Method for improving efficiency of site-directed insertion of genome by using small molecular compound

A small molecule compound, genome-targeted technology, applied in the field of genetic engineering, can solve problems such as low efficiency of HDR-mediated site-directed insertion

Active Publication Date: 2019-12-03
WENS FOOD GRP CO LTD
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the efficiency of DSB generation by CRISPR / Cas9 has been guaranteed to a certain extent, the efficiency of site-directed insertion mediated by HDR is still very low, so it is urgent to find ways to improve the efficiency of site-specific insertion in the genome

Method used

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  • Method for improving efficiency of site-directed insertion of genome by using small molecular compound
  • Method for improving efficiency of site-directed insertion of genome by using small molecular compound
  • Method for improving efficiency of site-directed insertion of genome by using small molecular compound

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Example 1: Screening of Small Molecular Compounds for Improving Genomic Site-Directed Insertion Efficiency ( figure 1 )

[0026] 1PX330-ACTB plasmid construction:

[0027] According to the gene sequence of ACTB (ENSG00000075624) provided by NCBI, a gRNA (https: / / crispr.cos.uni-heidelberg.de / index.html) was designed near its sixth exon, which is the stop codon. The primer sequences are as follows Shown: forward (F) SEQ ID No: 1: 5'-CACCCGTCCACCGCAAATGCTTCT-3'; reverse (R) SEQ ID No: 2: 5'-AAACAGAAGCATTTGCGGTGGACG-3'. After the primers were synthesized, the concentration was diluted to 10 μM, and 5 μl was mixed gently for annealing reaction. The reaction process was as follows: 95°C, 3min→10°C, 1min→95°C, 3min→10°C, 1min→95°C, 3min→10°C , 1min→95℃, 3min→4℃, 10min.

[0028] Plasmid PX330 (Addgene, #42230) was single-digested according to the instructions of ThermoFisher Company FastDigest BpiI (#FD1014), and the enzyme digestion reaction system is shown in Table 1:

[...

Embodiment 2

[0052] Example 2: Using Small Molecular Compounds to Improve the Efficiency of Genomic Site-directed Insertion

[0053] For the specific steps of plasmid construction and cell culture in the example, refer to Steps 1-3 of Example 1.

[0054] 4 Verification that small molecule compounds improve the efficiency of genome-directed insertion

[0055] At the same time, the optimal concentration of two candidate small molecule compounds for site-directed insertion efficiency in HEK293T cell lines was further verified, and two non-cell cycle / DNA damage compounds: Albendazole and Tedizolid. When the confluence of the cells in step 3 reaches 80-90%, co-transfect the PX330-ACTB and hACTB-T2A-GFP plasmids into HEK293T cells using Lipofectamine 3000 Reagent (#L3000015) from Shanghai Yingweijieji, the process is as follows : Prepare two 1.5mL centrifuge tubes, add 500μl Opti-MEM, 43.4μl Lipofectamine 3000 to one tube and mix well, add 14μg each of the two plasmids, 500μl Opti-MEM, 56μl P30...

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Abstract

The invention relates to the technical field of genetic engineering, and in particular to a method for improving efficiency of site-directed insertion of a genome by using a small molecular compound.The method is achieved by co-acting of the small molecular compound with a CRISPR / Cas9 system on a cell. Specifically, the CRISPR / Cas9 system comprises a gRNA fragment of a target gene, can identify the target gene and cause a double-strand break of the target gene, and at the break, the small molecular compound plays a role in performing homologous recombination repair to efficiently complete site-directed insertion of the genome. The efficiency of site-directed insertion of the genome is significantly improved in the cells treated with the small molecular compound.

Description

technical field [0001] The invention relates to the technical field of genetic engineering. Specifically, the present invention relates to a method for improving the efficiency of genome site-directed insertion by using small molecule compounds. Background technique [0002] With the completion of genome sequencing of more and more species, exploring genome functions or performing targeted transformation of genomes has become one of the focuses of scientific research. The traditional gene manipulation technology is to randomly insert the target gene into the genome. This random insertion method will bring great difficulties to the later study of gene-edited animal or human disease models. The genome-specific modification technology can realize the modification of specific genes. , has important application value in the study of gene function or breeding animal groups with specific traits, as well as the preparation of animal models of human diseases and the development of t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/90C12N9/22
CPCC12N9/22C12N15/907
Inventor 张献伟王豪强李国玲刘德武黄广燕李紫聪蔡更元吴珍芳杨化强
Owner WENS FOOD GRP CO LTD
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