Unlock instant, AI-driven research and patent intelligence for your innovation.

A base editing system based on Streptococcus pyogenes and its application 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: 2020-07-14
微光基因(苏州)有限公司
View PDF3 Cites 0 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A base editing system based on Streptococcus pyogenes and its application in gene editing
  • A base editing system based on Streptococcus pyogenes and its application in gene editing
  • A base editing system based on Streptococcus pyogenes and its application 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] Figures 1 to 4 Expression vector maps for more precise base editing systems for four Streptococcus pyogenes.

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

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

[0052] (1) Preparation of transcription vector for 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 a transcription template of Streptococcus pyogenes APOBEC1:Cas9:UGI containing the T7 promoter:

[0054]The transcription vector of HF1-BE2, HF1-BE3, HF2-BE2 or HF2-BE3 was cleaved with KpnI, and then the cleaved product was subjected to column purification with a PCR product purification kit (Axygen), followed by ...

Embodiment 2

[0066] Example 2 Preparation of rAPOBEC1:Cas9:UGI mRNA and gRNA components of the base editing system 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 Example 1 is as follows:

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

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

[0070] The transcription vector of HF1-BE2, HF1-BE3, HF2-BE2 or HF2-BE3 was prepared by plasmid extraction (independently developed), and then the transcription vector was digested with KpnI, and the reaction system was carried out according to the following table 1:

[0071] Table 1 Reaction system

[0072] Element Dosage mRNA transcription vector 2000ng 10X NEBuffer 1.1 5μl Kpn I 5μl ddH 2 O

Make up to 50μl

[0073] Digestion ove...

Embodiment 3

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

[0134] 1. In order to use a more precise base editing system based on Streptococcus pyogenes to achieve single-gene site-directed mutagenesis in mouse fertilized eggs, we designed two gRNAs (gRNA-1 and gRNA-2) for the Tyr gene. Site-directed mutagenesis of the Tyr gene results in the formation of a stop codon, which results in premature termination of the Tyr gene translation, which changes the coat color of the pups from black to white.

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

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 SF370 Cas9 and its application in gene editing of mammalian cells and embryos. Background technique [0002] The Human Genome Project completed in 2003 set off a wave of gene function research, making functional genomics the focus of life science research. A large number of sequencing results show the diversity of genes in the population, especially single nucleotide polymorphisms (SNVs). Studying the physiological functions of these SNVs will help to explain the physiological and even psychological relationships between people. Genetic basis of differences. Previously, the construction of animal or cell models of SNVs relied on gene targeting technology, which resulted in very low efficiency (<10%) due to the use of the cell's own spontaneous homologous recombin...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
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