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Set of base editing system based on suppurative streptococcus and application of set of basic group editing system in gene editing

A Streptococcus pyogenes, base editing technology, applied in the field of molecular biology, can solve problems such as hindering the application of base editing technology

Active Publication Date: 2017-11-24
微光基因(苏州)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, scientists have found that ordinary Cas9 proteins will bind to many sites similar to the target DNA sequence on the genome, which makes the base editing system based on ordinary Cas9 proteins have obvious off-target effects, hindering base editing technology Application in the field of disease model construction and gene therapy

Method used

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  • Set of base editing system based on suppurative streptococcus and application of set of basic group editing system in gene editing
  • Set of base editing system based on suppurative streptococcus and application of set of basic group editing system in gene editing
  • Set of base editing system based on suppurative streptococcus and application of set of basic group editing system in gene editing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Preparation method of base editing system components based on Streptococcus pyogenes

[0049] Figure 1~4 Expression vector map of a more precise base editing system for four species of Streptococcus pyogenes.

[0050] This example provides a preparation method for base editing system components rAPOBEC1:Cas9:UGI mRNA and gRNA based on Streptococcus pyogenes.

[0051] 1. Preparation of Streptococcus pyogenes rAPOBEC1:Cas9:UGI mRNA, the method is as follows:

[0052] (1) Prepare the transcription vector of HF1-BE2, HF1-BE3, HF2-BE2 or HF2-BE3, the sequence is shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 .

[0053] (2) Prepare the transcription template of Streptococcus pyogenes APOBEC1:Cas9:UGI comprising T7 promoter:

[0054]Use KpnI to cut the transcription vector of HF1-BE2, HF1-BE3, HF2-BE2 or HF2-BE3, and then use the PCR product purification kit (Axygen) to perform column purification on the digestion product, and then use nuclease-free...

Embodiment 2

[0066] Example 2 Preparation of base editing system components rAPOBEC1:Cas9:UGI mRNA and gRNA based on Streptococcus pyogenes

[0067] Specifically, the operation procedure of the preparation method of Streptococcus pyogenes APOBEC1:Cas9:UGI mRNA, APOBEC1:Cas9:UGI protein and gRNA described in the above-mentioned embodiment 1 is as follows:

[0068] 1. Preparation of APOBEC1:Cas9:UGI mRNA and gRNA transcription template DNA:

[0069] (1) Preparation of Streptococcus pyogenes APOBEC1:Cas9:UGI mRNA transcription template DNA

[0070] Prepare the transcription vector of HF1-BE2, HF1-BE3, HF2-BE2 or HF2-BE3 by plasmid extraction (independent research and development), and then digest the transcription vector with KpnI, and proceed according to the reaction system shown in Table 1 below:

[0071] Table 1 reaction system

[0072] Element

Dosage

mRNA transcription vector

2000ng

10X NEBuffer 1.1

5μl

Kpn I

5μl

wxya 2 o

Mak...

Embodiment 3

[0133] Example 3 Site-directed mutation of Tyr gene mediated by a more precise base editing system based on Streptococcus pyogenes

[0134] 1. In order to use a more accurate base editing system based on Streptococcus pyogenes to achieve single-gene site-directed mutation in mouse fertilized eggs, we designed two gRNAs (gRNA-1 and gRNA-2) for the Tyr gene. The site-directed mutation of the Tyr gene will form a stop codon, which will lead to the premature termination of the translation of the Tyr gene, so that the coat color of the pups will change from black to white.

[0135] First, we transcribed Tyr gRNA, then mixed Tyr gRNA (50ng / μl) and rAPOBEC1:Cas9:UGI(HF2-BE2) mRNA (100ng / μl) and injected them into 0.5-day-old mouse fertilized eggs. 48 hours after injection, the efficiency of site-directed mutation was detected. By combining PCR and Sanger sequencing detection, we found that: for gRNA-1, 11.6% of the mouse embryos were mutated, and for gRNA-2, 50% of the embryos were m...

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Abstract

The invention discloses a set of precise basic group editing system based on suppurative streptococcus Cas9 and an application of the set of precise basic group editing system in mammalian cells and / or embryonic gene editing. The basic group editing system is composed of the two parts of components of a rAPOBEC1 : Cas9 : UGI expression vector and a gRNA expression vector, the rAPOBEC1 : Cas9 : UGI expression vector is obtained by cloning an encoding gene of rAPOBEC1 : Cas9 : UGI into a pcDNA3.1 (-) vector through a method of gene synthesis and molecular cloning, and the gRNA expression vector is obtained by cloning a gRNA sequence into a pDR274 vector including a T7 promoter through the mode of restriction enzyme ligation. The set of precise basic group editing system can be applied to construction of gene modification, a mammalian cell model and an animal model and used for gene therapy of mammalian cells and embryos, and has a good application prospect.

Description

technical field [0001] The invention belongs to the technical field of molecular biology. More specifically, it relates to a set of precise base editing system (Base editing, BE) based on Streptococcus pyogenes (Streptococcus pyogenes SF370) Cas9 and its application in gene editing of mammalian cells and embryos. Background technique [0002] The completion of the Human Genome Project in 2003 set off an upsurge in gene function research, making functional genomics the focus of life science research. A large number of sequencing results have shown the diversity of genes in the population, especially single nucleotide polymorphisms (single nucleotide variations, SNVs). Studying the physiological functions of these SNVs will help to explain the physiological and even psychological differences between people. Genetic Basis of Difference. Prior to this, the construction of animal or cell models of SNV relied on gene targeting technology, which was very inefficient (<10 -5 )...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/90C12N9/22
CPCC12N9/22C12N15/113C12N15/907C12N2310/10C12N2800/80C12N2810/10
Inventor 黄军就松阳洲梁普平
Owner 微光基因(苏州)有限公司
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