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CRISPR/Cas9 system mediated wheat polygene knockout editing system

A multi-gene, gene technology, applied in genetic engineering, plant genetic improvement, angiosperm/flowering plants, etc., to achieve the effect of promoting research and speeding up complex traits

Active Publication Date: 2021-07-23
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For allohexaploid common wheat, the system and method of using CRISPR / Cas9 system to simultaneously target multiple target genes and improve multiple agronomic traits remains to be studied, expanded and applied

Method used

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  • CRISPR/Cas9 system mediated wheat polygene knockout editing system
  • CRISPR/Cas9 system mediated wheat polygene knockout editing system
  • CRISPR/Cas9 system mediated wheat polygene knockout editing system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Double-gene combination vector construction

[0053] 1.1 Construction of TaQsd1+TaNPT1 double gene combination knockout vector

[0054] First, the pBUE411-Ubi-NLS-Cas9-NLS-E9-Actin-PolyA-Nos vector was constructed on the pBUE411-Ubi-NLS-Cas9-NLS-E9 base vector. In the first round of PCR, two PCR systems were simultaneously amplified using the primer pair 414-M13-Actin-F / Nos-Hind-Act-R (Table 2) and Act-Hind-Nos-F / 414 -Ubi-Nos-R (Table 2), amplified with Actin sequence and Nos sequence as template, the second round of PCR using primer pair 414-M13-F / 414-Ubi-R (Table 2), with the first round of PCR The two PCR products obtained by PCR were used as templates to obtain the Actin-Nos expression cassette, and the expression cassette was digested with HindⅢ (NEB, Beijing, China) pBUE411-Ubi-NLS-Cas9-NLS-E9 basic vector, using the same Source recombinase (Quanshijin, Beijing, China) was connected to obtain the recombinant vector pBUE411-Ubi-NLS-Cas9-NLS-E9-Actin-Nos...

Embodiment 2 3

[0060] Example 2 Three-gene combination carrier construction

[0061] 2.1 Construction of TaQsd1+TaARE1+TaNPT1 triple gene combination knockout vector

[0062] Construction of pBUE411-Ubi-NLS-Cas9-NLS-E9-Actin-tRNA-gRNA1(TaQsd1)-tRNA-gRNA2(TaARE1)-tRNA-gRNA3(TaNPT1)-tRNA-PolyA-Nos three gene combination knockout vector. The first round of PCR uses tRNA as a template, and uses the primer pair 9-Actin-tRNA-F / 9-TaQsd1-tRNA-R to amplify, and the second round of PCR uses the tracrRNA-tRNA fragment as a template, respectively, using the primer pair 9-TaQsd1 -TrRNA-F / 9-TaAER1-tRNA-R and 9-TaARE1-TrRNA-F / PolyA-Hind-tRNA-R were amplified, and in the third round of PCR, the primer pair Actin-w-F0 / Poly- t-R0, using the three PCR products of the first round and the second round as templates to amplify to obtain the tRNA-gRNA1(TaQsd1)-tRNA-gRNA2(TaARE1)-tRNA fragment, which was then utilized by homologous recombinase ( Whole gold, Beijing, China) was cloned into the pBUE411-Ubi-Cas9-E9-A...

Embodiment 3 4

[0065] Example 3 Four-gene combination vector construction

[0066] 3.1 Construction of TaQsd1+TaARE1+TaNPT1+TaIPA1 Four-gene Combination Knockout Vector

[0067] The first round of PCR uses the tracrRNA-tRNA fragment as a template, and uses primers to amplify tRNA-TaIPA-F0 / PolyA-Hind-tRNA-R, and the second round of PCR uses the first round of PCR product as a template, using primers tRNA-TaIPA -F / PolyA-Hind-tRNA-R was amplified to obtain the gRNA4(TaIPA1)-tRNA fragment, which was digested with pBUE411-Ubi-NLS-Cas9-NLS-E9 with HindⅢ (NEB, Beijing, China) -Actin-tRNA-gRNA1(TaQsd1)-tRNA-gRNA2(TaARE1)-tRNA-gRNA3(TaNPT1)-tRNA-PolyA-Nos vector, using homologous recombinase (Quanshijin, Beijing, China) to obtain TaQsd1+ TaARE1+TaNPT1+TaIPA1 four-gene combination knockout vector pBUE411-Ubi-NLS-Cas9-NLS-E9-Actin-tRNA-gRNA1(TaQsd1)-tRNA-gRNA2(TaARE1)-tRNA-gRNA3(TaNPT1)-tRNA-gRNA4( TaIPA1)-tRNA-PolyA-Nos. The sequence of the TaQsd1+TaARE1+TaNPT1+TaIPA1 four-gene combination knockout...

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Abstract

The invention discloses a CRISPR / Cas9 system mediated wheat polygene knockout editing system. The system comprises a recombinant vector, wherein the recombinant vector contains a skeleton structure, the skeleton structure is obtained by connecting a Ubi promoter, a nuclear localization sequence, a Cas9 gene, a nuclear localization sequence, an E9 terminator, an Actin promoter, a tRNA coding gene, n DNA fragments 1, a PolyA sequence and a Nos terminator, N is a natural number between 2 and 5, and the DNA fragment 1 is obtained by connecting a gRNA coding gene and a tRNA coding gene. According to the method, a CRISPR / Cas9 gene editing technology and a strategy of simultaneously releasing a plurality of sgRNAs through tRNA self-cleavage are utilized, knockout editing of 5 genes simultaneously targeting 2, 3, 4 and up to 15 genomes is realized in wheat, and polymerization of a plurality of excellent alleles is realized only in one generation.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a wheat multi-gene knockout editing system mediated by a CRISPR / Cas9 system. Background technique [0002] The third-generation gene editor CRISPR / Cas9 system has become an effective molecular tool for crop genetic improvement and breeding research due to its advantages of simple operation and high efficiency. The system uses Cas9 protein and guide RNA to locate the cleavage target site within the genome of the target organism, and then Cas9 protein cleavage causes genomic DNA double-strand breaks, which are then repaired by mainly non-homologous end joining or low-efficiency homologous recombination Introduce mutations. When performing non-homologous end joining repair, it usually causes the deletion or insertion of small fragments of nucleotides, which changes the expression product of the target gene, loses its function, and finally causes the change of the corresponding traits o...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/29A01H5/00A01H6/46
CPCC07K14/415C12N15/8261C12N15/8216
Inventor 夏兰琴罗金满马有志李少雅徐佳竞闫磊
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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