vector plasmid as a template, introducing mutations into one or more coding amino acid sites of Arabidopsis transcription factor AtSOG1 to obtain the expression vector;

[0009] The mutations include at least one of the following: S350D, S356D, S372A, S430D, S436D, and S372D.

[0010] The present invention also provides an expression vector constructed using the above-described construction method.

[0011] The present invention also provides a method for improving the efficiency of plant genome gene editing, comprising the following steps: mixing the above expression vector and genome gene editing vector, and co-transfecting gene editing material to obtain gene-edited plants.

[0012] The present invention also provides a gene editing vector containing the expression element of Arabidopsis thaliana transcription factor AtSOG1, the nucleotide sequence of which is shown in SEQ ID No. 1.

[0013] In a preferred embodiment of the present invention, the expression element of the Arabidopsis transcription factor AtSOG1 is obtained by amplification, and the expression vector is used as a template during the amplification.

[0014] The present invention also provides a plant genome gene editing method, comprising the following steps: transfecting plants using the above-mentioned gene editing vector.

[0015] This invention also provides the application of the above-mentioned gene editing vector or the above-mentioned plant genome gene editing method in constructing T-DNA-free gene-edited T0 generation plants.

[0016] Beneficial Effects: This invention reveals that the AtSOG1 transcription factor has multiple effects on gene editing. Co-transforming target plants with expression vectors and gene editing vectors constructed using the AtSOG1 transcription factor, or constructing the expression element of the AtSOG1 transcription factor into a gene editing vector and then transforming the target plant, can improve the efficiency of plant genome editing. The method described in this invention can not only promote targeted gene knock-in based on PE (prime editor) and HDR-mediated knock-in, but also improve the knockout efficiency of long fragments. Furthermore, in the embodiments of this invention, experiments were designed to show that AtSOG1 can promote T-DNA non-integration knock-in events, directly obtaining T-DNA-free knock-in plants in the T0 generation. Attached Figure Description

[0017] Figure 1This diagram illustrates different phosphorylation sites of AtSOG1. In the diagram, SQ represents a phosphorylation site; DQ represents a persistent phosphorylation site; and AQ represents a dephosphorylation site.

[0018] Figure 2 To edit the spectrum of the vector pNBGE32-MPK5-HiBiT-AtSOG1;

[0019] Figure 3 To edit the spectrum of the vector pCBGE32-MPK5-HiBiT-AtSOG1;

[0020] Figure 4 This diagram illustrates the process of knocking the HiBiT protein tag sequence into the UBI10 promoter of rice using PE32-HiBiT and explains the detection principle of the HiBiT protein tag. In the diagram, LgBiT represents the large subunit of NanoLUC luciferase, and HiBiT represents the small subunit of NanoLUC luciferase.

[0021] Figure 5 The transcriptional levels of homologous recombination-related proteins in pNBGE32-MPK5-HiBiT-AtSOG1-P T1 generation leaves were detected by RT-qPCR (n=3).

[0022] Figure 6 The image shows the results of measuring the luminescence signal using a microplate reader after mixing the PE32-HiBiT editing vector with various expression vectors in rice protoplasts. In the image, PE32 is the control vector without pegRNA.

[0023] Figure 7 The results are shown in the figure below. Bioluminescence detection of AtSOG1 after being mixed with the editing vector pWDV32-MPK5-HiBiT and transfected into protoplasts, and bioluminescence detection of AtSOG1 transcription factor expression element fusion editing vectors pCBGE32-MPk5-HiBiT and pNBGE32-MPK5-HiBiT-SOG1 after being transfected into protoplasts.

[0024] Figure 8 The image shows the RT-qPCR results after the editing vector pCBGE32-MPk5-HiBiT, which contains the AtSOG1 transcription factor expression element fusion, was transfected into protoplasts.

[0025] Figure 9 Bioluminescence detection results of callus tissue containing the editing vector pNBGE32-MPK5-HiBiT, which fuses AtSOG1 transcription factor expression elements;

[0026] Figure 10 The image shows the detection results of T-DNA integration in AtSOG1T0 generation transgenic plants;

[0027] Figure 11 Image showing the detection results of editing events in AtSOG1T0 generation transgenic plants;

[0028] Figure 12 Figure 1 shows the detection results of the HiBiT knock-in event in AtSOG1 T-DNA free plants. Figure A: Genotyping of AtSOG1 T0 generation transgenic plants; Actin primers were used to detect the quality of genome extraction; Cas9 primers were used to detect T-DNA integration; KI primers were used to detect the full-length knock-in of the HiBiT sequence at the target site; Figure B: Genotyping of AtSOG1 callus tissue without hygromycin selection; Figure C: Genotyping of AtSOG1 T0 generation plants without hygromycin selection; Figure D: Sanger sequencing results of HiBiT knock-in events in all AtSOG1 T-DNA free samples.

[0029] Figure 13 The image shows the Sanger sequencing results at the target sites of transgenic plants SOG1#7, SOG1(DDADD)#12, and SOG1(S372A)#19.

[0030] Figure 14 This describes the knockout of a large fragment in the AtSOG1 knockout vector pRGEB32-MPK1-MPK5;

[0031] Figure 15 This document describes the knockout of a large fragment in the AtSOG1 knockout vector pRGEB32-MPK1-MPK5-SOG1.

[0032] Figure 16 For large fragment knockout cases without the AtSOG1 knockout vector pRGEB32-CPK4-CPK18;

[0033] Figure 17 The knockout of a large fragment in the AtSOG1 knockout vector pRGEB32-CPK4-CPK18-SOG1;

[0034] Figure 18 This is a plasmid map of the pPE32 vector. Detailed Implementation

[0035] This invention provides the application of Arabidopsis transcription factor AtSOG1 in improving the efficiency of plant genome editing, and the nucleotide sequence of the Arabidopsis transcription factor AtSOG1 is shown in SEQ ID No. 1.

[0036] The nucleotide sequence of the transcription factor AtSOG1 described in this invention is shown in SEQ ID No. 1.

[0037] The transcription factor AtSOG1 described in this invention can affect the expression of downstream genes, including the expression of exonucleases FEN1A (related to terminal digestion), DNA recombinases RAD51A, RAD51B, and RAD54, as well as the expression of poly-ADP-ribose polymerase PARP2A. Simultaneously, AtSOG1 can influence T-DNA integration in rice cells to a certain extent and promote T-DNA non-integration knock-in events, directly obtaining T-DNA-free knock-in plants in the T0 generation. AtSOG1 described in this invention may regulate multiple repair pathways and improve the knockout efficiency of long fragments.

[0038] The plant genome editing described in this invention includes gene editing based on PE editors and / or gene editing based on HDR, and the genome editing includes gene knock-in and / or knockout.

[0039] The present invention also provides a method for constructing an expression vector of Arabidopsis transcription factor AtSOG1, comprising using a 35S::SOG1-GFP vector plasmid as a template, and introducing mutations into one or more coding amino acid sites of Arabidopsis transcription factor AtSOG1 to obtain the expression vector;

[0040] The mutations include at least one of the following: S350D, S356D, S372A, S430D, S436D, and S372D.

[0041] The AtSOG1 transcription factor described in this invention contains Figure 1 The five phosphorylation sites shown are phosphorylated sequentially during the DNA damage response, thereby initiating the expression of downstream genes. However, the expression of downstream genes regulated by different numbers of phosphorylation sites is not entirely consistent. The expression of most genes related to DNA damage repair increases with the number of phosphorylation sites, but the expression of some proteins related to the HDR repair pathway reaches its highest level at four phosphorylation sites (S350, S356, S430, and S436). SQ represents the original serine sequence, where the amino acid can be phosphorylated by upstream kinases. DQ indicates the conversion of the original serine to aspartic acid, resulting in sustained phosphorylation at this site. AQ indicates the mutation of the original serine to alanine, preventing further phosphorylation at this site.

[0042] In the embodiments of this invention, AtSOG1 expression vectors containing different numbers of phosphorylation sites were constructed based on the specificity of AtSOG1 phosphorylation sites. During construction, the 35S::SOG1-GFP vector plasmid was used as a template, and base mutations were introduced according to the primers shown in Table 1 to simulate phosphorylation or dephosphorylation of specific phosphorylation sites, ultimately obtaining expression vectors 35S::SOG1-P-GFP, 35S::SOG1(DDADD)-GFP, and 35S::SOG1(S372A)-GFP. The 35S::SOG1-GFP vector described in this invention is based on a previously published article (Xuanpeng Wang, Lili Wang, Yongchi Huang, Zhiping Deng, Cunliang Li, Jian Zhang, Mingxi Zheng, Shunping Yan. A plant-specific module for homologous recombination repair. Proc Natl Acad Sci U SA. 2022 Apr19;119(16):e2202970119. doi:10.1073 / pnas.2202970119.).

[0043] Table 1 Primer information for introducing mutations in expression vector construction.

[0044]

[0045]

[0046] The present invention also provides an expression vector constructed using the above-described construction method.

[0047] The expression vector 35S::SOG1-P-GFP described in this invention corresponds to Figure 1 SOG1-P contains 5 DQs; the expression vector 35S::SOG1(DDADD)-GFP corresponds to Figure 1 SOG1-(DDADD) contains 4 DQ and 1 AQ; the expression vector 35S::SOG1(S372A)-GFP corresponds to Figure 1 SOG1 (S372A) contains 4 SQs and 1 AQ.

[0048] The present invention also provides a method for improving the efficiency of plant genome gene editing, comprising mixing the above-mentioned expression vector and genome gene editing vector, and co-transfecting gene editing material to obtain gene-edited plants.

[0049] The gene editing described in this invention includes gene knock-in and gene knockout. In one embodiment of this invention, in order to evaluate the effect of the Arabidopsis NAC-type transcription factor AtSOG1 on PE editor-mediated gene editing, a PE vector pPE32-HiBiT was constructed to knock in the protein tag HiBiT (33bp) after the strong constitutive promoter UBI10 in rice. The PE vector pPE32-HiBiT and the above expression vector were mixed and co-transformed into plants to obtain gene knock-in plants. The examples verified that the AtSOG1 transcription factor can improve the efficiency of PE editor-mediated short sequence knock-in to a certain extent.

[0050] The plasmid map of the pPE32 vector described in this invention is as follows: Figure 18 As shown, when constructing the pPE32 vector, pENTR11-Cas9 (published in the article: Kabin Xie, Yinong Yang. RNA-guided genome editing in plants using a CRISPR-Cas system. Mol Plant. 2013 Nov; 6(6):1975-83. doi:10.1093 / mp / sst119.) was used as a template. Base mutations were introduced through primers pRGEB32-stop-mF and Cas9-H480A to obtain the vector pENTR11-nCas9(H840A) with a mutated stop codon and the mutated H840A. Using pUC57-OsMMLV (the OsMMLV sequence shown in SEQ ID No. 60 was inserted into the EcoRV site of pUC57 by GenScript) as a template, the OsMMLV fragment was amplified using primers Gib-MMLV-F and Gib-MMLV-R. This fragment was then cloned into the ECORI site of pENTR11-nCas9 (H840A) to obtain the vector pENTR11-nCas9-MMLV. pENTR11-nCas9-MMLV was reacted with p1300-32 (Ding et al 2018)LR to obtain the vector pPE32 (named pRGEB32).

[0051] The method for constructing pPE32-HiBiT according to the present invention includes assembling the PTG structure of HiBiT using pGTR as a template and the Golden Gate method, and ligating it into the pRGEB32 vector with T4 ligase to obtain the vector pPE32-HiBiT.

[0052] Table 2 Primer names and sequences for PE vector construction

[0053]

[0054] In one embodiment of this invention, a HDR-targeting knock-in vector pWDV32-MPK5-HiBiT was constructed. The expression vector and pWDV32-MPK5-HiBiT were co-transfected into rice protoplasts. The results showed that AtSOG1 had a certain promoting effect on HDR-mediated targeting knock-in. The pWDV32-MPK5-HiBiT described in this invention has been previously published (Chen Yache. Research on labeling the rice MPK5 gene using targeted gene insertion technology. [Master's Thesis]. Huazhong Agricultural University. 2021). The vector name pRGEB32-MPK5-HiBiT in the paper was changed to pWDV32-MPK5-HiBiT; both refer to the same vector.

[0055] In one embodiment of the present invention, an HDR gene knockout vector was also constructed, that is, the expression element described in the present invention was inserted into a gene editing vector such as CRISPR / Cas9.

[0056] The present invention also provides a gene editing vector containing the expression element of Arabidopsis thaliana transcription factor AtSOG1, the nucleotide sequence of which is shown in SEQ ID No. 1.

[0057] The expression element of the Arabidopsis transcription factor AtSOG1 described in this invention is obtained by amplification, and the above-mentioned expression vector is used as a template during the amplification.

[0058] In one embodiment of the present invention, the AtSOG1 expression element is cloned into an editing vector. The AtSOG1 expression element includes OsSOG1, SOG1, SOG1-P, SOG1(DDADD), and SOG1(S372A) expression elements, which are respectively cloned into the Sbf cells of the knock-in vectors pNBGE32-MPK5-HiBiT and pCBGE32-MPK5-HiBiT. At site I, and during the cloning process, the following vector plasmids were used as templates: 35S::OsSOG1-GFP, 35S::SOG1-GFP, 35S::SOG1-P-GFP, 35S::SOG1(DDADD)-GFP, and 35S::SOG1(S372A)-GFP. SOG1-related expression elements were amplified using primers SOG1-MPK5-HiBiT-F2 and SOG1-MPK5-HiBiT-R2. These expression elements were then seamlessly cloned into the SbfI site of the backbone vector, thereby constructing an editing vector containing AtSOG1-acting elements. The resulting editing vector pNBGE32-MPK5-HiBiT-AtSOG1 is shown in the diagram below. Figure 2 As shown, the pCBGE32-MPK5-HiBiT-AtSOG1 spectrum is as follows: Figure 3As shown. The knock-in vectors pNBGE32-MPK5-HiBiT and pCBGE32-MPK5-HiBiT described in this invention have both been disclosed in articles (Chen Yache. Research on labeling rice MPK5 gene using targeted gene insertion technology. [Master's Thesis]. Library of Huazhong Agricultural University, 2021), and the vector p32-N-WDV1-MPK5-HiBiT in the paper is the vector pNBGE32-MPK5-HiBiT of this invention, and the vector p32-C-WDV1-MPK5-HiBiT is the vector pCBGE32-MPK5-HiBiT of this invention.

[0059] This invention also constructs a knockout vector with an AtSOG1 action element, which can target and knock out the target gene, particularly the knockout between two gRNA spacer sequences of the target gene. In constructing the knockout vector, knockout vectors 32-7 (created by this invention, Ding Dan) and 32-CPK4 / CPK18 were used as base vectors, and... Figure 2 The pNBGE32-MPK5-HiBiT-AtSOG1 vector plasmid shown is used as a template. Primers SOG1-MPK5-HiBIT-F2 (SEQ ID No. 8) and SOG1-MPK5-HiBiT-R2 (SEQ ID No. 9) are used to amplify the AtSOG1 expression element and clone it into the SbfI site of the knockout vector. The knockout vector 32-7 described in this invention has been disclosed in an article (Dan Ding, Kaiyuan Chen, Yuedan Chen, Hong Li, Kabin Xie. Engineering Introns to Express RNA Guides for Cas9-andCpf1-Mediated Multiplex Genome Editing. Mol Plant. 2018 Apr 2; 11(4):542-552. doi:10.1016 / j.molp.2018.02.005.), and the vector PTG7 in that article is the knockout vector 32-7 described in this invention. The construction method of the knockout vector 32-CPK4 / CPK18 described in this invention is completely identical to the construction method of the knockout vector 32-7, except for the four gRNAs listed in Table 5.

[0060] The present invention also provides a plant genome gene editing method, including transfecting plants using the above-mentioned gene editing vector.

[0061] The present invention does not specifically limit the transfection method, such as using Agrobacterium-mediated genetic transformation.

[0062] This invention also provides the application of the above-mentioned gene editing vector or the above-mentioned plant genome gene editing method in constructing T-DNA-free gene-edited T0 generation plants.

[0063] The AtSOG1 described in this invention can regulate multiple repair pathways and promote the occurrence of T-DNA nonintegration knock-in events, directly obtaining T-DNA-free knock-in plants in the T0 generation.

[0064] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, of a method for improving plant genome editing efficiency based on transcription factor AtSOG1 and its utilization, should not be construed as limiting the scope of protection of the present invention.

[0065] The names and sequences of the primers used to detect gene editing in this embodiment of the invention are shown in Table 3.

[0066] Table 3. Primer names and sequences used in PCR detection of gene editing.

[0067]

[0068]

[0069] The genetic transformation methods used in the embodiments of this invention include rice protoplast transformation, referring to the isolation and transformation of rice protoplasts (Jiang Yunhe, Cheng Ke, Zhao Xiaobo, Ouyang Yidan. (2018). Isolation and transformation of rice protoplasts. Bio-101e1010125. Doi:10.21769 / BioProtoc.1010125.) and Agrobacterium-mediated genetic transformation of japonica rice, referring to Agrobacterium tumefaciens-mediated genetic transformation of japonica rice (Chen Qiuhong, Chen Taiyu, Lin Yongjun, Chen Hao. (2018). Agrobacterium tumefaciens-mediated genetic transformation of japonica rice. Bio-101e1010174. Doi:10.21769 / BioProtoc.1010174.).

[0070] Example 1

[0071] Construction of AtSOG1 expression vectors containing different phosphorylation sites

[0072] according to Figure 1The AtSOG1 phosphorylation sites shown were specifically constructed to contain AtSOG1 expression vectors with different numbers of phosphorylation sites. Using the 35S::SOG1-GFP vector plasmid as a template, base mutations were introduced through primers S350D, S356D, S372A, S430D, S436D, and S372D as shown in Table 1, so that specific phosphorylation sites were simulated by phosphorylation or dephosphorylation. Finally, the expression vectors 35S::SOG1-P-GFP, 35S::SOG1(DDADD)-GFP, and 35S::SOG1(S372A)-GFP were obtained.

[0073] Example 2

[0074] The impact of AtSOG1 on PE editor-mediated gene editing

[0075] This invention is based on Figure 4 As shown in the diagram, a PE vector PE32-HiBiT was constructed that targets the strong constitutive promoter UBI10 in rice and knocks in the protein tag HiBiT (33 bp). After the HiBiT tag is successfully knocked into the rice genome, it can be translated and expressed under the action of the UBI10 promoter. When the HiBiT tag protein fuses with the large subunit LgBiT, it forms the luciferase NanoLUC. When the substrate furimazine is added, it produces strong bioluminescence. The knock-in status of HiBiT is quantified by detecting the luminescence signal.

[0076] Rice protoplasts were transfected with the knock-in vector PE32-HiBiT and the expression vector 35S::AtSOG1-GFP and cultured in the dark for 18 h. After adding lytic lysate to lyse the cells, exogenous large subunit LgBiT and substrate furimazine were added. The reaction was carried out at 600 r / min for 10 min, and the luminescence signal was detected by microplate reader. PE32-empty vector (without pegRNA) was used as a negative control to determine the luminescence background, and the repair protein RAD54, which is not related to the editing principle of PE editor, was used as another control.

[0077] The results of the luminescence detection are as follows Figure 6 As shown, compared with the control vector PE32-HiBiT, the addition of AtSOG1 transcription factor increased the bioluminescence detected in cells by 1.5 times, and the HDR-related repair protein RAD54 did not affect the PE editor-mediated short sequence knock-in; the results indicate that AtSOG1 transcription factor can improve the efficiency of PE editor-mediated short sequence knock-in to a certain extent.

[0078] Example 3

[0079] 3.1 The impact of AtSOG1 on HDR-based targeted knock-in

[0080] To evaluate the impact of AtSOG1 on HDR-based targeted knock-in, this invention co-transfected rice protoplasts with the expression vector constructed in Example 1 and the editing vector pWDV32-MPK5-HiBiT. This vector targets the rice endogenous gene OsMPK5 and attempts to knock in the tag protein HiBiT at the three amino acids before the stop codon of the sixth exon. After the knock-in is completed, the HiBiT knock-in status can be determined by PCR, qPCR and bioluminescence measurement.

[0081] Eighteen hours after co-transfection, the bioluminescence of rice protoplasts was detected using a microplate reader. The results showed that... Figure 7 As shown, the luminescence signal was increased by 1.2 times in the sample transfected with AtSOG1, indicating that AtSOG1 has a certain promoting effect on HDR-mediated targeted knock-in.

[0082] Consistent with the detection results in the PE editor, fully phosphorylated AtSOG1-P actually reduced the luminescence signal of HiBiT. This may be because the continuous phosphorylation of AtSOG1 has an important impact on other pathways in the cell, thereby reducing DNA damage repair.

[0083] Next, this invention fuses the AtSOG1 transcription factor expression element into the editing vectors pCBGE32-MPK5-HiBiT and pNBGE32-MPK5-HiBiT, targeting the endogenous gene OsMPK5 knock-in tag protein HiBiT, similar to the pWDV32-MPK5-HiBiT vector. After transfecting rice protoplasts with the fusion vectors for 18 hours, HiBiT bioluminescence was measured. The results showed that the AtSOG1 transcription factor increased the luminescence signal by 1.3-fold and 15.7-fold, respectively, in the two samples.

[0084] 3.2 Construction of an editing carrier with AtSOG1 active element

[0085] To better examine the effect of the AtSOG1 transcription factor on gene editing, this invention clones the AtSOG1 expression element into the editing vector, wherein the expression elements OsSOG1, SOG1, SOG1-P, SOG1(DDADD), and SOG1(S372A) are cloned into the SbfI site of the knock-in vectors pNBGE32-MPK5-HiBiT and pCBGE32-MPK5-HiBiT, respectively.

[0086] Using the 35S::OsSOG1-GFP, 35S::SOG1-GFP, 35S::SOG1-P-GFP, 35S::SOG1(DDADD)-GFP, and 35S::SOG1(S372A)-GFP vector plasmids as templates, SOG1-related expression elements were amplified using primers SOG1-MPK5-HiBiT-F2 and SOG1-MPK5-HiBiT-R2. These expression elements were then seamlessly cloned into the SbfI site of the backbone vector, thus constructing the vectors as shown below. Figure 2 The editing vector pNBGE32-MPK5-HiBiT-AtSOG1 shown, and as... Figure 3 The pCBGE32-MPK5-HiBiT-AtSOG1 shown is an example.

[0087] The modified AtSOG1 expression element was cloned into the editing vector pCBGE32-MPK5-HiBiT. Genomic DNA was extracted 18 hours after transfection of rice protoplasts. The copy number of HiBiT knock-in in the target site genome was detected using 3' primers HiBiT-qPCR-F and MPK5-qPCR-R. The qPCR results are shown below. Figure 8 As shown, under the action of the AtSOG1 transcription factor, the relative copy number of HiBiT was 3.52 times that of the control; however, the re-engineered AtSOG1 constitutive phosphorylated protein did not further promote the knock-in of HiBiT as expected.

[0088] This invention also produced transgenic materials. Various editing vectors were transferred into rice callus tissue via Agrobacterium-mediated genetic transformation. The callus tissue was then ground and mixed with an appropriate amount of lysis buffer to measure bioluminescence. The results are as follows: Figure 9 As shown, the HiBiT emission signal was increased by 7.7 times in the sample containing AtSOG1.

[0089] 3.3 Expression detection of genes related to downstream homologous recombination of AtSOG1

[0090] This invention is based on transfection Figure 2 The expression of downstream homologous recombination genes of AtSOG1 was detected in protoplasts and stably transformed plants of the plasmid pNBGE32-MPK5-HiBiT-AtSOG1, including the expression of exonucleases FEN1A, RAD51A, RAD51B and RAD54 related to terminal digestion, and poly-ADP ribose polymerase PARP2A.

[0091] Table 4. Primer information used in the detection

[0092]

[0093] Leaf RNA was collected from pNBGE32-MPK5-HiBiT-AtSOG1-PT1 generation plants. RT-qPCR was used to identify the expression of exonuclease FEN1A (related to terminal digestion), DNA recombinases RAD51A, RAD51B, and RAD54, and poly-ADP-ribose polymerase PARP2A. RT-qPCR results are shown below. Figure 5 As shown, the transcriptional level of homologous recombination-related proteins was indeed increased in samples expressing phosphorylated SOG1.

[0094] Example 4

[0095] The effect of AtSOG1 on gene knockout

[0096] To investigate whether the AtSOG1 transcription factor can promote gene knockout, especially the knockout between two gRNA spacer sequences targeting a gene, this invention selected knockout vectors 32-7 and 32-CPK4 / CPK18 for testing.

[0097] 4.1 Construction of a knockout vector with an AtSOG1 active element

[0098] Knockout vectors 32-7 and 32-CPK4 / CPK18 both use pRGEB32 as their vector backbone and contain two gRNA spacers targeting the target genes. Using the pNBGE32-MPK5-HiBiT-SOG1 vector plasmid as a template, primers SOG1-MPK5-HiBIT-F2 and SOG1-MPK5-HiBiT-R2 were used to amplify the AtSOG1 expression element, which was then cloned into the SbfI site of the knockout vector.

[0099] Table 5 Target site sequences for CRISPR knockout

[0100]

[0101] To investigate whether the AtSOG1 transcription factor can promote gene knockout, this invention generated transgenic materials. Various knockout vectors were transferred into rice callus tissue through Agrobacterium-mediated genetic transformation. DNA was extracted from the callus tissue and the results showed that long-fragment knockout could be achieved through AtSOG1.

[0102] Two gRNAs were designed targeting exons 1 and 3 of OsMPK5. Without AtSOG1, a large knockout was detected in one of 17 T0 generation strains, with a knockout efficiency of approximately 6%. Figure 14 Sequencing results showed that the large fragment knockout event was completed through the NHEJ repair pathway; with the addition of AtSOG1, two large fragment knockouts were detected in 12 callus samples. Figure 15The knockout efficiency of large fragments was about 16.7%. Sequencing results showed that the 5' ends of the two cleavage sites were clearly removed, and five bases in the knockout sequence could correspond to two genome segments at the same time. It is speculated that these five bases are the micro-homologous annealing sequences in the MMEJ (micro-homologous end joining) pathway.

[0103] Two gRNAs were designed targeting exons 1 and 4 of OsCPK18. Without AtSOG1, no large fragment knockout was detected in the T0 generation, indicating a large fragment knockout efficiency of 0%. Figure 16 ); with the addition of AtSOG1, knockout of large fragments was detected in 3 out of 12 callus samples, with a knockout efficiency of approximately 25%. Figure 17 The knockout sequence was detected by Sanger sequencing. No sequence change occurred at gRNA2, resulting in a knockout of a large fragment of nearly 2000 bp. However, 16 bp was knocked in at the same time (with the same sequence 144 bp upstream of gRNA1). It is speculated that this may be due to bridging repair. During DNA repair, one strand of the genome is cut into a short single-stranded fragment, and this fragment happens to be homologous to the genome at both ends of the cleavage site.

[0104] Example 5

[0105] 5.1 The coexistence of AtSOG1 and Rep protein may affect T-DNA integration.

[0106] Using pNBGE32-MPK5-HiBiT as a control, expression elements SOG1, SOG1-P, SOG1(DDADD), and SOG1(S372A) were incorporated into the genetic transformation mediated by Agrobacterium. Leaf genomes were extracted from the obtained T0 generation transgenic plants, and genotypes were identified. The quality of genome extraction was first assessed using Actin primers, and T-DNA integration was detected using Cas9 primers. PCR results are shown below. Figure 10 As shown, 11 out of 16 T0 generation plants of pNBGE32-MPK5-HiBiT were Cas9 positive (11 / 16, 68.75%), 5 out of 21 T0 generation plants of SOG1 were Cas9 positive (5 / 21, 23.81%), 11 out of 29 T0 generation plants of SOG1-P were Cas9 positive (11 / 29, 37.93%), 9 out of 26 T0 generation plants of SOG1(S372A) were Cas9 positive (9 / 26, 34.62%), and 3 out of 20 T0 generation plants of SOG1(DDADD) were Cas9 positive (3 / 20, 15%). Statistical data indicate that the Cas9 positivity rate in the T0 generation plants of AtSOG1 was significantly lower, which may suggest that AtSOG1 affects the integration of T-DNA in rice cells to some extent.

[0107] HiBiT knock-in was identified by detecting 5' and 3' knock-in in all Cas9-positive plants and by nested PCR. The results are as follows: Figure 11 As shown, in pNBGE32-MPK5-HiBiT, 9 / 11 (81.82%) of the 5' end was knocked in, and 8 / 11 (72.73%) of the 3' end was knocked in; in SOG1, 2 / 5 (40%) of the 5' end was knocked in, and 1 / 5 (20%) of the 3' end was knocked in; in SOG1-P, 10 / 11 (90.91%) of the 5' end was knocked in, and 9 / 11 (81.82%) of the 3' end was knocked in; in SOG1(S372A), 4 / 9 (44.44%) of the 5' end was knocked in, and 4 / 9 (44.44%) of the 3' end was knocked in; in SOG1(DDADD), 2 / 3 (66.67%) of the 5' end was knocked in, and 2 / 3 (66.67%) of the 3' end was knocked in. DNAPAGE results showed that no HiBiT knock-in (325 bp) was detected in any of the AtSOG1 samples.

[0108] Table 6 Summary of Genotyping Results for Generation AtSOG1T0

[0109]

[0110] 5.2 AtSOG1 can promote T-DNA non-integration knock-in events.

[0111] During the detection of AtSOG1T0 generation plants, a clear HiBiT knock-in band was detected in Cas9-negative plants, and the HiBiT sequence knock-in in the OsMPK5 genome was confirmed by Sanger sequencing. However, this phenomenon was not found in the control ID-1T0 generation Cas9-positive plants. Figure 12 (A) It is speculated that although T-DNA enters rice cells and does not integrate into the genome, it successfully expresses Cas9 protein, gRNA, and WDV replicons and completes knock-in editing before being recognized and digested by rice cells. Therefore, the editing event can be detected in Cas9-negative T0 generation plants. Inspired by the idea that T-DNA-free T0 generation edited plants can be directly obtained using AtSOG1, this invention re-transforms pNBGE32-MPK5-HiBiT and pNBGE32-MPK5-HiBiT-AtSOG1 into Agrobacterium without using hygromycin selection to obtain all infected T0 generations. T-DNA-free HiBiT knock-in was successfully detected in the callus obtained without hygromycin selection. The brightness comparison between the knock-in band (325bp) and the wild-type band (297bp) strongly suggests a heterozygous knock-in event. Figure 12(Middle B). Therefore, the sample size was expanded to attempt to obtain T-DNA-free HiBiT knock-in plants in the T0 generation. DNAPAGE gel analysis and Sanger sequencing confirmed the existence of T-DNA-free knock-in chimeras in the T0 generation plants obtained without hygromycin selection. Figure 12 (C, D). Therefore, AtSOG can promote T-DNA non-integration knock-in events.

[0112] 5.3 AtSOG1 may regulate multiple repair pathways

[0113] In all Cas9-positive plants, the DNAPAGE gel results were as follows: Figure 13 As shown, no HiBiT knock-in (325 bp) was detected in any of the AtSOG1 samples, but many short sequence deletions that were not indels were observed. This indicates that AtSOG1 also regulates other DSB-related repair pathways, which may lead to small fragment deletions in the genome during the repair process. Ligating the PCR products into the pEASY-T1 vector and performing Sanger sequencing attempted to identify the pattern of fragment deletions. The sequencing results strongly suggest that MMEJ (microhomological end joining) repair is the most likely cause.

[0114] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of Arabidopsis transcription factor AtSOG1 in improving the efficiency of plant genome editing, characterized by, The nucleotide sequence of the Arabidopsis transcription factor AtSOG1 is shown in SEQ ID No.

1.

2. The application according to claim 1, characterized in that, The plant genome editing includes PE editor-based gene editing and / or HDR-mediated gene editing.

3. The application according to claim 1 or 2, characterized in that, The genome editing includes gene knock-in and / or knockout.

4. A method for constructing an expression vector for the Arabidopsis thaliana transcription factor AtSOG1, characterized in that, Includes the following steps: Using the 35S::SOG1-GFP vector plasmid as a template, the expression vector was obtained by introducing mutations into one or more coding amino acid sites of the Arabidopsis transcription factor AtSOG1. The mutations include at least one of the following: S350D, S356D, S372A, S430D, S436D, and S372D.

5. The expression vector constructed using the construction method of claim 4.

6. A method for improving the efficiency of plant genome gene editing, characterized in that, The process includes the following steps: mixing the expression vector and the genome gene editing vector as described in claim 5, and then co-transfecting the gene editing material to obtain a gene-edited plant.

7. A gene editing vector containing the expression element of the Arabidopsis transcription factor AtSOG1, characterized in that, The nucleotide sequence of the Arabidopsis transcription factor AtSOG1 is shown in SEQ ID No.

1.

8. The gene editing vector according to claim 7, characterized in that, The expression element of the Arabidopsis transcription factor AtSOG1 is obtained by amplification, and the expression vector of claim 5 is used as a template during the amplification.

9. A method for editing plant genome genes, characterized in that, The procedure includes the following steps: transfecting plants using the gene editing vector described in claim 7 or 8.

10. The application of the gene editing vector of claim 7 or 8 or the plant genome gene editing method of claim 9 in constructing T-DNA-free gene-edited T0 generation plants.

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