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Co-editing marker ben-1sgRNA target site, CRISPR/Cas9 co-editing system and application of target site

A technology of ben-164sgrna and target sites, applied in the direction of DNA / RNA fragments, applications, recombinant DNA technology, etc., can solve problems such as inability to co-edit marker sites, achieve growth advantages, normal phenotypes, and save time Effect

Inactive Publication Date: 2019-10-08
福建上源生物科学技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The first purpose of the present invention is to find a new co-editing marker site, which can be conveniently used as a co-editing marker gene site for gene editing on the nematode II chromosome, so as to solve the problem that the dyp-10sgRNA co-editing marker site cannot be used as Issues with co-editing marker loci for gene editing on chromosome 2

Method used

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  • Co-editing marker ben-1sgRNA target site, CRISPR/Cas9 co-editing system and application of target site
  • Co-editing marker ben-1sgRNA target site, CRISPR/Cas9 co-editing system and application of target site
  • Co-editing marker ben-1sgRNA target site, CRISPR/Cas9 co-editing system and application of target site

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Finding effective ben-1 sgRNA target sites

[0049] The sgRNA site as a co-editing marker must itself be editable, and the editing efficiency is not lower than 10%. In order to find editable high-efficiency sgRNA sites in the ben-1 gene, the inventors selected 4 sgRNA target sites in the process of realizing the present invention, compared the editing efficiencies of these 4 sites, and searched for sites that can be regarded as co-editing marked site.

[0050] 1) Find the sgRNA site: download the coding sequence of ben-1 from the wormbase website, and check the sgRNA efficiency prediction website (such as http: / / crispor.tefor.net / ) to find specific sgRNA target sites with low off-target effects in the coding sequence of the first exon. In this example, the following 3 sgRNA sites were selected.

[0051] ben-1 29 sgRNA target sites:

[0052] ben-1 64 sgRNA target sites:

[0053] ben-1 112 sgRNA target sites:

[0054] The superscripted number i...

Embodiment 2

[0075] Example 2: Screening of F33A8.4 (Q82-, C83A) gene-edited nematodes by random screening method

[0076] Screening for F33A8.4(Q82-,C83A) gene editing by random screening

[0077] The purpose of F33A8.4 (Q82-, C83A) gene editing is to mutate the 82nd amino acid of the gene from glutamine to a stop codon, and to mutate the 83rd amino acid from cysteine ​​to alanine. Such as Figure 4 As shown, the codons corresponding to the 82nd and 83rd amino acids are marked with upper bars. After editing, a Hind III restriction site is generated as shown in the box. Random screening methods such as Figure 5 As shown, the specific screening steps are as follows:

[0078] 1) Select the sgRNA target site: search for the sgRNA target site near the target mutation site Q83 and C84 on the http: / / crispor.tefor.net / website, select the sgRNA site with high specificity and high efficiency, Find the following sgRNA target sites, namely:

[0079] F33A8.4-sgRNA target site: CAAGTTCCGCAGTGCT...

Embodiment 3

[0095] Embodiment three: ben-1 64 sgRNA co-editing for screening F33A8.4(Q82-,C83A) gene editing

[0096] use ben-1 64 sgRNA co-editing method to screen F33A8.4(Q82-,C83A) gene-edited nematodes.

[0097] Compared with the random screening method, using ben-1 64 The sgRNA co-editing system screens heterozygous or homozygous target editing gene mutant nematodes, the steps are as follows:

[0098] 1) Preparation of raw materials: The same raw materials as in Example 1 random screening method include: Cas9 plasmid, sgRNA plasmid that specifically recognizes the target mutation site of F33A8.4, reporter gene plasmid, repair template required to obtain the target mutation of F33A8.4, hermaphrodite Caenorhabditis elegans, common nematode culture plate. In addition, additional preparations are required: ben-1 64 sgRNA co-editing plasmids and nematode culture plates at a final concentration of 14 μM benomyl. ben-1 64 The sgRNA co-editing method for screening target editing metho...

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Abstract

The invention discloses a nematode CRISPR / Cas9 co-editing marker site based on a ben-1sgRNA target site, a co-editing system of the site and an application of the site, relates to the technical fieldof nematode gene editing, in particular to an efficient CRISPR / Cas9 co-editing system built in a nematode by the ben-1sgRNA target site and a method for selecting a target editing gene mutation nematode. The marker site has the advantages that (1) the site overcomes the shortcoming that co-editing and marking of genes on chromosomes II are unfavorably implemented by sgRNA target sites such as dyp-10; (2) the number of available co-editing marker sgRNA sites in the nematode CRISPR / Cas9 co-editing system is increased; (3) the ben-1sgRNA serves as a co-editing site, compared with a wild nematode,the edited nematode has more growth advantages and is normal in phenotype and conveniently selected, growth states cannot be affected, research is rapidly and efficiently completed, and time is saved; (4) by the aid of the ben-1sgRNA co-editing system, compared with random screening, screening efficiency is improved by 25 times or more, workload is reduced by 1 / 5, and screening time is greatly saved.

Description

technical field [0001] The present invention relates to the technical field of nematode gene editing, and more specifically relates to the establishment of an efficient CRISPR / Cas9 co-editing system in nematodes using the sgRNA target site of the ben-1 gene, and using this system to efficiently screen for heterozygous or pure The application method of the mutant nematode of the target gene. Background technique [0002] Caenorhabditis elegans (Caenorhabditis elegans, hereinafter referred to as nematode) is a kind of nematode that feeds on bacteria, lives in soil, is non-toxic and harmless, and can survive independently. Its characteristics are: (1) the number of cells is fixed, there are 959 cells in the whole body of hermaphroditic nematodes, and 1031 somatic cells in the whole body of male nematodes; (2) there are two sexes, hermaphroditic and male, and both hybridization and selfing can be carried out; ( 3) Genome sequencing has been completed; (4) the life cycle is shor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/113C12N15/85C12N15/90A01K67/033
CPCC12N15/113C12N15/902A01K67/0336C12N2310/10A01K2227/703C07K14/4354C12N15/8509C12N2310/20A01K2207/15A01K2217/075
Inventor 赵培康雅虹黄河
Owner 福建上源生物科学技术有限公司
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