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Gene base editor

A technology of editing and residues, applied in chemical instruments and methods, antibody mimics/scaffolds, hybrid peptides, etc., can solve the problem of ineffective correction, limited effective editing sites of base editors, and deletion of RNA splicing sites And other issues

Inactive Publication Date: 2018-11-16
SHANGHAI TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, rA1-based base editors cannot efficiently edit base C in the GpC site, thus limiting the effective editing sites of existing base editors
For example, the mutation from GpT to GpC can lead to the deletion of RNA splicing sites, which can cause a variety of human diseases; while the existing rA1-based base editor cannot effectively correct the mutation from GpT to GpC

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0102] Example 1 The FANCF site of the human genome uses the hA3A-BE base editor to realize efficient base editing of cytosine at the GpC site

[0103] A gene base editor, including hA3A-BE expression vector and sgRNA expression vector. The sequence of the hA3A-BE expression vector is SEQ ID NO:16.

[0104] The preparation and base editing method of the above-mentioned base editing system are as follows:

[0105] 1.1 Experimental materials

[0106] 1.1.1 Reagents and plasmids

[0107] Primers were synthesized from Suzhou Jinweizhi Biological Co., Ltd.; restriction enzymes, DNA ligase, high-fidelity DNA polymerase Purchased from NEB Company; Plasmid Recombination Kit Clone Purchased from Vazyme; pCMV-BE3 from addgene website, the article number is 73021; DNA gel recovery kit was purchased from Corning; transfection reagent LTX, Available from Thermo Fisher; QuickExtract TM Genomic DNA extraction reagents were purchased from Illumina.

[0108] 1.1.2 Cell lines

[...

Embodiment 2

[0129] Example 2 The human genome HEK293-Site4 site uses the hA3A-BE base editor to realize efficient base editing of cytosine at the GpC site

[0130] A gene base editor, including hA3A-BE expression vector and sgRNA expression vector. The sequence of the hA3A-BE expression vector is SEQ ID NO:16.

[0131] The preparation and base editing method of the above-mentioned base editing system are as follows:

[0132] 2.1 Experimental materials

[0133] 2.1.1 Reagents and plasmids: Same as Example 1.

[0134] 2.1.2 Cell lines: Same as Example 1.

[0135] 2.2 Experimental method

[0136] 2.2.1 Construction of hA3A-BE expression vector: same as Example 1.

[0137] 2.2.2 Construction of sgRNA expression plasmid

[0138] Anneal the following primers 28 and 29, and connect the annealed product into the sgRNA expression vector pGL3-u6-sgRNA-puro digested with the restriction endonuclease BsaI to obtain the sgRNA expression plasmid targeting the HEK293-Site4 site of the human genome...

Embodiment 3

[0153] Example 3 The human genome HEK293-Site3 site uses the hA3A-BE-muts base editor to achieve high-precision and high-efficiency base editing

[0154] A gene base editor, including hA3A-BE expression vector, hA3A-BE-Y130F expression vector, hA3A-BE-Y132D expression vector and sgRNA expression vector. The sequence of the hA3A-BE expression vector is SEQ ID NO: 16; the sequence of the hA3A-BE-Y130F expression vector is SEQ ID NO: 17; the sequence of the hA3A-BE-Y132D expression vector is SEQ ID NO: 18.

[0155] The preparation and base editing method of the above-mentioned base editing system are as follows:

[0156] 3.1 Experimental materials

[0157] 3.1.1 Reagents and plasmids: Same as Example 1.

[0158] 3.1.2 Cell lines: Same as Example 1.

[0159] 3.2 Experimental method

[0160] 3.2.1 Construction of hA3A-BE expression vector: same as Example 1.

[0161] 3.2.2 Construction of hA3A-BE-Y130F expression vector

[0162] Using hA3A-BE as a template, PCR was performed ...

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Abstract

The invention provides a gene base editor, in particular relates to fusion protein. The fusion protein is characterized by comprising two fragments. The first fragment comprises APOBEC3A and the second fragment comprises CRISPR-related Cas protein. The base editor is capable of performing base edition in DNA and performing deamination on cytosine to be uracil, and the base editor still has higherediting efficiency even if cytosine is located at a GpC site or in a hypermethylated state.

Description

technical field [0001] The invention relates to a gene base editor. Background technique [0002] Genome editing technology refers to a genetic engineering technology that uses designable nucleases (molecular scissors) to modify specific segments of the genome DNA of an organism through base insertion, deletion or substitution to achieve editing of the target gene. Using genome editing technology to genetically manipulate cells can be widely used in basic life science research, biotechnology development, agricultural technology development, and pharmaceutical research and development. For example: directly correcting the genetic mutations that cause genetic diseases in the body will be able to treat genetic diseases fundamentally; carry out precise genetic engineering on crops to increase their yield or resist environmental pollution or pathogen infection; precise microbial genome transformation, thereby promoting the development of renewable bioenergy, etc. [0003] Since...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N9/22C12N15/10
CPCC12N9/22C12N9/78C12N15/102C12P19/30C07K2319/00
Inventor 陈佳杨力黄行许杨贝王潇李佳楠
Owner SHANGHAI TECH UNIV
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