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Method for improving site-specific modification efficiency of cellular genome by utilizing modified ssODN

A genome-targeted and efficient technology, applied in the field of genetic engineering, can solve problems such as low efficiency of fixed-site modification

Active Publication Date: 2019-12-13
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • 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 modification mediated by HDR is still very low, so it is urgent to find new ways to improve the efficiency of genome site-directed modification

Method used

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  • Method for improving site-specific modification efficiency of cellular genome by utilizing modified ssODN
  • Method for improving site-specific modification efficiency of cellular genome by utilizing modified ssODN
  • Method for improving site-specific modification efficiency of cellular genome by utilizing modified ssODN

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Method for Improving the Efficiency of Site-directed Modification of Human Cell Genome

[0026] In this example, human-derived cells (293T) were used as a tool cell line for experiments.

[0027] 1. Plasmid construction

[0028] Use the online website (https: / / crispr.cos.uni-heidelberg.de / index.html) to design the pig ROSA26 gene gRNA sequence (the gene sequence is shown in SEQ ID No:1 and SEQ ID No:2), by Hua Big Genomics is artificially synthesizing. After the synthetic primers were annealed, they were ligated with the linearized PX330 plasmid after digestion with BpiI, and the successfully constructed plasmid after ligation was named PX330-ROSA26 (the gene sequence of the plasmid is shown in SEQ ID No: 3). Simultaneously designed and synthesized ssODN-GFP reporter carrier by Nanjing GenScript Biotechnology Co., Ltd. (the gene sequence of the reporter carrier is shown in SEQ ID No: 4): a sequence homologous to the ROSA26 gene is inserted in the middle of ...

Embodiment 2

[0039] Example 2: Method for Improving the Efficiency of Site-directed Modification of Mouse Cell Genome

[0040] In this example, hamster kidney cells (BHK cells) were used as a tool cell line for experiments.

[0041] Wherein, the specific implementation steps of plasmid construction, cell recovery and culture, and tool cell line screening in the embodiment refer to steps 1-3 in the first embodiment.

[0042] 4. Results of co-transfection of PX330-ROSA26 plasmid and ssODN donor in hamster kidney cells

[0043] Transfection was started when the hamster kidney cells in the 24-well plate grew to 60%-80%. For the transfection process, refer to Thermofisher’s Instructions for LTX&Plus Reagent protocol, each well was transfected with 1 μg / well PX330-ROSA26 plasmid and 400ng ssODN. Among them, the transfected ssODNs were the unmodified blank group and the experimental group modified by BIO, PHO, THS, THO and AMN, respectively. After 4-6 hours, replace with fresh culture medium...

Embodiment 3

[0045] Example 3: Method for Improving the Efficiency of Site-directed Modification of Porcine Cell Genome

[0046] In this example, pig-derived cells (PK-15 / PEF cell line) were used as a tool cell line for experiments.

[0047] Wherein, the specific implementation steps of plasmid construction, cell recovery and culture, and tool cell line screening in the embodiment refer to steps 1-3 in the first embodiment.

[0048] 4. Pig-derived cells co-transfected with PX330-ROSA26 plasmid and ssODN donor results

[0049] Transfection starts when the pig-derived cells (PK-15 / PEF cell line) in the 24-well plate grow to 60%-80%. The transfection process refers to Thermo fisher's Instructions for LTX&Plus Reagent protocol, each well was transfected with 1 μg / well PX330-ROSA26 plasmid and 400ng ssODN. Among them, the transfected ssODNs were the unmodified blank group and the experimental group modified by BIO, PHO, THS, THO and AMN, respectively. After 4-6 hours, replace with fresh cu...

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Abstract

The invention discloses a method for improving the site-specific modification efficiency of a cellular genome by utilizing modified ssODN. The method is realized through combined action of a CRISPR / Cas9 system and modified ssODN on cells. Particularly, the CRISPR / Cas9 system comprises a target gene gRNA fragment, the target gene can be subjected to site-specific recognition, the target gene is subjected to double-strand breakage, the ssODN and a target gene base are subjected to complementary pairing at a breakage position so as to efficiently complete the genome site-specific modification, and the modified ssODN can significantly improve the site-specific modification efficiency.

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 site-directed modification of cell genome by using modified ssODN. Background technique [0002] At present, the most widely used gene editing tools mainly include zinc finger endonuclease (Zinc-finger endonuclease, ZFN), transcription activator-like effector nuclease (Transcription activator-like effector nuclease, TALEN), regularly clustered interval short palindrome Repeat (Clustered regularly interspaced short palindromic repeats, CRISPR), in which the CRISPR / Cas9 system consists of an exogenous single-stranded guide RNA (sgRNA) and Cas9 protein. The sgRNA recognizes the genomic sequence through the principle of complementary base pairing and guides the Cas9 protein to bind to the target Genomic double strand break (Double strand break, DSB). At the same time, cells activate two different rep...

Claims

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Application Information

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IPC IPC(8): C12N15/90C12N9/22C12N15/65
CPCC12N9/22C12N15/65C12N15/907
Inventor 吴珍芳李国玲张献伟王豪强李紫聪蔡更元刘德武杨化强
Owner SOUTH CHINA AGRI UNIV
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