Controllable vector elimination method as well as easy-to-use CRISPR-Cas9 tool

A carrier and tool technology, applied in the field of genetic engineering, can solve problems such as large workload and time, and achieve the effect of easy elimination and rapid genetic modification

Active Publication Date: 2017-12-22
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this strategy is step-by-step, and the work

Method used

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  • Controllable vector elimination method as well as easy-to-use CRISPR-Cas9 tool
  • Controllable vector elimination method as well as easy-to-use CRISPR-Cas9 tool
  • Controllable vector elimination method as well as easy-to-use CRISPR-Cas9 tool

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1. Design, construction and application of controllable carrier elimination gene circuit

[0058] 1. Materials and methods

[0059] 1. Strains, plasmids and culture methods

[0060] All strains and plasmids used in this example are listed in Table 1. Among them, Escherichia coli NEB10β was used as a cloning strain and the phenotypic verification of the gene circuit. All E. coli were cultured in LB medium. Except for the strain containing plasmid pKD46 which was grown at 30°C, the other strains were grown at 37°C. The composition of LB medium is: peptone 10g / L, yeast powder 5g / L, NaCl 10g / L. Add 15g / L agar to the solid medium.

[0061] Antibiotics were added to the above strains as needed: 100 μg / mL ampicillin, 50 μg / mL kanamycin, 20 μg / mL chloramphenicol, and 200 μg / mL erythromycin. L-arabinose, as an inducer, was added to the medium according to the concentration indicated in the text.

[0062] The strains were cultured statically in an incubator, or vib...

Embodiment 2

[0163] Example 2, the establishment and application of the easy-to-use CRISPR-Cas9 system in Escherichia coli

[0164] 1. Materials and methods

[0165] 1. Strains, plasmids and culture conditions

[0166] The strains and plasmids used in this example are listed in Table 5, and the culture conditions are the same as those described in Step 1 of Example 1. Wild-type E.coli MG1655 was used to construct atrazine-degrading strains. L-arabinose and IPTG were used as inducers, and were added to the medium according to the concentrations indicated in the text. 5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) was coated on the surface of LB solid plate for blue and white spot screening.

[0167] Table 5 The strains and plasmids used in this study

[0168]

[0169] 2. Sequence Analysis

[0170] Sequence analysis is the same as Step 1-2 of Example 1. Determine the N20 sequence of the sgRNA by searching the genome.

[0171] 3. DNA manipulation

[0172] For the DNA oper...

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Abstract

The invention discloses controllable vector elimination method as well as an easy-to-use CRISPR-Cas9 tool. The invention provides a controllable vector elimination tool, which is composed of a control module and an elimination module, the control module including: a core zone which contains an elaborately-controlling promoter and a homing endonuclease encoding gene, two terminators respectively at two ends of the core zone, and homing endonuclease recognition sequences on the outer ends of the two terminators; and the elimination module including: an antibiotic resistance gene expression cassette, and homing endonuclease recognition sequences on the two ends; the tool can easily, quickly and high-effectively remove a plasmid vector in one step. A further developed easy-to-use CRISPR-Cas9 system can achieve one-step quick elimination to Cas9 and sgRNA encoding plasmids from a host cell. The easy-to-use CRISPR-Cas9 system is very convenient and practical, greatly saves experiment time, reduces experiment intensity, and is a very practical gene editing tool.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and relates to a controllable carrier elimination method and an easy-to-use CRISPR-Cas9 gene editing tool. Background technique [0002] Vector is an important molecular biology operation platform, and most gene manipulation tools are developed based on the carrier platform. These genetic manipulation tools, such as gene editing, large fragment chromosome integration, and experimental evolution, have greatly facilitated genetic manipulation. However, how to quickly eliminate the vector in which it is located from the host cell after the genetic modification is completed is a difficult problem that restricts subsequent genetic modification or application. [0003] The tool carrier remains in the host cell, which will cause unnecessary metabolic burden to the cell and hijack cell resources. And some genetic manipulation tools, whose functional elements are toxic to host cells, need to be elimin...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/55
CPCC12N9/16C12N15/70C12N2800/80C12N2810/10
Inventor 刘双江汤强姜成英
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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