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Method used for specifically increasing of CRISPR-CAS system gene editing efficiency

A gene editing and efficient technology, applied to other methods of inserting foreign genetic materials, genetic engineering, biochemical equipment and methods, etc., can solve the problems of low homologous recombination probability and affect precise editing, etc., to improve gene editing efficiency, The effect of improving efficiency

Active Publication Date: 2017-12-15
江西汉氏联合干细胞科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013] The purpose of the present invention is to provide a method for improving the gene editing efficiency of the CRISPR-cas system, which effectively overcomes the technical defects of the prior art that the probability of homologous recombination in animals is low and thus affects the precise editing, especially for gene editing of stem cells defect

Method used

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  • Method used for specifically increasing of CRISPR-CAS system gene editing efficiency
  • Method used for specifically increasing of CRISPR-CAS system gene editing efficiency
  • Method used for specifically increasing of CRISPR-CAS system gene editing efficiency

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Example 1, cloning the potentiating protein CREnhancer1.0 and constructing the vector

[0032] The CREnhancer1.0 gene was cloned, and the gene sequence described in SEQ ID NO: 1 was obtained by the method of whole gene synthesis. Using this sequence as a template, according to the sequences of the upstream and downstream primers, they were 5'-ATGCAGGAGAACCTGGCCCCCTG-3', 5 '-CAGGCAGCTCACGCTCCTCTCG-3', primers and whole genome were synthesized by Shanghai Sangong Co., Ltd. The target gene fragment of CREnhancer1.0 gene was amplified by PCR reaction. The amplification reaction system was as follows: 95°C, 40s, 57°C, 1min, 72°C, 1min, 72°C, 10min, cycled 35 times, and the PCR product was produced by Shanghai Shenggong Co., Ltd. Sequencing was performed, and the binding was a complete match to SEQ ID NO:1 by sequencing. Subsequently, the target gene amplified by PCR was connected to the empty vector lentiviral vector pHIV-CS-CDF-CG-PRE, and the recombinant lentiviral vector...

Embodiment 2

[0033] Example 2 Application Analysis of CRISPR / Cas9 in Bone Marrow Mesenchymal Stem Cells

[0034] CRISPR / Cas9 editing vector based on pBGN plasmid containing BSD-fsEGFP fusion gene

[0035] (1) BSD-fsEGFP fusion gene: use conventional PCR to amplify the known BSD gene, 5'-PCR primers with HindIII sites, and 3'-PCR primers to introduce I-SceI and EcoRI sites. Insert the PCR product (BSD) into the HindIII and EcoRI positions between the CMV driver and the EGFP coding region in the EGFP plasmid (the EGFP nucleotide sequence is a sequence known in the art, such as shown in sequence 1 and sequence 2 in CN105647968A) Point, generate the plasmid pBGN containing the BSD-fsEGFP fusion gene, the nucleotide sequence of the BSD-fsEGFP fusion gene is as shown in sequence 3 and sequence 4 in CN105647968A). The fusion gene is driven by CMV driver or PGK driver, but EGFP is inactive due to frameshift, so it is called fsEGFP.

[0036] 5'-PCR primers are

[0037] CTCAAGCTTAACTAAACCATGGCCAA...

Embodiment 3 Embodiment 2

[0044] Example 3 Efficiency Analysis of the System of Example 2 in Human U2OS Cells

[0045] The system prepared according to Example 2 was transformed into human U2OS cells, and the human U2OS cells were respectively introduced or not introduced with the synergistic protein described in Example 1 as a control. HPRT-inactivated cells will develop resistance to 6-mercaptoguanine (6-TG) due to frameshifts or mutations caused by repair in human U2OS cells. After 4 weeks of screening using the known 6-TG screening method, 6-TG resistant cell clones will form, and the ratio of the formation frequency to the cell clone formation frequency without 6-TG selection represents true 6-TG resistant cells Clonogenic efficiency. This efficiency represents the efficiency with which the endogenous HPRT gene is edited. The result is as image 3 As shown, in U2OS cells, the endogenous HPRT gene editing efficiency mediated by the intracellular sgRNA without the synergist protein is 52.6%, and ...

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Abstract

The invention provides a novel enhancin CREnhancer 1.0 capable of increasing in vivo CRISPR / Cas9 gene editing efficiency obviously, provides a novel approach used for increasing gene editing efficiency in vivo, and provides a genome editing system with higher efficiency. When the enhancing and CRISPR / Cas9 are used together, CRISPR / Cas9 genome editing efficiency can be increased obviously.

Description

technical field [0001] The invention provides a method for improving the gene editing efficiency of the CRISPR-cas system, and in particular relates to a new synergistic factor that can significantly increase the probability of homologous recombination. Combining the synergistic factor with the CRISPR-cas system can significantly improve Genome Editing Efficiency. Background technique [0002] The CRISPR cluster is a special DNA repeat sequence family widely present in the genomes of bacteria and archaea, and its sequence consists of a leader (Leader), multiple short and highly conserved repeat sequence regions (Repeat) and multiple spacers ( Spacer) composition. The leader region is generally located upstream of the CRISPR cluster and is an AT-rich region with a length of 300-500 bp, which is considered to be the promoter sequence of the CRISPR cluster. The repeat sequence region is 21-48bp in length, contains palindromic sequence, and can form a hairpin structure. The r...

Claims

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Application Information

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IPC IPC(8): C07K14/47C12N15/113C12N15/90
CPCC07K14/4705C12N15/113C12N15/907C12N2310/10
Inventor 杨骏刘语方张立平
Owner 江西汉氏联合干细胞科技有限公司
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