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Base editing molecule and applications thereof

A base editing and gene editing technology, applied in the field of fusion proteins

Pending Publication Date: 2020-07-28
SHANGHAI TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the Cas9 protein mainly recognizes the PAM sequence in the guanine G/cytosine C rich region

Method used

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  • Base editing molecule and applications thereof
  • Base editing molecule and applications thereof
  • Base editing molecule and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Example 1 Human genome DYRK1A target uses a series of dCas12a-hA3A base editors to achieve efficient and high-precision C-T base editing

[0085] 1.1 Experimental materials

[0086] Reagents and plasmids: primers synthesized from Sangon Bioengineering (Shanghai) Co., Ltd.; restriction enzymes, DNA ligase, high-fidelity DNA polymerase Purchased from NEB Company; Plasmid Recombination Kit Clone Purchased from Vazyme Company; 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.

[0087] Cell line: Human fetal kidney cells HEK293T were cultured in DMEM medium (Gibco) supplemented with 10% fetal bovine serum (Gbico) and 1% double antibody (Gibco).

[0088] 1.2 Experimental method

[0089] Construction of dCas12a-hA3A-BE expression vector:

[0090] Using puc57-hA3A (synthesized by GenScript Biotechnology Co., Ltd.) as a template, using...

Embodiment 2

[0126] Example 2 The human genome SITE6 target uses a series of dCas12a-hA3A base editors to achieve efficient and high-precision C-T base editing

[0127] 2.1 Experimental materials

[0128] Reagents and plasmids: same as Example 1.

[0129] Cell line: same as Example 1.

[0130] 2.2 Experimental method

[0131] Construction of dCas12a-hA3A-BE expression vector: same as Example 1.

[0132] Construction of dCas12a-hA3A-BE-W98Y expression vector: same as Example 1.

[0133] Construction of dCas12a-hA3A-BE-W104A expression vector: same as Example 1.

[0134] Construction of dCas12a-hA3A-BE-P134Y expression vector: same as Example 1.

[0135] Construction of dCas12a-hA3A-BE-W98Y-W104A expression vector: same as Example 1.

[0136] Construction of dCas12a-hA3A-BE-W98Y-P134Y expression vector: same as Example 1.

[0137] Construction of dCas12a-hA3A-BE-W104A-P134Y expression vector: same as Example 1.

[0138] Construction of dCas12a-hA3A-BE-W98Y-W104A-Y130F expression vector...

Embodiment 3

[0156] Example 3 Human genome RUNX1 target uses a series of dCas12a-hA3A base editors to achieve efficient and high-precision C-T base editing

[0157] 3.1 Experimental materials

[0158] Reagents and plasmids: same as Example 1.

[0159] Cell line: same as Example 1.

[0160] 3.2 Experimental method

[0161] Construction of dCas12a-hA3A-BE expression vector: same as Example 1.

[0162] Construction of dCas12a-hA3A-BE-W98Y expression vector: same as Example 1.

[0163] Construction of dCas12a-hA3A-BE-W104A expression vector: same as Example 1.

[0164] Construction of dCas12a-hA3A-BE-P134Y expression vector: same as Example 1.

[0165] Construction of dCas12a-hA3A-BE-W98Y-W104A expression vector: same as Example 1.

[0166] Construction of dCas12a-hA3A-BE-W98Y-P134Y expression vector: same as Example 1.

[0167] Construction of dCas12a-hA3A-BE-W104A-P134Y expression vector: same as Example 1.

[0168] Construction of dCas12a-hA3A-BE-W98Y-W104A-Y130F expression vector: ...

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Abstract

The invention relates to the technical field of biology and especially relates to a fusion protein and applications thereof. The fusion protein includes a hA3A fragment, a dCas12a fragment and a UGI fragment. The problems that existing base editors cannot precisely and efficiently perform base editing at A/T enrichment areas can be overcome; a novel base editor can be developed by utilizing CRISPR/Cas protein Cas12a(Cpf1) capable of identifying A/T enrichment PAM sequences and human APOBEC3A (hA3A), so that high-efficiency and high-precision directed base editing can be realized at the A/T enrichment areas, and precise and efficient base editing can be realized at the more extensive loci in the genome of each species; and applications of the base editor can be greatly expanded, especiallyto the precise gene treatment of related diseases in the field of medical treatment.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a fusion protein and its application. 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 promoti...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/90C12N9/22C12N5/10
CPCC12N15/90C12N9/22C12Y305/04001C12N9/78C07K2319/00
Inventor 陈佳杨力黄行许杨贝王潇于文霞丁诚峰
Owner SHANGHAI TECH UNIV
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