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Target polynucleotide editing method and application thereof

A technology for targeting polynucleotides and oligonucleotides, which is applied in genome editing methods and its applications, and in the field of targeted cutting of genomes, which can solve the problems of restricting the application of artificial endonucleases and the limitation of RGEN targets

Pending Publication Date: 2018-02-13
NANJING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the RGEN target is limited by the need to have a PAM sequence that can be recognized by Cas9 (Mojica FJ, etal. Microbiology 2009, 155:733-740.)
In summary, DNA sequence specificity limits the application of artificial endonucleases in DNA editing

Method used

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  • Target polynucleotide editing method and application thereof
  • Target polynucleotide editing method and application thereof
  • Target polynucleotide editing method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1 Construction and expression of recombinant structure recognition endonuclease

[0059] In the present invention, a recombinant structure recognition endonuclease (Structure-guided endonuclease, SGN) is constructed, which is composed of FEN-1 that recognizes the 3'-Flap structure and the Fok I (Fn1) cleavage domain that cuts the DNA chain . Wherein, the sequence is encoded by the following gene: Fok I (196 amino acid residues) at the C-terminal, glycine-serine repeat sequence at the middle link, and FEN-1 enzyme. This sequence was inserted into the prokaryotic expression vector pET28a(+) to form pET28a(+)-SGN. The coding sequence and amino acid sequence of SGN are shown in figure 1 , Figure 3-4 The plasmid map of pET28a(+)-SGN is shown in figure 2 shown in . In the pET28a(+)-SGN construct, the SGN gene is located downstream of the T7 promoter.

[0060] CaCl for pET28a(+)-SGN 2 Heat shock method for transformation into the host bacterial strain Arctic E...

Embodiment 2

[0064] Example 2 SGN cuts single-stranded DNA in vitro

[0065] To test whether the engineered SGN can cleave DNA strands, we incubated 1 ng SGN, 10 pmol substrate single-stranded DNA (ssDNA) (S-1), and 10 pmol gDNA-1 in a 10-μL reaction (all ssDNA and DNA The sequence of the oligonucleotide gDNA is shown in Table 1), and the 10-μL reaction system also included MOPS (10mM), 0.05% Tween-20, 0.01% nonidet P-40 and MgCl 2 (7.5mM). Among them, the 5' end of S-1 is labeled with a fluorescent Cy5 group. And before SGN was added, the mixture was first incubated at 95°C for 5 minutes and at 55°C for 10 minutes. Then add SGN and react at 37°C for 2 hours.

[0066] The gDNA-1 and S-1 form a 3'-Flap structure. SGN was added to the mixture for reaction, and then the mixture was separated by denaturing-PAGE and imaged by fluorescence. The specific steps are that the product obtained by the reaction is analyzed by PAGE under denaturing conditions. Loading buffer contained 90% formamid...

Embodiment 3

[0072] Example 3 SGN cleavage activity is not dependent on the target sequence

[0073] In order to prove whether SGN has DNA sequence preference, ssDNAs with different sequences (S-2, S-3, see Table 1 for sequence) were used as the substrate of SGN for reaction. The 10-μL reaction system and conditions are the same as those described above with S-1 as the substrate.

[0074] The results showed that, when guided by gDNA-2 or gDNA-3, SGN could cut S-2 or S-3 respectively (see Image 6 b and Image 6 Lane 5 of c). while only containing S plus SGN ( Image 6 b and Image 6 Lane 1 of c), S plus gDNA ( Image 6 b and Image 6 Lane 2 of c), S plus Fok I and gDNA ( Image 6 b and Image 6 Lane 3 of c), or S plus FEN-1 and gDNA ( Image 6 b and Image 6 No cleavage occurred in the reaction of lane 4) of c. This result indicates that SGN cleavage activity is independent of the target sequence but recognizes the 3'-Flap structure.

[0075] To demonstrate the importance of un...

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Abstract

The invention relates to a target polynucleotide editing method. The target polynucleotide editing method comprises the specific steps: designing a pair of DNA (Deoxyribonucleic Acid) oligonucleotidesingle chains aiming at a target gene and enabling the DNA oligonucleotide single chains to be complementary with a sense strand and an antisense strand of the target gene, so as to generate a structure which can be identified by nuclease with a target sequence identification structure and cutting activity; DNA of a target genome is cut through the nuclease to realize editing of the genome, wherein the structure which can be identified by the nuclease with the target sequence identification structure and the cutting activity is a 3'-Flap structure; a 3' tail end of each DNA oligonucleotide single chain is not complementary with the target gene; a recombinant structure is used for identifying endonuclease and contains a structure identification function region, a DNA cutting function region, a peptide section for connecting the two regions and a nuclear localization signal.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to a genome editing method and its application, in particular to a technology for targeted cutting of the genome. Background technique [0002] DNA editing is crucial in many molecular biology experiments in vitro or in vivo. Type II restriction endonucleases (REases) are indispensable tools in these experiments due to their delicate and precise cleavage of target nucleotides. So far, 3700 type II REases have been developed, but only 262 different nucleic acid sequences can be recognized by these enzymes ( S, et al. Proc Natl Acad Sci U S A 2007, 104:10358-10363.). Therefore, these limited sequences limit various demands in DNA editing operations. To overcome this limitation, several approaches have been developed as follows. The first method involves mutating the amino acid sequence of existing REases, such as the Not I mutant, and using protein structure informatics to help inc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/113
CPCC12N15/113C12N15/63C12N2310/10C12N2800/80
Inventor 周国华赵庆顺俆澍曹莎莎邹秉杰岳芸芸
Owner NANJING UNIV
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