Molecular cloning method based on CRISPR/Cas9 and homologous recombination of saccharomyces cerevisiae cell endogenous genes

A technology of Saccharomyces cerevisiae cells and molecular cloning, applied in the field of molecular cloning based on CRISPR/Cas9 and endogenous homologous recombination of Saccharomyces cerevisiae cells, which can solve problems that have not been proposed, that have not considered the efficiency of multi-fragment splicing, and have not been mentioned.

Active Publication Date: 2016-06-01
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods do not mention whether multiple target fragments can be operated on at the same time, nor do they consider the efficiency of multi-fragment splicing
[0006] At present, there is no in vitro molecular cloning method that uses the CRISPR / Cas9 system as a molecular biology tool to target multiple targets on the carrier or target multiple DNA fragments of interest.

Method used

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  • Molecular cloning method based on CRISPR/Cas9 and homologous recombination of saccharomyces cerevisiae cell endogenous genes
  • Molecular cloning method based on CRISPR/Cas9 and homologous recombination of saccharomyces cerevisiae cell endogenous genes
  • Molecular cloning method based on CRISPR/Cas9 and homologous recombination of saccharomyces cerevisiae cell endogenous genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1-3

[0166] Embodiment 1-3: Insert 1-3 target fragments into different positions of the initial vector respectively, and replace a replaced fragment at another position of the initial vector with another target segment at the same time; wherein, the homology arm sequence is located adjacent to be connected flank of one of the fragments

Embodiment 1

[0167] Example 1: Insert a target fragment into the initial vector, and replace the replaced fragment at another position of the initial vector with another target fragment

[0168] The purpose of this example is to insert the target fragment mRFP (SEQ.ID.NO: 4) into site IV of the initial vector, and at the same time, insert the fragment ura3 (SEQ.ID.NO: 6 ) was replaced by the target fragment trp1 (SEQ.ID.NO:2).

[0169] (1) Positioning: designed for image 3 The sgDNA sequences of sites I, II and IV (sgDNA-1, sgDNA-2, sgDNA-3, as shown in Table 1). Each sgDNA was ligated into the sgRNA in vitro transcription template (SEQ.ID.NO: 7) by overlapping PCR, and the primers used in overlapping PCR were shown in Table 1.

[0170] The PCR purification kit (Omega-D6492) was used to recover and purify the obtained sgRNA in vitro transcription template.

[0171] The Epicenter ASF3257 commercial kit was used to perform in vitro transcription using T7 RNA polymerase to obtain sgRNAs (...

Embodiment 2

[0185] Example 2: Insert two target fragments into different positions of the initial vector, and replace the replaced fragment at another position of the initial vector with another target fragment

[0186] The purpose of this example is to insert the target fragments mRFP (SEQ.ID.NO: 4) and lacZ (SEQ.ID.NO: 3) into site IV and site III of the initial vector respectively, and at the same time, insert the site The fragment ura3 between I and II was replaced by the target fragment trp1.

[0187] (1) Positioning: by a method similar to that described in Example 1, design and synthesize targeting image 3 The sgRNA sequences of site I, site II, site IV and site III (sgRNA-1, sgRNA-2, sgRNA-3 and sgRNA-4, as shown in Table 1). All obtained sgRNAs were mixed at equal concentrations.

[0188] (2) Digestion: By a method similar to that described in Example 1, use Cas9 nuclease and the sgRNA obtained in step (1) to digest the initial vector in vitro, so that site I, site II, site D...

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Abstract

The invention relates to a novel molecular cloning method, and in particular to a method for obtaining recombinant vectors by modifying initial vectors through combined utilization of a CRISPR/Cas9 system and a saccharomyces cerevisiae cell endogenous gene homologous recombination system; only through one-time transformation and selection operations, the method can simultaneously complete insertion of ore or more target DNA fragments into the initial vectors, deletion of one or more DNA fragments from the initial vectors and/or substitution of one or more DNA fragments on the vectors for one or more target DNA fragments. The invention further relates to a reagent kit for conveniently and effectively applying the method provided by the invention.

Description

technical field [0001] The present invention relates to a new molecular cloning method, in particular to a method for transforming an initial vector by combining the CRISPR / Cas9 system and the endogenous homologous recombination system of Saccharomyces cerevisiae cells to obtain a recombinant vector, said method Inserting one or more target DNA fragments into the initial vector, deleting one or more DNA fragments from the initial vector and / or replacing one or more DNA fragments on the vector with a or multiple target DNA fragments. The present invention also relates to a kit for conveniently and effectively implementing the molecular cloning method described in the present invention. Background technique [0002] Molecular cloning is a molecular biology experimental method that assembles the target DNA fragment and carrier DNA into a recombinant vector and then introduces it into a host cell or organism to make it replicate in the host. During the host cell amplification ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12N15/81C12N1/19
Inventor 娄春波赵学金
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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