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Method of achieving high-accuracy site-directed gene knock-out in coprinus cinereus

A high-accuracy, gene-knock-out technology, applied in the field of genetic engineering, achieves the effects of simple technical methods, reduced labor, and reduced material expenditures

Active Publication Date: 2017-07-14
ZHEJIANG UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The key innovation in the design of the present invention - the structure of "linear 3' protruding long sticky end double-stranded DNA" and its application have not been reported yet

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  • Method of achieving high-accuracy site-directed gene knock-out in coprinus cinereus

Examples

Experimental program
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Embodiment 1

[0025] Step 1: 1000 bp homologous sequence cloning of the upstream (sequence A1) and downstream (sequence B1) of the endocellulase gene.

[0026]The polymerase chain reaction (PCR) method was used for cloning, the DNA polymerase used was high-fidelity DNA polymerase (Phusion DNA Polymerase) produced by NEB, the amplification template was Coprinus cinerea LT2 genomic DNA, and the primers were based on Coprinus cinerea. Oligonucleotides designed and synthesized from the genome sequence of okayama7#130, PPF1, PPR1, PTF1 and PTR1, the sequence information is as follows:

[0027] PPF1:5'-CTCGGTCTATTCTTTTGATTTAGATGGCACCGTTCTTGTCGGCTG-3'

[0028] PPR1:5'-GAAGATGCGGCCGCTGTTAC AGCTGGTCTACAATGTTCGAT-3'

[0029] PTF1: 5'-GTAACAGCGGCCGCATCTTC AAGGGTGAGTTCGGTTCTAT-3'

[0030] PTR1:5'-GCCGAAATCGGCAAAATCCCTTAGCTGTGGTAGGCCCCTAATCAGG-3'

[0031] Primers PPF1 and PPR1 are used to clone the 1kb region upstream of the endocellulase, and PTF1 and PTR1 are used to clone the 1kb region downstream...

Embodiment 2

[0055] Step 1: Cloning of 100 bp homologous sequences upstream (sequence A2) and downstream (sequence B2) of the endocellulase gene.

[0056] The cloning method is the same as in Example 1, the primers are PPF2, PPR1, PTF1 and PTR2, and the sequence information is as follows:

[0057] PPF1:5'-CTCGGTCTATTCTTTTGATTTAGATGGCACCGTTCTTGTCGGCTG-3'

[0058] PPR2:5'-GAAGATGCGGCCGCTGTTAC TGGGCCTGGTCATTGGGCA-3'

[0059] PTF2: 5'-GTAACAGCGGCCGCATCTTC GTTATCTTCAAGCGCCTA-3'

[0060] PTR1:5'-GCCGAAATCGGCAAAATCCCTTAGCTGTGGTAGGCCCCTAATCAGG-3'

[0061] Primers PPF1 and PPR2 are used to clone the 100bp region upstream of the endocellulase, PTF2 and PTR1 are used to clone the 100bp region downstream of the endocellulase, the PCR reaction system is except that the primer PPR2 replaces PPR1, PPF2 replaces PPF1, the amount of each corresponding component With embodiment 1. The PCR amplification conditions were the same as in Example 1 except that the extension time was changed to 20 sec, and the...

Embodiment 3

[0075] Step 1: Cloning of 500 bp homologous sequences upstream (sequence A3) and downstream (sequence B3) of the endocellulase gene.

[0076] The cloning method is the same as in Example 1, the primers are PPF3, PPR1, PTF1 and PTR3, and the sequence information is as follows:

[0077] PPF1:5'-CTCGGTCTATTCTTTTGATTTAGATGGCACCGTTCTTGTCGGCTG-3'

[0078] PPR3: 5'-GAAGATGCGGCCGCTGTTAC GTAAAGACGCCTGGTGGAAGTGTCTG-3'

[0079] PTF3: 5'-GTAACAGCGGCCGCATCTTC TGCACTTATGCGCGAGCCTGGGTC-3'

[0080] PTR1:5'-GCCGAAATCGGCAAAATCCCTTAGCTGTGGTAGGCCCCTAATCAGG-3'

[0081] Primers PPF1 and PPR3 are used to clone the 500bp region upstream of the endocellulase, and PTF3 and PTR1 are used to clone the 500bp region downstream of the endocellulase. The PCR reaction system replaces PPR1 with primer PPR3 and replaces PPF1 with PPF3. With embodiment 1. The PCR amplification conditions were the same as in Example 1 except that the extension time was changed to 30 sec, and the PCR product purification metho...

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Abstract

The invention provides a method of achieving high-accuracy site-directed gene knock-out in coprinus cinereus. The method includes the steps of: 1) acquiring upstream and downstream sequences of a target knock-out gene through methods of polymerase chain reaction or total gene synthesis, and connecting the upstream and downstream sequences in the opposite direction of the original gene, and performing insertion onto a vector plasmid to construct an annular clone vector; 2) performing linear treatment to the annular clone vector to obtain linear double-chain DNA; and 3) processing the linear double-chain DNA through a 5'-to-3' exonuclease to form a linear 3'-terminal protruded long cohesive end double-chain DNA, thereby converting cells of coprinus cinereus. In the method, through structure and type of a special exogenous DNA, high-accuracy site-directed gene knock-out is achieved in the coprinus cinereus with site-directed gene knock-out accuracy rate reaching 25-70%. The method greatly reduces work load of the site-directed gene knock-out of the coprinus cinereus and has huge application potential in the fields of functional gene researching of coprinus cinereus and construction of a gene engineering bacterial strain.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and mainly relates to a high-precision fixed-point gene knockout method of Coprinus cinerea. Background technique [0002] Coprinus cinereus belongs to the Basidiomycetes subphylum Coprinus family Coprinus fungus, the whole genome sequence is known, and it is a large filamentous fungal model organism. The research group, represented by Professor Lorna, an academician of the Royal Academy of Sciences, has carried out a lot of research work on sex recognition, developmental mechanism and exogenous gene expression of filamentous fungi based on Coprinus cinereus. Coprinus cinereus targeted gene knockout refers to the study of introducing exogenous DNA into Coprinus cinereus to knock out or inactivate certain functional genes. At present, the targeted gene knockout efficiency of Coprinus cinereus is very low, generally around 5%. Over the years, the low efficiency of site-specific gene knockout ha...

Claims

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Application Information

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IPC IPC(8): C12N1/15C12R1/645
CPCC12N15/80C12N1/145C12R2001/645
Inventor 柳永刘士旺鲍文娜
Owner ZHEJIANG UNIVERSITY OF SCIENCE AND TECHNOLOGY
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