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Cloning method for series parallel expression of a plurality of sgRNA based on grading assembling and application

A cloning method and expression vector technology, applied in the field of genetic engineering, can solve the problem of difficulty in transferring plasmids into the same cell, etc.

Inactive Publication Date: 2017-06-20
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0010] The technical purpose of the present invention is to develop a rapid cloning method for concatenating multiple sgRNAs into the same plasmid, which can achieve the purpose of simultaneously transfecting the expression cassettes of multiple sgRNAs into cells to solve the problem of co-transfection of multiple sgRNA expression plasmids. Difficulty transferring all plasmids into the same cell

Method used

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  • Cloning method for series parallel expression of a plurality of sgRNA based on grading assembling and application
  • Cloning method for series parallel expression of a plurality of sgRNA based on grading assembling and application
  • Cloning method for series parallel expression of a plurality of sgRNA based on grading assembling and application

Examples

Experimental program
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Embodiment 1

[0035] Example 1: A vector-independent cloning method based on hierarchical assembly of multiple sgRNAs expressed in series and in parallel, taking connection of 20 sgRNAs as an example, prepared by the following steps ( figure 2 ):

[0036] (1) First, the promoters expressing small RNAs (mU6, hU6, H1, 7SK, etc.) and the basic skeleton of sgRNAs were amplified from the vectors expressing a single sgRNA (basic sgRNA vector psgRNA2.0 or modified sgRNA) by PCR. The 3' end of the primer for amplifying the promoter and the 5' end of the primer for amplifying the sgRNA each have a Type IIs restriction enzyme site (BsmBI). Pure DNA fragments were recovered by DNA agarose gel electrophoresis and gel purification kit.

[0037] (2) Annealing the two strands of the synthetic targeting pair primer. The 4-base overhang at the 5' end of the positive strand is complementary to the cohesive end after digestion of the promoter, and the 4-base overhang at the 5' end of the negative strand is...

Embodiment 2

[0051] Example 2: The application of serial and parallel expression of multiple sgRNAs based on hierarchical assembly in gene editing, taking the green fluorescent protein sfGFP cut and integrated into the genome of mammalian cells as an example to illustrate ( image 3 ):

[0052] (1) Use the sgRNA online design tool to search for targetable sgRNA sequences in the gene coding frame of sfGFP, and select the 5 sgRNAs with the highest scores (see attached table 3 below for the sequences).

[0053] (2) According to the conventional sgRNA cloning method, five separate sgRNAs were obtained.

[0054] (3) Cloning five sgRNAs into the same vector according to the method described in Example 2 above.

[0055] (4) In a six-well plate of cells stably expressing sfGFP, use the transfection reagent chemifect to co-transfect 1 μg of the plasmid px330 expressing Cas9 and 1 μg of the sgRNA vector expressed alone or 1 μg of the vector expressing five sgRNAs in tandem. As a control, 1 μg of p...

Embodiment 3

[0060] Example 3: The application of serial and parallel expression of multiple sgRNAs based on hierarchical assembly in the coordinated regulation of a single gene, using 20 sgRNAs to down-regulate the endogenous gene SOX2 in mammalian cells as an example ( Figure 4 ):

[0061] (1) Use the sgRNA online design tool to find targetable sgRNA sequences in the promoter region of SOX2, and select the 20 highest-scoring sgRNAs (see attached table 4 below for the sequences).

[0062] (2) According to the conventional sgRNA cloning method, 20 separate sgRNA expression vectors were obtained as controls.

[0063] (3) Cloning 20 sgRNAs into the same vector according to the method described in Example 1 above.

[0064] (4) In the six-well plate of MDA-MB-231 cells, use the transfection reagent chemifect to co-transfect 1 μg of the plasmid pHAGE-EF1α-dCas9-KRAB expressing dCas9-KRAB and 1 μg of the sgRNA vector expressed alone or 1 μg of 20 tandem expressions sgRNA carrier. As a contro...

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Abstract

A CRISPR / Cas9 system has an ultrahigh parallel capacity. In order to meet the requirement for expressing a plurality of sgRNA in some cases, the invention provides a quick assembling method for a plurality of parallel expressed sgRNA. The invention utilizes grading Golden Gate reaction to develop a multi-turn amplifying method based on polymerase chain reaction and independent of a carrier, so as to realize the quick connecting assembling for 20 sgRNA within one week. The method for serially assembling a plurality of sgRNA developed by the invention has the advantages of time-saving and labor-saving effect, flexibility, high efficiency and multifunction. The method can be used for quickly assembling 2-20 sgRNA in different quantity onto one carrier. A plurality of parallel expressed sgRNA are utilized to target to a section of DNA, so that the functions of marking and tracking unrepeated sequence chromatin locus in living cells, cooperatively activating or restraining a single gene, simultaneously editing a plurality of genes and simultaneously up-regulating and down-regulating the genes can be achieved. The method can be widely used for editing the gene and understanding the organization structure and dynamic change of chromatin.

Description

[0001] The Hierarchical Assembly Method of Multiplexed sgRNA Expression and Its Applications Technical field [0002] The invention relates to a cloning method for hierarchical assembly of multiple sgRNAs for serial and parallel expression and its application in genome editing, gene expression regulation and chromosome marking. Belongs to the field of genetic engineering. Background technique [0003] The nucleus of mammalian cells is a highly complex self-assembled structure, of which chromatin is the main component. Chromatin is composed of DNA, RNA and proteins that interact with them. They jointly regulate basic cellular life activities such as transcription, DNA replication, repair and RNA splicing. Studying which genes control certain specific processes in cells, that is, the relationship between phenotype and genotype, requires the ability to specifically interfere with the expression of certain genes. This includes permanent inactivation of genes and transient up- ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/66C12N15/65C12N15/85
CPCC12N15/65C12N15/66C12N15/85C12N2800/107C12N2810/10
Inventor 邵世鹏常蕾孙雨傲孙育杰
Owner PEKING UNIV
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