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A dna fragment with promoter function and its application

A promoter and fragment technology, applied in the field of DNA fragments, can solve the problems of high construction cost and technical difficulty, and achieve the effect of simple structure

Active Publication Date: 2019-04-09
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The CRISPR-Cas9 system of Aspergillus niger uses a "hammerhead" technology to initiate the correct transcription of gRNA, but the "hammerhead" technology is expensive to construct and technically difficult
So far, no gRNA promoter derived from Aspergillus niger has been found to correctly guide the synthesis of gRNA in vivo

Method used

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  • A dna fragment with promoter function and its application
  • A dna fragment with promoter function and its application
  • A dna fragment with promoter function and its application

Examples

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Embodiment 1

[0035] 1. Acquisition of Cas9 expression plasmid

[0036] will contain the PFC330 plasmid (already in the literature " C S, Nielsen J B, Kogle M E, et al.ACRISPR-Cas9system for genetic engineering of filamentous fungi[J].PloS one,2015,10(7):e0133085."public) Escherichia coli strains (Escherichia coli mech1T1, purchased (from takara company) was activated, inoculated in liquid LB+Amp (final concentration of ampicillin 100ug / ml) at 37°C, 200rpm and cultured for 10h, and then the plasmid was extracted to obtain the Cas9 expression plasmid.

[0037] 2. Expression of Cas9 protein in Aspergillus niger CBS513.88

[0038] (1) Obtaining of CBS513.88ΔpyrG strain:

[0039]The Aspergillus niger CBS513.88 wild strain was purchased from the CBS Culture Collection Center of the Netherlands. Using the Aspergillus niger CBS513.88 genome as a template, the 1500 bp fragment upstream of the pyrG coding region was amplified using F-pyrG-up and R-pyrG-up primers, and named It is pyrGup1500bp; u...

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Abstract

The invention discloses a DNA fragment with functions of a promoter and application of the DNA fragment. The DNA fragment is any one of the following sequences of (a) a nucleotide sequence shown as SEQ ID NO.1 or SEQ ID NO.2 or a complementary sequence of the nucleotide sequence; (b) a nucleotide sequence which is obtained by conducting substitution or deletion or addition of one or more nucleotides on the nucleotide sequence shown as SEQ ID NO.1 or SEQ ID NO.2 and has the functions same as those of the nucleotide sequence shown as SEQ ID NO.1 or SEQ ID NO.2 as the promoter or a complementary sequences of the nucleotide sequence. The DNA fragment has the functions of the promoter and has the very high specific expression activity, expression of gRNA in a CRISPR-Cas9 system can be achieved on the condition that an inducer does not need to be added, and therefore a U6 promoter from aspergillus niger can be applied in a CRISPR-Cas9 system of the aspergillus niger.

Description

technical field [0001] The invention relates to a DNA segment, in particular to a DNA segment with promoter function and its application. Background technique [0002] Gene editing technology is an important tool for functional genome research. It can be used to achieve precise modification in a variety of species, and has the advantages of precision and efficiency. Zinc finger nuclease technology (ZFNS), transcription activator-like nuclease technology (TALENS) and CRISPR-Cas9 system are three mainstream genome editing technologies developed recently. [0003] The principles of the above three genome editing technologies are to create DNA breakage damage at specific sites in the biological genome, thereby activating the body's own DNA damage repair mechanism, and causing various mutations in the process. ZFNS is the earliest developed general genome editing technology, which can be used to implement site-specific knockout and site-specific knock-in mutations, but the devel...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/63C12N1/15C12R1/685
CPCC07K14/38C12N15/63C12N2800/80
Inventor 潘力董宏智
Owner SOUTH CHINA UNIV OF TECH
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