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A kind of engineering bacterium containing ncas3 single-stranded endonuclease, preparation method and application

A technology of endonuclease and single-stranded nucleic acid, which is applied in the field of preparation of engineering bacteria containing nCas3 single-stranded endonuclease, can solve the problem of only reaching the editing efficiency, and achieve the effect of improving transformation efficiency and editing efficiency

Active Publication Date: 2022-03-01
武汉睿嘉康生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the article also pointed out that when using this system to knock out large fragments of genes, the editing efficiency only reached 50%.

Method used

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  • A kind of engineering bacterium containing ncas3 single-stranded endonuclease, preparation method and application
  • A kind of engineering bacterium containing ncas3 single-stranded endonuclease, preparation method and application
  • A kind of engineering bacterium containing ncas3 single-stranded endonuclease, preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Preparation and function of protein and its coding gene.

[0046] nCas3 single-stranded endonuclease refers to the introduction of mutations in the helicase functional domain of the wild-type Cas3 protein, making it lose the helicase activity, thereby transforming into nCas3 protein with only single-stranded nuclease activity. The endonuclease activity can act on a single strand of the target DNA duplex by endonucleating 1,4-phosphodiester bonds.

[0047] The amino acid sequence of the wild type (wide type) Cas3 protein involved in this embodiment of the present invention is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2. Three kinds of nCas3 single-stranded endonucleases with different sequences are specifically provided in this embodiment, named K458A respectively (the lysine at position 458 of the wild-type Cas3 protein is replaced with alanine, the amino acid sequence is the same as that of SEQ ID NO .1, the difference is only t...

Embodiment 2

[0070] Example 2 Using circular DNA as a substrate to verify protein function

[0071] The pL2R plasmid (Zheng et al, 2019) was selected as the reaction substrate, and the total length of the plasmid was 3283bp. The reaction system contains 150ng pL2R circular plasmid; 2mM MgCl2; 0.5mM ATP; 250nM Cascade protein; 250nM one of the above purified Cas3 or nCas3 protein variants; 32nt crRNA. The above crRNA was synthesized by GenScript Biotechnology Co., Ltd., and its sequence is shown in SEQ ID NO.5. After reacting at 30°C for 15, 30, and 60 minutes respectively, the reaction products were run on agarose gel electrophoresis.

[0072] The result is as figure 2 as shown, figure 2 The leftmost lane in the center represents nucleic acid molecular weight standards (5.0, 3.0, 2.0kb); the rightmost Reaction time axis represents the reaction duration (15, 30, 60min); the right OC, L, SC represent circular DNA molecules after the reaction state, [OC (open circle); L (linear); SC (neg...

Embodiment 3

[0073] Embodiment 3 comprises the preparation of the engineering bacterium of nCas3 single-stranded endonuclease

[0074] In this example, D608A was taken as an example to prepare engineering bacteria containing nCas3 single-stranded endonuclease.

[0075] 1. Design and construct single base editing plasmid

[0076] (1) According to the experimental requirements, it was decided to use the endogenous I-F type CRISPR-Cas editing system in the ZM4 strain to edit the target site on the genome.

[0077] like image 3 As shown, through careful analysis of the Cas3 protein coding gene on the genome of the ZM4 strain, it was found that there was a 5'-TCC-3' PAM sequence in the cas3 gene sequence, so the 32-nt sequence downstream of it was designed as a protospacer sequence . In order to prevent the CRISPR-Cas system from destroying the transferred donor plasmid, and to complete the in situ replacement of 676,661 adenine nucleotides to cytosine nucleotides in the ZMO0681 gene, a tot...

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Abstract

The invention belongs to the field of biotechnology, and in particular relates to an engineering bacterium containing nCas3 single-stranded endonuclease, a preparation method and an application. This application finds the functional structure division of its Cas3 protein by comparing the differences in the coding gene sequences of endogenous CRISPR-related proteins between the ZM4 strain and other strains that also carry the endogenous type I CRISPR-Cas system. And by exploring the working principle of the I‑F CRISPR‑Cas system, an amino acid in the helicase domain of the Cas3 protein in the ZM4 strain was designed and replaced. The adenine nucleotide number 676,661 in the ZMO0681 gene encoding the Cas3 protein on the original ZM4 strain was replaced with a cytosine nucleotide in situ, so that the Cas3 protein lost the helicase activity and transformed into a single-stranded DNA endonuclease The enzymatically active nCas3 protein was obtained to obtain the modified strain DRM2.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an engineering bacterium containing nCas3 single-stranded endonuclease, a preparation method and an application. Background technique [0002] CRISPR-Cas is a prokaryotic adaptive immune system widely present in most bacteria and archaea, which is used to prevent the invasion of exogenous genetic materials such as viruses or plasmids. [0003] The system is guided by RNA, and the genetic information stored in the CRISPR sequence will transcribe specific RNA, which will specifically bind to the site of the foreign invasion genetic material after combining with the Cas nuclease, causing a double-strand break and stimulating the host's repair mechanism , including homologous recombination repair (HDR) and non-homologous end joining (NHEJ). This immune repair mechanism retained by bacteria has now been developed as a gene editing tool commonly used in the life sciences. [...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/74C12N15/55C12R1/01
CPCC12N9/22C12N15/74
Inventor 彭文舫郝怡乐
Owner 武汉睿嘉康生物科技有限公司
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