Random nucleic acid editing composition and method

A composition and nucleic acid technology, applied in the field of molecular biology, can solve the problems of poor targeting specificity of Fn1 domain, off-target genomic DNA, low SGN editing efficiency, etc., and achieve the effect of improving gene editing efficiency and strong versatility

Active Publication Date: 2020-05-19
CHINA PHARM UNIV
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  • Abstract
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  • Claims
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Problems solved by technology

[0006] But the fly in the ointment is that the editing efficiency of the first-generation SGN is low, only 1-10% in zebrafish cells. Our research believes that the reason for the low efficiency of the first-generation SGN is that the target DNA needs to be combined with gDNA to form 3 Only after the 'flap structure can be combined by SGN, this two-st

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  • Random nucleic acid editing composition and method
  • Random nucleic acid editing composition and method
  • Random nucleic acid editing composition and method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1

[0049] In order to verify whether AfuFEN1 (the nucleotide sequence is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2) can successfully cut target DNA substrates with different sequences, we designed two sequences with completely different DNA substrates S1 and S2, the 5′ end of the target DNA substrate is labeled with fluorescent Cy5, and each substrate matches the corresponding common probe gDNA-1, gDNA-2, and the stem-loop structure hpDNA-1 (referred to as Hp-1) and hpDNA-2 (referred to as Hp-2). The reaction conditions were as follows: 5pmoL hpDNA, 5pmoL target nucleic acid substrate and Mix (10mmol / L MOPS pH 7.5, 0.05% Tween-20, 0.05% Nonidet P40) were thoroughly mixed, and the volume was made up to 10 μL with ultrapure water. Hybridize with the program of "95°C 5min, 55°C 10min", add AfuFEN1 and react at 37°C for 2h. Such as image 3 As shown, when hpDNA and AfuFEN1 exist at the same time, there are obvious cutting...

Embodiment 2

[0051] Embodiment 2

[0052] In order to prove that AfuFEN1 and hpDNA can form a complex independent of the target substrate, the 5' end of Hp-Cy5 was labeled with the fluorescent group Cy5, and incubated with an appropriate amount of AfuFEN1 at 37°C for 0.5h. The mixture was separated by 12% native electrophoresis and imaged by fluorescence. Figure 4 Electrophoretic mobility assay (EMSA) results in demonstrated that AfuFEN1 and Hp-Cy5 can form a complex in the absence of target DNA.

[0053]

Embodiment 3

[0054] Embodiment 3

[0055] We compared the efficiency of SGN+gDNA and AfuFEN1+hpDNA ( Figure 5 -A)). Such as Figure 5 As shown in -B, 1.78 and 0.89 pmol of SGN and gDNA-3 can only cut part of S3, while 0.45, 0.22, 0.11, 0.05 and 0.02 pmol of SGN and gDNA-3 hardly cut S3. At the same time, if Figure 5 As shown in -C, 1.78, 0.89, 0.45, 0.22 pmol of AfuFEN1 and Hp-3 cut S3 almost completely, while 0.11, 0.05 and 0.02 pmol of AfuFEN1 and Hp-3 could cut a part of S3. The results showed that compared with SGN+gDNA, the amount of nuclease required for AfuFEN1+hpDNA to cleave the target protein was reduced by about 40 times ( Figure 5 -D), demonstrating a significant increase in cleavage efficiency.

[0056]

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Abstract

The present invention discloses a random nucleic acid editing composition and method. The composition for targeted cleavage of random nucleic acids comprises two main components: a component A: an oligonucleotide probe consists of two parts, one part is complementary to a target substrate, and the other part has a secondary structure of nucleic acids; and a component B: an endonuclease molecule: the endonuclease molecule can recognize the secondary structure of the nucleic acids of the oligonucleotide probe to bind with the probe, and can also cleave a DNA or RNA substrate. The secondary hairpin structure (stem-loop structure) on the oligonucleotide probe can be directly recognized by the endonuclease molecule to form a complex molecule. Probability that the complex molecule collides withthe target substrate is greater than probability that two independently diffused molecules of the probe and the enzyme simultaneously collide with the target substrate, thereby improving gene editingefficiency.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and relates to a composition and method for arbitrary nucleic acid editing. Background technique [0002] The completion of the "Human Genome Project" has provided the possibility to elucidate genome functions and establish causal links between genetic variation and biological phenotypes, and an efficient, simple and precise gene editing system is an important tool to realize this possibility . Many gene editing systems for this purpose have been developed and applied, such as zinc finger ribonuclease (ZFN) system, transcription activator-like effector nuclease (TALEN) system and clustered regularly interspaced short palindromic repeat system (CRISPR-Cas ) system, etc. [0003] As an important gene editing tool, the CRISPR-Cas system has the characteristics of simple design and high cutting efficiency. It has been widely used in genome DNA editing of cells, animals and plants, and has...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N9/22C12N15/90
CPCC12N15/113C12N9/22C12N15/902
Inventor 徐澍周国华邹秉杰
Owner CHINA PHARM UNIV
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