Efficient DNA editing method
A DNA sequence and editing technology, applied in the field of efficient DNA editing, can solve problems such as inability to cut, and achieve the effect of improving the ability of mismatched bases
Inactive Publication Date: 2018-07-24
李燕强
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Problems solved by technology
First of all, the cutting of the target sequence by the Cas9 protein does not only rely on the matching of the crRNA sequence, there must be some small prospacer sequence adjacent motifs (PAM) near the target sequence, if there is no PAM around the target sequence or cannot be strictly matched, then Cas9 protein cannot cut arbitrary sequences
Finally, like ZFN and TALEN technologies, CRISPR / Cas9 also faces the problem of how to control non-homologous end-joining repair after double-strand breaks, which may randomly produce cytotoxicity
Method used
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Embodiment 1
[0018] An efficient DNA editing method, knock out the co-GFAP gene containing the co-GFAP plasmid in 293T cells. Specifically through the following methods:
[0019] 1) According to the sequence of SEQ ID NO.1, express and purify the fusion protein in prokaryote;
[0020] 2) Add the oligonucleotides of Table 1 to the above purified protein. The mixture is then added to the cultured cells.
[0021] 3) The co-GFAP gene can be knocked out by incubating for 72 hours again.
[0022] Table 1 Oligonucleotide sequence
[0023]
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The invention discloses an efficient DNA editing method, T7 endonuclease I is connected to a DNA binding structural domain by a genetic engineering method, and when a foreign DNA containing a specificrecognition characteristic of the DNA binding structural domain is bound to a target DNA to be edited, the endoribonuclease of the T7 endonuclease I of a fusion protein actively recognizes a base mismatch site, and cuts the target DNA to be edited. The method realizes the specific and efficient editing of the target DNA to be edited, can be used for changing the sequence of the DNA in vitro and in vivo, and is simple and practical.
Description
Technical field [0001] The invention belongs to the technical field of genetic engineering, and relates to a method for high-efficiency DNA editing, specifically a method for sequence modification of target DNA based on T7 endonuclease I combined with a DNA binding domain. Background technique [0002] DNA editing technology refers to the ability to allow humans to modify the sequence of target DNA to achieve knockout and addition of specific DNA fragments. [0003] In the past few years, the sequence-specific nuclease technology represented by ZFN (zinc-finger nucleases) and TALEN (transcriptionactivator-like effector nucleases) has been able to efficiently perform targeted genome editing, and is used in basic research, gene therapy and Genetic improvement has shown great potential. Since the advent of CRISPR / Cas9 technology, it has had unparalleled advantages over other gene editing technologies. After continuous improvement of the technology, it is believed to be able to "edit"...
Claims
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IPC IPC(8): C12N15/63
CPCC12N15/63C12N2800/80
Inventor 李燕强
Owner 李燕强
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