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Construction method and application of CRISPR/Cas9 mediated plant polygene editing vector

A construction method and gene editing technology, applied in the field of genetic engineering, can solve problems such as difficulty in construction, decreased transcription level of sgRNAs, and impact on editing efficiency, and achieve broad application prospects, efficient and specific multi-gene editing, and fast construction methods

Pending Publication Date: 2021-10-29
SHANGHAI NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the limitations of the "Golden Gate" cloning method, it becomes more and more difficult to construct as the number of DNA fragments increases. Generally, no more than 8 sgRNAs are connected to a transcription unit, and due to the influence of promoter activity, as the length of the transcription unit increases, It will lead to a decrease in the transcription level of sgRNAs far from the promoter, thereby affecting the editing efficiency

Method used

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  • Construction method and application of CRISPR/Cas9 mediated plant polygene editing vector
  • Construction method and application of CRISPR/Cas9 mediated plant polygene editing vector
  • Construction method and application of CRISPR/Cas9 mediated plant polygene editing vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] This embodiment provides a method for quickly constructing multiple sgRNAs into pMK(1-12) vectors, including the following steps:

[0063] (1)获取OsLEA1、OsLEA2、OsLEA4、OsLEA5、OsLEA6、OsLEA7、OsLEA9、OsLEA10、OsLEA11、OsLEA13、OsLEA15、OsLEA17、OsLEA18、OsLEA19、OsLEA20、OsLEA21、OsLEA22、OsLEA23、OsLEA24、OsLEA27 / 28、OsLEA30、OsLEA31、 Sequence information of OsLEA33 and OsLEA34 genes. The target site of the target gene was designed according to the database CRISPR-GE (http: / / skl.scau.edu.cn / ).

[0064] The design of the target site should avoid the off-target of the core region (9-20) (2) the GC content is 40-70% (3) it should not contain the GGTCTC sequence (4) avoid the duplication of the designed linker. The designed target sites, primer names and sequences are shown in Table 1 below.

[0065] (2) Using the pGTR (Xie et al. ProcNatlAcadSciUSA, 2015, 112:3570-3575) plasmid as a template, use the designed primers to amplify the clone containing the target site by gold mix DNA polymerase...

Embodiment 2

[0102] This embodiment will provide a method for constructing a multi-gene editing vector that connects the OsU6 / 3 expression cassette on multiple pMK-PTG intermediate vectors to pMMK-Cas9, including the following steps:

[0103]The pMK1-PTG, pMK3-PTG, pMK5-PTG, pMK7-PTG, pMK9-PTG, pMK10-PTG intermediate vectors obtained in Example 1 were connected in a certain order by the Golden Gate assembly method, and the OsU6 / 3 expression cassette on the intermediate vector was used with II Type restriction endonuclease AarI (Thermofisher company) and T4 DNA ligase (NEB company) carry out enzyme cutting-ligation cycle reaction and connect to the pMMK-Cas9 gene editing vector, and the connection reaction system is shown in Table 7.

[0104] Take 10 uL of the reaction solution to transform Escherichia coli competent Dh5a (Shanghai Weidi Biotechnology Co., Ltd.), and use a concentration of 100 ng / mL kanamycin resistance plate to screen. After the resistant colonies were picked, PCR was used...

Embodiment 3

[0117] Example 3. Transformation of rice callus with pMMK-PTG multigene editing vector

[0118] Through the Agrobacterium-mediated transformation method, the multi-gene editing vector pMMK-PTG obtained in Example 2 was transformed into Agrobacterium (EHA105), and then the rice callus was infected. The transformed variety is "Xiushui 134". Detailed transformation steps and references for various medium formulations used: Komari T. Efficient transformation of rice (Oryzasativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. [J]. Plant Journal for Cell&Molecular Biology , 2010, 6(2):271-282.

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Abstract

The invention discloses a method for constructing a plant polygene editing vector which is simple, convenient and efficient. The plant polygene editing vector comprises a pMK-(1-12) intermediate vector and a final vector pMMK-Cas9. The vector construction method comprises the following steps: a plurality of sgRNAs are connected onto a pMK (1-12) intermediate vector in a polycistron tRNA-gRNA manner, 1-8 sgRNAs can be connected to each intermediate vector to obtain a pMK (1-12)-PTG vector; U6 / U3 expression cassettes on the plurality of pMK (1-12)-PTG vectors are connected to a pMMK-Cas9 vector, the pMMK-Cas9 can be connected with the U6 / U3 expression cassettes on 2 to 7 pMK (1-12)-PTG vectors to obtain a pMMK-PTG vector, and multi-gene editing of 2-56 target sites can be obtained according to different target site and small vector combinations. The whole process can be completed only through one-time PCR reaction and two-time "golden-gate" linked system, and the process is simple and rapid. The invention also takes the vector knocking out 25 members of the rice LEA gene family as an example to verify the high efficiency of the vector multi-gene editing.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a highly efficient and rapid multi-gene editing vector for plants and its construction method and application. Background technique [0002] As the third-generation gene editing technology, CRISPR / Cas9 system has the characteristics of simple construction and high labeling efficiency, so it is widely used in gene editing research. CRISP / Cas9 mainly includes two core components: Cas expression cassette and sgRNA expression cassette. Cas expression cassette is composed of strong promoters such as RNA polymerase II (Ubi, 35S, etc.) and terminators such as NOS; Driven by enzyme III promoter (U6 or U3, etc.), more than 6 consecutive polybase T terminations. In research, it is often necessary to edit multiple genes at the same time. For multi-gene editing in animals, it is only necessary to co-transfect the plasmids of multiple sgRNA expression cassettes to obt...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/29C12N15/66A01H5/00A01H6/46
CPCC07K14/415C12N15/8216
Inventor 张辉汪冲罗鹏宇洪政张亚军
Owner SHANGHAI NORMAL UNIVERSITY