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Reagent and method for repairing FBN1T7498C mutation by utilizing basic group compiling

A base editing and kit technology, applied in the field of gene repair, can solve problems such as disease in offspring

Active Publication Date: 2018-11-06
SHANGHAI TECH UNIV
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although some patients can be treated with surgery, there is still a risk of disease for future generations, and genetically treating the mutation that causes the disease will be the fundamental method

Method used

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  • Reagent and method for repairing FBN1T7498C mutation by utilizing basic group compiling
  • Reagent and method for repairing FBN1T7498C mutation by utilizing basic group compiling
  • Reagent and method for repairing FBN1T7498C mutation by utilizing basic group compiling

Examples

Experimental program
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Effect test

Embodiment 1

[0059] In this example, Cas9 / sgRNA combined with ssODN was used to make mutant FBN1 on the cell line T7498C mutant cell lines, and use the base editing system to repair the mutant strains ( figure 2 ).

[0060] 1.1 Plasmid construction

[0061]Near the mutation site, design a mutant mt-sgRNA (SEQ ID NO.1) and synthesize oligos. The upstream sequence is: 5'-taggCGCCAATGGTGTTAACACAT-3' (SEQ ID NO.(14)), and the downstream sequence is: 5'- aaacATGTGTTAACACCATTGGCG-3'(SEQ ID NO.(15)), the upstream and downstream sequences passed the program (95°C, 5min; 95°C-85°C at-2°C / s; 85°C-25°C at-0.1°C / s; hold at 4°C) and ligated to the PUC57-T7 sgRNA vector (addgene: 51132) linearized with BsaI (NEB: R0539L). The linearization system is as follows: PUC57-T7sgRNA 2μg; buffer (NEB: R0539L) 6μL; BsaI 2μL; ddH 2 O to make up to 60 μL. Digest overnight at 37°C. The homologous template ssODN (SEQ ID NO.2) used was synthesized by Sangon Biotech (http: / / www.sangon.com / ) by PAGE purification....

Embodiment 2

[0086] In this example, the base editing system was used to repair the mutation in human embryos ( Figure 7 ).

[0087] 2.1 Plasmid construction

[0088]Near the mutation site, according to the characteristics of base editing, design and repair re-sgRNA (SEQ ID NO.3) and synthesize oligos. The upstream sequence is 5'-taggCTACGTGTTAACACCATTGG-3' (SEQ ID NO.18), and the downstream sequence is 5'-aaacCCAATGGTGTTAACACGTAG-3' (SEQ ID NO. 19). The upstream and downstream sequences were annealed through the program (95°C, 5min; 95°C-85°C at-2°C / s; 85°C-25°C at-0.1°C / s; hold at 4°C), and connected to the DNA through BsaI (NEB: R0539L ) on the linearized PUC57-T7 sgRNA vector. The linearization system and procedure are as above. The ligation system is as follows: T4 ligation buffer (NEB: M0202L) 1 μL, linearized vector 20 ng, annealed oligo fragment (10 μM) 5 μL, T4 ligase (NEB: M0202L) 0.5 μL, ddH 2 Make up to 10 μL with O. Ligate overnight at 16°C. The connected vector was tra...

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Abstract

The invention provides a reagent and a method for repairing FBN1T7498C mutation by utilizing basic group compiling. A kit for efficiently repairing the FBN1T7498C mutation is characterized by comprising a basic group compiling system and a repairing re-sgRNA specific to an FBN1T7498C site. By utilizing the basic group compiling technology, the FBN1T7498C mutation can be repaired accordingly through accurate CT single base group mutation, so that an efficient safe method is provided for treating Marfan's syndrome caused by the kind of mutation.

Description

technical field [0001] The present invention relates to the field of gene repair, more specifically, using base editing to repair FBN1 related to Marfan syndrome T7498C method of mutation. Background technique [0002] So far, nearly 10,000 genetic diseases have been identified in medicine, which has caused a huge burden on families and society. However, only about 6% of genetic diseases can currently be treated (Austin and Dawkins, 2017). Diagnosis and treatment of genetic diseases has become an important research content in medicine. The development of high-throughput sequencing technology has made the diagnosis of genetic diseases easier. Although preimplantation genetic diagnosis (PGD) can block the occurrence of some genetic diseases, for some genetic diseases such as homozygous mutations, more effective genetic treatments need to be developed (Dunbar et al., 2018). [0003] Gene editing technology, especially CRISPR / Cas9, has been widely used in gene manipulation an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/90C12N5/10
CPCC12N15/113C12N15/907C07K14/47C12N2310/10C12N2310/20C12N2320/34C12N9/22A61K48/005C07K14/78
Inventor 黄行许李广磊李佳楠
Owner SHANGHAI TECH UNIV
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