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Engineering bacterium containing nCas3 single-stranded endonuclease, preparation method and application

A single-stranded nucleic acid, engineering bacteria technology, applied in the biological field, can solve the problem of only reaching the editing efficiency, and achieve the effect of improving the transformation efficiency and editing efficiency

Active Publication Date: 2021-10-22
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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  • Engineering bacterium containing nCas3 single-stranded endonuclease, preparation method and application
  • Engineering bacterium containing nCas3 single-stranded endonuclease, preparation method and application
  • 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] Such as 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 ...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to an engineering bacterium containing nCas3 single-stranded endonuclease as well as a preparation method and application of the engineering bacterium. The functional structure division of the Cas3 protein of the ZM4 strain is found by comparing the difference of the coding gene sequences of the ZM4 strain, other strains which also carry an endogenous I-type CRISPR-Cas system and endogenous CRISPR-related proteins. An amino acid in a Cas3 protein helicase structural domain in the ZM4 strain is designed and replaced by exploring the working principle of the I-F type CRISPR-Cas system. According to the method, adenine nucleotides 676 and 661 in ZMO0681 genes for coding Cas3 protein on an original ZM4 strain are replaced with cytosine nucleotides in situ, so that the Cas3 protein loses helicase activity and is converted into nCas3 protein only carrying single-stranded DNA endonuclease activity, and therefore, a modified strain DRM2 is obtained.

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 Applications(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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