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Gene editing technique taking Argonaute nuclease as core

A gene editing and nuclease technology, applied in the field of targeted gene editing technology, can solve the problems of low system efficiency and off-target, and achieve the effects of controllable dose, simple transfection of cells, and simple design

Inactive Publication Date: 2016-04-13
ZHEJIANG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the Cas9 system is inefficient for GC-rich gene sequences because RNA easily forms secondary structures
Moreover, because the cells themselves produce a large number of RNA fragments, Cas9 has the possibility of binding to these non-specific RNAs and being misguided to cut non-target genomic sites; and Cas9 has a higher tolerance to mismatches between guide RNA and target DNA sequences ( 5 nucleotide mismatches do not prevent cleavage), so Cas9 has some off-target defects

Method used

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  • Gene editing technique taking Argonaute nuclease as core
  • Gene editing technique taking Argonaute nuclease as core
  • Gene editing technique taking Argonaute nuclease as core

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: DNA plasmid endolysis in vitro

[0030] 1. The complete reading frame of Argonaute (NgAgo for short) obtained from Natronobacterium gregoryi SP2 bacteria is cloned on the pcDNA3.1 plasmid, and its 5' end and 3' end are accompanied by FLAG and HA tag sequences respectively. The amino acid sequence of NgAgo is shown in SEQ ID NO:1.

[0031] 2. synthesize a section of 5' phosphorylated guide DNA 5'P-TGCTTCAGCCGCTACCCCGACCAC-3', which is complementary to a section of the GFP gene on the pACYCDuet-GFP targeting plasmid ( Figure 1a ).

[0032] 3. Co-transfect the constructed FLAG-NgAgo-HA-pcDNA3.1 plasmid and the guide fragment in step 2 above into human 293T cells.

[0033] 4. Use FLAG, HATandemAffinityPurificationKit Kit (Sigma-Aldrich Company) to purify the FLAG-NgAgo-HA protein bound to the guide DNA from the transfected cells.

[0034] 5.5 μg of purified NgAgo protein combined with guide DNA and 400ngpACYCDuet-GFP plasmid were placed in a 50ul reaction syst...

Embodiment 2

[0037] Example 2: Argonaute-mediated plasmid cleavage in mammalian cells and comparison with CRISPR / Cas9 system-mediated cleavage efficiency

[0038] 1. A nuclear localization signal (NLS) is added to the N-terminus of the NgAgo protein, and the NLS-NgAgo gene sequence of the improved protein is constructed on the pcDNA3.1 plasmid. NLS-NgAgo-pcDNA3.1 mainly distributed in the nucleus after expressed in Hela cells.

[0039] 2. Hela cells were co-transfected with EGFP-N1 target plasmid and NLS-NgAgo-pcDNA3.1 expression plasmid. In addition, the cells were also transfected with 4 guide DNAs (G1-G4) for the target plasmid CMV promoter region and GFP coding region ( Figure 2a ). Transfection conditions: 200ngNLS-NgAgo-pcDNA3.1 plasmid and 100ng guide DNA were transfected into 2×10 5 Intracellular (in 24-well plate).

[0040] 3. Another group of cells were co-transfected with the EGFP-N1 target plasmid, the Cas9 expression plasmid SpCas9-pCDNA3.1, and the guide RNA (sgRNA1) tar...

Embodiment 3

[0050]Example 3: Argonaute-mediated cleavage of GC-rich sequences in the genome and comparison with CRISPR / Cas9 system-mediated cleavage efficiency in mammalian cells

[0051] 1.293T cells were transfected with the NLS-NgAgo expression plasmid and the guide DNA for the GC-rich region in the HBA2 gene and GATA4 gene ( Figure 3a ).

[0052] 2. As a comparison, some 293T cells were transfected with Cas9 expression plasmid and guide RNA targeting the same region.

[0053] 3. Because the double-strand breaks in the mammalian genome are mainly repaired by the cell's spontaneous indel-forming non-homologous endjoining (NHEJ) pathway, we use the T7 endonuclease I (T7EI) detection method to detect the cleavage efficiency of the genomic target sequence. In each T7EI reaction, 500 ng of PCR product was reacted with T7 endonuclease I, which cleaves unpaired DNA sequences. The products were detected by denaturing PAGE electrophoresis and silver staining. Band density was analyzed by Im...

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Abstract

The invention discloses a gene editing technique taking Argonaute nuclease as a core. In the presence of guide DNA (Deoxyribose Nucleic Acid), Argonaute endonuclease can cut any locus of a targeted DNA sequence, including a genome of a mammal to cause breakage of DNA double chains, thereby fulfilling an editing aim. Editing means include incision, deletion, mutagenesis induction, exogenous sequence insertion, fragment substitution and the like. Editing effects include gene inactivation, gene mutation, exogenous gene induction and the like. The protein can serve as an important tool for genome editing. A gene editing tool taking the protein as the core has the characteristics of easiness in operation, high efficiency, low target missing rate and high fidelity. Moreover, almost any genome locus can be effectively targeted and cut by the Argonaute nuclease. By using the technique, high-specificity and high-efficiency genome targeted editing is helpfully realized.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a simple, efficient, precise targeted gene editing technology with Argonaute endonuclease as the core. Background technique [0002] In-depth understanding of genetic information and gene therapy require precise and efficient genome editing tools. Gene or genome editing is a genetic engineering technique. This technology uses nucleases to mutate, insert, and replace specific sites in the genome. The process of gene editing includes three steps: first, a specially designed guide DNA or RNA (gene guidance element) is combined with an endonuclease (gene cutting element), and the former guides the latter to a specific region of the targeted gene; second, the nucleic acid The endonuclease cuts a specific region of the targeted gene, forming a gap on each of the two DNAs, causing a double-strand break (DSB); finally, using the phenomenon that the DSB activates the inherent repair mechani...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/102
Inventor 沈啸韩春雨
Owner ZHEJIANG UNIV
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