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Repair of GALC associated with Krabbe disease using base editing C1586T Reagents and methods for mutagenesis

A base editing and mutation site technology, applied in the field of gene repair, can solve problems such as breakage, low HDR efficiency, and imprecise gene editing

Active Publication Date: 2020-11-24
国家卫生健康委科学技术研究所
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AI Technical Summary

Problems solved by technology

CRISPR / Cas9-mediated gene editing is when sgRNA (single guided RNA) guides Cas9 protein to position and cut double-stranded DNA through target sequence complementarity, resulting in double-strand breaks (DSB), under the condition of no template , non-homologous end joining repair occurs, resulting in frameshift mutation (frameshift mutation), leading to gene knockout (knockout); under the condition of template, it is repaired by homologous recombination to realize gene knockin (knockin), due to HDR Inefficient (integration rarely occurs) and non-homologous end-joining mechanisms are prone to random insertions and deletions (indels), making it possible to randomly introduce new bases near breakpoints, leading to imprecise gene editing

Method used

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  • Repair of GALC associated with Krabbe disease using base editing <sup>C1586T</sup> Reagents and methods for mutagenesis
  • Repair of GALC associated with Krabbe disease using base editing <sup>C1586T</sup> Reagents and methods for mutagenesis
  • Repair of GALC associated with Krabbe disease using base editing <sup>C1586T</sup> Reagents and methods for mutagenesis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] In this example, Cas9 / sgRNA combined with ssODN was used to make mutant GALC on the cell line C1586T For mutant cell lines, this method will be implemented using the RNA-bound ssODN format of Cas9 mRNA and sgRNA, see Figure 1A-Figure 1E .

[0061] 1.1 Plasmid construction

[0062] Near the mutation site, design a mutant mt-sgRNA (SEQ ID NO.3) and synthesize oligos. The upstream sequence is: 5'-taggGTTGAGAACTTGGCGTAGCG-3' (SEQ ID NO.(10)), and the downstream sequence is: 5'- aaacCGCTACGCCAAGTTCTCAAC-3' (SEQ ID NO.(11)), 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 carrier (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.4) used was synthesized by Sangon Biotech...

Embodiment 2

[0081] In this example, Cas9 / sgRNA combined with ssODN was used to make mutant GALC on the cell line C1586T For mutant cell lines, this method will use Cas9 protein and sgRNA RNA to form RNP combined with ssODN.

[0082] 1.1 Plasmid construction

[0083]Near the mutation site, design a mutant mt-sgRNA (SEQ ID NO.3) and synthesize oligos. The upstream sequence is: 5'-taggGTTGAGAACTTGGCGTAGCG-3' (SEQ ID NO.(10)), and the downstream sequence is: 5'- aaaccGCTACGCCAAGTTCTCAAC-3' (SEQ ID NO.(11)), 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 carrier (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.4) used was synthesized by Sangon Biotech (http: / / www.sangon.com / ) by PAGE pur...

Embodiment 3

[0093] In this example, for the obtained homozygous mutant cell line, the base editing system was used to C1586T Implement the fix. In this example, spCas9-ABE and KKH SaCas9-ABE mRNA will be combined with the corresponding repair sgRNA to repair the mutation site, see Figure 2A-2E as well as Figure 3A and Figure 3B .

[0094] 1.1 Plasmid construction

[0095] Near the mutation site, design and repair re-sgRNA and synthesize oligos. If spCas9-ABE is used, the upstream sequence is: 5'-taggAGCATGAAGTGATGCTCGCC-3'(SEQ ID NO.(15)), and the downstream sequence is: 5'-aaacGGCGAGCATCACTTCAtGCT- 3'(SEQ ID NO.(16)), if KKH SaCas9-ABE is used, the upstream sequence is: 5'-taggTAGCATGAAGTGATGCTCGC-3'(SEQ ID NO.(17)), the downstream sequence is: 5'-aaacGCGAGCATCACTTCAtGCTA-3' (SEQ ID NO. (18)). 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: R05...

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Abstract

The present invention provides a reagent and method for repairing the GALCC1586T mutation associated with krabbedisease using base editing. A kit for efficient repair of the GALCC1586T mutation includes a base editing system and repair re-sgRNA directed against the GALCC1586T site. The base editing technology is utilized to repair the mutation of GALCC1586T through precise AG single base mutationto provide the efficient safe method for treating the krabbedisease caused by the mutation.

Description

technical field [0001] The present invention relates to the field of gene repair, more specifically, the use of base editing to repair GALC associated with Krabbe disease C1586T (Krabbe Disease)-associated mutations reagents and methods. Background technique [0002] Krabbe Disease, also known as Globoidleukodystrophy, is an autosomal recessive, neurodegenerative disorder caused by a defect in the gene encoding the hydrolase galactosylceramidase (Galactocerebrosidase, GALC). lysosomal storage disease (Suzuki and Suzuki, 1970). The GALC gene is encoded by the gene GALC located on chromosome 14q31, which consists of 17 exons and encodes 669 amino acids. KD pathogenic variants occur throughout the protein. So far, more than 200 mutations have been reported in the human gene mutation database, including missense, nonsense, deletion and insertion (Tappino et al., 2010). GALC is responsible for the lysosomal degradation of important myelin (including galactosylceramide), and t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/90C12N9/22C12N15/85
Inventor 马旭李广磊金孝华王鑫杰
Owner 国家卫生健康委科学技术研究所
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