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Reagent and method for repairing TGM1C607T mutation related to lamellar ichthyosis by base editing

A TGM1C607T, base editing technology, applied in the field of gene repair, can solve the problems of not being covered by medical insurance, no treatment methods, and few opportunities for patients to obtain drugs

Active Publication Date: 2021-08-10
肇庆市瑞思元生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, less than 5% of rare diseases can be effectively intervened or treated, and Chinese patients have fewer opportunities to obtain drugs, and many drugs for rare diseases are not covered by medical insurance
There is still no effective treatment for the disease, and the use of base editors to repair the disease-causing mutation will be a potential strategy for the treatment of the disease

Method used

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  • Reagent and method for repairing TGM1C607T mutation related to lamellar ichthyosis by base editing
  • Reagent and method for repairing TGM1C607T mutation related to lamellar ichthyosis by base editing
  • Reagent and method for repairing TGM1C607T mutation related to lamellar ichthyosis by base editing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] In this example, BEmax-AID, BEmax-A3A-Y130F(, AncBE4max, and AncBE4max-NG combined with mutant sgRNA were used to prepare TGM1 on cell lines C607T mutant cell lines.

[0056] 1.1 Plasmid construction

[0057] Near the mutation site, design two pairs of mutant mt-sgRNA (SEQ ID NO.1-2) and synthesize oligos. The upstream sequence of mt-sgRNA1 is: 5'-accgTGGGCAGAATCTGAACCTGC-3', and the downstream sequence is: 5'-aaacGCAGGTTCAGATTCTGCCCA- 3'; the upstream sequence of mt-sgRNA2 is: 5'-accgGTGGGCAGAATCTGAACCTG-3', and the downstream sequence is: 5'-aaacCAGGTTCAGATTCTGCCCAC-3'. The upstream and downstream sequences were annealed (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 then connected to BsaI (NEB, R0539L) for linearization On the pGL3-U6-sgRNA-EGFP vector (Addgene, 107721). The linearization system is: pGL3-U6-sgRNA-EGFP 2 μg; buffer (NEB, R0539L) 6 μL; BsaI 2 μL; ddH 2 Make up to 60 μL with O, digest overnight at 37°C. The vector was recover...

Embodiment 2

[0063] In this example, in TGM1 C607T On the mutant cell line, the base editing system ABEmax-NG was used to repair the mutant strain.

[0064] 2.1 Plasmid construction

[0065] In mimics of pathogenic TGM1 C607T On the mutant cell line, according to the characteristics of ABEmax-NG base editing, design repair re-NG-sgRNA (SEQ ID NO.3-6) of different lengths, synthesize oligos, and the upstream sequence of re-NG-sg20 is 5'- accgCAGATTTCTACCCACTGGCCT-3', the downstream sequence is 5'-aaacAGGCCAGTGGGTAGAATCTG-3'; the upstream sequence of re-NG-sg19 is 5'-accgAGATTCTACCCCACTGGCCT-3', the downstream sequence is 5'-aaacAGGCCAGTGGGTAGAATCT-3'; the upstream sequence of re-NG-sg18 The upstream sequence of re-NG-sg17 is 5'-accgATTCTACCCCACTGGCCT-3', and the downstream sequence is 5'-aaacAGGCCAGTGGGTAGAAT-3'. The upstream and downstream sequences were annealed (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 then connected to BsaI (NEB, R0539L) for linearizatio...

Embodiment 3

[0071] In this example, in TGM1 C607T On the mutant cell line, the base editing system Sc-ABEmax was used to repair the mutant strain.

[0072] 3.1 Plasmid construction

[0073] against TGM1 C607T Mutation site, according to the characteristics of Sc-ABEmax base editing function, design repair re-Sc-sgRNA (SEQ ID NO.7-10) of different lengths, and synthesize oligos, the upstream sequence of re-Sc-sg20 is 5'-accgTCAGATTCTACCACTGGCC -3', the downstream sequence is 5'-aaacGGCCAGTGGGTAGAATCTGA-3'; the upstream sequence of re-Sc-sg19 is 5'-accgCAGATTCTACCCACTGGCC-3', the downstream sequence is 5'-aaacGGCCAGTGGGTAGAATCTG-3'; the upstream sequence of re-Sc-sg18 is 5'-accgAGATTCTACCCACTGGCC-3', the downstream sequence is 5'-aaacGGCCAGTGGGTAGAATCT-3'; the upstream sequence of re-Sc-sg17 is 5'-accgGATTCTTACCCCACTGGCC-3', the downstream sequence is 5'-aaacGGCCAGTGGGTAGAATC-3'. The upstream and downstream sequences were annealed (95°C, 5min; 95°C-85°C at-2°C / s; 85°C-25°C at-0.1°C / s; ho...

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Abstract

The invention provides a reagent and a method for repairing TGM1C607T mutation by base editing. A kit for efficiently repairing the TGM1C607T mutation is characterized in that the kit comprises a basic group editing system and repairing re-sgRNA aiming at a TGM1C607T locus. According to the invention, a base editing technology is utilized, and through accurate TC single base mutation, TGM1C607T mutation can be repaired, so that an efficient and safe method is provided for treating lamellar ichthyosis caused by the mutation.

Description

technical field [0001] The present invention relates to the field of gene repair, more specifically, using base editing to repair TGM1 related to lamellar ichthyosis C607T method of mutation. Background technique [0002] At present, there are more than 7000 kinds of rare diseases confirmed in the world, about 80% of which are caused by genetic defects, and half of the patients with rare diseases develop the disease at birth or in childhood, and society and families bear a huge burden. However, less than 5% of rare diseases can be effectively intervened or treated, and Chinese patients have fewer opportunities to obtain drugs, and many drugs for the treatment of rare diseases are not covered by medical insurance. Although preconception screening, prenatal screening and prenatal diagnosis, and newborn screening are critical to reducing the birth rate of children with rare diseases, precise gene therapy strategies still need to be developed (Dunbar et al., 2018). [0003] Ge...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/90C12N5/10
CPCC12N15/1137C12N15/907C12N9/1044C12Y203/02013C12N2310/20
Inventor 党露李广磊黄行许
Owner 肇庆市瑞思元生物科技有限公司
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