Base editing system based on bimolecular deaminase complementation and application thereof

A technology of base editing and deaminase, applied in the directions of hydrolase, peptide, chemical instruments and methods, etc., can solve the problems of increasing safety hazards, uncertainty of clinical application, etc., and achieve the effect of high-efficiency target activity

Active Publication Date: 2022-07-01
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003]However, in recent years, a number of international independent studies in organisms such as rice and mice have found that traditional base editing systems (such as the BE3 system) will Genome-wide triggers a large number o

Method used

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  • Base editing system based on bimolecular deaminase complementation and application thereof
  • Base editing system based on bimolecular deaminase complementation and application thereof
  • Base editing system based on bimolecular deaminase complementation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Example 1 Evaluation of the on-target and off-target efficiencies of a bimolecular deaminase complementation-based cytosine base editing system in the monocot model plant rice.

[0072] 1. Experimental materials: The wild-type rice variety used in this example is japonica Zhonghua 11 (Oryza sativa L.ssp.japonica ZH11).

[0073] 2. Construction of base editing related rice protoplast transient expression vector

[0074] The expression vector pHBT-rAPOBEC1-nCas9-UGI of the base editor "N-BE3" (or called "BE3") was independently constructed by our laboratory. The promoter used is the maize ZmUbi-1 promoter and the terminator used is NOS terminator.

[0075]Cytosine base editor "Split-AID10" based on bimolecular deaminase complementation is composed of "Split-AID10-N" expression vector and "Split-AID10-C" expression vector; by Shanghai Sangon Biotechnology Co., Ltd. (SangonBiotech.Co., Ltd.) commercialized the synthesis of a polynucleotide sequence encoding a polypeptid...

Embodiment 2

[0116] Example 2 Efficient base editing was also achieved in transgenic Arabidopsis plants using the bimolecular deaminase complementation-based cytosine base editing system Split-AID10.

[0117] 1. Experimental materials

[0118] The wild-type Arabidopsis used in this example is the Col-0 ecotype (Arabidopsis thaliana Col-0).

[0119] 2. Arabidopsis genetic transformation

[0120] For the construction of the Arabidopsis genetic transformation vector used in this example, refer to the published article [15] In accordance with the method described in Example 1, respective genetic transformation vectors were constructed for two different targets (see Table 1 for guide RNA target sequence information). The aforementioned vectors were respectively transformed into Agrobacterium tumefaciens strain GV3101 by electroporation, and Arabidopsis plants were transformed by pollen tube introduction method. Specifically, the GV3101 bacterial solution containing the target vector was in...

Embodiment 3

[0125] Example 3 The cytosine base editing systems Split-AID10 and Split-BE3 based on bimolecular deaminase complementation also exhibited good on-target editing and very low genome-wide random off-targets in unicellular eukaryotic yeast.

[0126] 1. Experimental materials

[0127] Saccharomyces cerevisiae BY4741 was used in this example.

[0128] 2. Construction of base editing related yeast expression vector

[0129] Reference published articles for base editing related expression vector construction [16] , to obtain expression vectors pGAL1-rAPOBEC1-nCas9-UGI(N-BE3), pGAL1-Split-AID10 and pGAL1-Split-BE3. Wherein, the amino acid sequences of Split-AID10-N and Split-AID10-N contained in the pGAL1-Split-AID10 expression vector are as described above, and the amino acid sequence of Split-BE3-N contained in the pGAL1-Split-BE3 expression vector is as shown in SEQ ID NO: 8, the amino acid sequence of Split-BE3-C is shown in SEQ ID NO: 9. The yeast U6 promoter was directly ...

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Abstract

The invention relates to a base editing system based on bimolecular deaminase complementation and application thereof, and belongs to the technical field of gene engineering. The system mainly comprises nCas9 and nucleobase deaminase double complementary base editing fusion proteins A and B and guide RNA (Ribonucleic Acid). Compared with a traditional cytosine base editing system, the cytosine base editing system based on bimolecular deaminase complementation disclosed by the invention has the advantages that Cas9 dependent type and Cas9 independent type off-target on a genome is greatly reduced while the target efficiency in efficient cytosine base editing is maintained; the invention further provides an adenine base editing system based on bimolecular deaminase complementation, wherein the adenine base editing system can work efficiently. The cytosine and adenine base editing system based on bimolecular deaminase complementation is generally suitable for eukaryotes from fungi and animals to plants, and has wide application prospects in the fields of crop genetic breeding, animal variety improvement, clinical treatment of human genetic diseases and the like.

Description

technical field [0001] The invention relates to a base editing system based on bimolecular deaminase complementation and its application, and belongs to the technical field of genetic engineering. Background technique [0002] Numerous crop agronomic traits and human genetic diseases are determined by single nucleotide polymorphisms (SNPs) in the genome [1,2] . The base editing technology derived from the CRISPR / Cas9 gene editing technology has the characteristics of not generating DNA double-strand breaks and accurately realizing single-base substitution at the target site, which greatly improves the accuracy and efficiency of gene editing. Because of its safety, it is widely used in the fields of gene function research, crop genetic improvement and clinical treatment of human genetic diseases. [3] . Base editing systems are mainly composed of base editors (Baseeditors, BEs) and guide RNAs (single-guide RNA, sgRNA). The most commonly used base editor BE3 by researchers ...

Claims

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Application Information

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IPC IPC(8): C12N9/22C12N9/78C07K19/00C12N15/113
CPCC12N9/22C12N9/78C12N15/113C12Y305/04005C12Y305/04004C07K2319/00C07K2319/09C12N2310/20
Inventor 李剑峰贺雄雷熊翔宇刘科辉黎镇祥
Owner SUN YAT SEN UNIV
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