Gene editing application of prokaryotic Argonaute protein

Abstract

The invention discloses a gene editing application of a prokaryotic Argonaute protein, which comprises the following steps: constructing a prokaryotic expression vector of the Argonaute protein derived from bacteria, detecting the degradation of the expression vector and a co-transformation vector of the Argonaute protein after transforming a host cell, and detecting genome sites. It is found that the gene has the ability of being activated by a specific transcript and targeting a specific DNA target sequence, and gene editing with the inDel frequency of 3% is carried out on a genome target sequence under the participation of guide RNA transcribed in eukaryotic cells. Experimental results show that the prokaryotic Argonaute protein can be used for developing intracellular gene editing tools.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and relates to the nuclease characteristic of prokaryotic Argonaute (pAgo) protein and the development of an intracellular gene editing tool realized therethrough. Background technique [0002] Gene editing tools commonly used in gene editing technologies include ZFNs (Kim, Skowron, and zybalski 1996; Bitinaite et al. 1998) and TALENs (Cermak et al. 2011), as well as CRISPR / Cas9 (Bikard et al. 2012). CRISPR / Cas9 technology uses artificial nucleases to introduce double strand breaks (Double strand breaks, DSBs) at the target position of genomic DNA, using the cell's own repair pathways, such as non-homologous end joining (NHEJ), And homologous recombination (homologous recombination, HR) mediated by homologous sequences to realize the modification of the target gene sequence, which can more accurately modify the specific target gene locus of the organism genome. However, CRISPR / Cas9 t...

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Problems solved by technology

[0007] However, most of the published pAgos are derived from thermophilic bacteria, and the reaction temperature in the extracellular cleavage experiment is higher than 65°C, even reaching 85°C or 98°C (Swarts et al.2014; Swarts et al.2015; Zander et al. 2017), under such temperature conditions, the double-strand DNA is in an unwinding state, and the experimental results cannot fully reflect its effect on double-stranded DNA

Extracellular cleavage experiments on thermophile-derived Argonaute proteins at room temperature or low temperature have demonstrated that they cannot meet the conditions to be engineered for genome editing in mammalian cells, for example, showing low activity of cleavage and lack of targeting sex

And the prokaryotic Argonaute protein of other sources reported at present, for example, the Argonaute (CbAgo) protein derived from Clostridium butyricum and the Argonaute (NgAgo) protein derived from Natronobacterium gregoryi, which Targeted DNA cleavage in cells was not obtained under more representative physiological conditions (e.g., 37°C)

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