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Base editing mimics, repairs man2b1 associated with mannosidosis c2248t Mutation reagents and methods

A MAN2B1C2248T, base editing technology, applied in the direction of cells modified by introducing foreign genetic material, recombinant DNA technology, introduction of foreign genetic material using vectors, etc., can solve the problems of low HDR efficiency, inaccurate gene editing, breakage, etc.

Active Publication Date: 2020-08-04
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, causing double-strand breaks (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 the HDR efficiency Low (integration rarely occurs), and the non-homologous end-joining mechanism is prone to random insertions and deletions (indels), making it possible to randomly introduce new bases near the breakpoint, resulting in imprecise gene editing 8

Method used

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  • Base editing mimics, repairs man2b1 associated with mannosidosis <sup>c2248t</sup> Mutation reagents and methods
  • Base editing mimics, repairs man2b1 associated with mannosidosis <sup>c2248t</sup> Mutation reagents and methods
  • Base editing mimics, repairs man2b1 associated with mannosidosis <sup>c2248t</sup> Mutation reagents and methods

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] In this example, Cas9 / sgRNA combined with ssODN was used to make mutant MAN2B1 on the cell line C2248T For mutant cell lines, this method will be implemented using the RNA-bound ssODN format of Cas9 mRNA and sgRNA, see Figure 1A-Figure 1D .

[0048] 1.1 Plasmid construction

[0049]Near the mutation site, design a mutant mt-sgRNA (SEQ ID NO.1) and synthesize oligos. The upstream sequence is: 5'-taggCTACACAGACAGCAATGGCC-3' (SEQ ID NO.(5)), and the downstream sequence is: 5'- aaacGGCCATTGCTGTCTGTGTAG-3'(SEQ ID NO.(6)), 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

[0067] In this example, the base editing system CBEs (Cytosine base editors) was used to make the mutant MAN2B1 on the cell line C2248T For mutant cell lines, this method will be implemented using the corresponding BE3 mRNA and sgRNA RNA forms, see Figures 2A-2C .

[0068] 1.1 Plasmid construction

[0069] sgRNA vector construction: near the mutation site, design a mutant mt-sgRNA (SEQ ID NO.3) and synthesize oligos. The upstream sequence is: 5'-taggTGGCCGGGAGATCCTGGAGA-3' (SEQ ID NO.(10)), and the downstream sequence is : 5'-aaacTCTCCAGGATCTCCCGGCCA-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) annealed 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 make up to 60 μL. Digest overnight at 37°C. The ligation system is as fol...

Embodiment 3

[0093] In this example, for the obtained homozygous mutant cell line, the MAN2B1 C2248T Implement the fix. In this example, the combination of XABE and ABE plasmid repair sgRNA will be used to repair the mutation site, see Figure 3A-3D .

[0094] 1.1 Plasmid construction

[0095]Near the mutation site, design and repair re-sgRNA, synthesize oligos, the upstream sequence is: 5'-accgCTCCCAGCCATTGCTGTCTG-3' (SEQ ID NO. (17)), the downstream sequence is: 5'-aaacCAGACAGCAATGGCtGGGAG-3' (SEQ ID NO.(18)), the upstream and downstream sequences are 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) annealing, ligation onto the PGL3-U6 sgRNA vector linearized with BsaI (NEB: R0539L) on SEQ ID NO. (19) (addgene: 51133). The linearization system is as follows: PGL3-U6sgRNA 2μg; buffer (NEB: R0539L) 6μL; BsaI 2μL; ddH 2 O to make up to 60 μL. Digest overnight at 37°C. The ligation system is as follows: T4 ligation buffer (NEB: M0202L) 1...

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Abstract

The invention provides a reagent and method for base edition, simulation and repair of MAN2B1C2248T mutation related to mannoside storage syndrome. A kit for efficiently simulating the MAN2B1C2248T mutation comprises a base editing system and mutant mt-sgRNA aiming at the MAN2B1C2248T site; a kit for efficiently repairing the MAN2B1C2248T mutation comprises a base editing system and mutant re-sgRNA aiming at the MAN2B1C2248T site. According to the invention, a base editing technology is utilized, and the mutation of the MAN2B1C2248T can be simulated and repaired through accurate CT or AG single base mutation, so that an efficient and safe method is provided for treating mannoside storage diseases caused by the mutation.

Description

technical field [0001] The present invention relates to the field of gene repair, more specifically, using base editing to simulate and repair MAN2B1 related to mannosidosis in human cells C2248T Reagents and methods for (α-mannosidosis)-associated mutations. Background technique [0002] α-mannosidosis (α-mannosidosis) is an autosomal recessive genetic metabolic disease, mainly due to mutations in the gene MAN2B1 encoding mannosidase, resulting in rare lysosomes caused by α-mannosidase deficiency storage disease 1 . The incidence rate of this disease is about 1 / 500000-1 / 1000000, and it occurs worldwide. Its clinical symptoms are diverse, and there is no unified and unique clinical phenotype, mainly manifested as immune deficiency, hearing loss, facial distortion, abnormal bone development, mental retardation and other complications 2 . At present, early symptomatic treatment is mainly used for α-mannosidosis, including hematopoietic stem cell transplantation and enzyme...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N5/10
Inventor 马旭李广磊金孝华刘馨怡
Owner 国家卫生健康委科学技术研究所
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