Multi-target editing vector and its construction method and application

A construction method and multi-target technology, applied in the field of gene editing, can solve the problems of cumbersome experimental operation steps, limited gene editing efficiency, easy to produce off-target effects, etc., to expand the scope of editing, simplify experimental design steps, and reduce experimental costs Effect

Active Publication Date: 2022-02-01
GENEWIZ INC SZ
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] Therefore, the technical problem to be solved by the present invention is to overcome the high difficulty of gene editing, limited gene editing efficiency, easy off-target effects, and high experimental cost and experimental operation steps existing in the multi-target editing system for gene editing in the prior art. cumbersome problems; thereby providing a CRISPR / Cpfl multi-target editing vector that can significantly reduce the off-target effect of gene editing, improve gene editing efficiency and simplify experimental steps, and its construction method and application

Method used

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  • Multi-target editing vector and its construction method and application
  • Multi-target editing vector and its construction method and application
  • Multi-target editing vector and its construction method and application

Examples

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Embodiment 1

[0048] This embodiment provides a method for constructing a Cpf1 recombinant expression plasmid, wherein Cpf1 is codon-optimized AsCpf1 or LbCpf1, specifically comprising the following steps:

[0049] 1. Codon optimization

[0050] Cpf1 (AsCpf1 for short) derived from Acidaminococcus sp.BV3L6 was selected using codon optimization software (stored in Suzhou Jinweizhi Biotechnology Co., Ltd.), and human and mouse were selected as optimized species, according to the preference of human and mouse Codons, the codons in the original sequence of Cpf1 are replaced with the codon sites with different codon usage preferences from humans and mice, and the optimized AsCpf1 is obtained, and its nucleotide sequence is as shown in SEQ ID NO.1 shown.

[0051] Cpf1 (abbreviated as LbCpf1) derived from Lachnospiraceae bacterium ND2006 was optimized using the above codon optimization method to obtain an optimized sequence whose nucleotide sequence is shown in SEQ ID NO.2.

[0052] The optimize...

Embodiment 2

[0062] This embodiment provides a multi-target editing vector and its construction method. The core of the multi-target editing vector includes a Cpfl protein expression element and four sgRNA transcription elements, specifically including the following steps:

[0063] 1. Determine the target target and design the sgRNA sequence

[0064] Using the DNMT1, HPRT1, CCR5 and AAVS1 genes on the human genome as target genes, use the online design tool (http: / / rgenome.net) to find the PAM sites of Cpf1 on the coding and non-coding strands of the above four genes 5'-TTTN-3', and design the corresponding guide sequence (that is, sgRNA sequence) according to the PAM site, wherein the sgRNA sequence of DNMT1 is shown in SEQ ID.3, and the sgRNA sequence of HPRT1 is shown in SEQ ID.4, The sgRNA sequence of CCR5 is shown in SEQ ID.5, and the sgRNA sequence of AAVS1 is shown in SEQ ID.6.

[0065] 2. Design primers and synthesize targeted short fragments

[0066] Design paired primers for th...

Embodiment 3

[0084] This example provides a multi-target editing vector pEGFP-AsCpf1-ABCG1 and its construction method. The difference between pEGFP-AsCpf1-ABCG1 constructed in this example and pEGFP-AsCpf1-DNMT1-HPRT1-CCR5-AAVS1 constructed in Example 2 In this embodiment, three different sites on the ABCG1 gene are used as target sites to design sgRNA sequences respectively, wherein the sgRNA sequence of site 1 (ABCG1-g1) is shown in SEQ ID.7, site 2 ( The sgRNA sequence of ABCG1-g2) is shown in SEQ ID.8, and the sgRNA sequence of site 3 (ABCG1-g3) is shown in SEQ ID.9.

[0085] The difference between the construction method provided in this example and the construction method provided in Example 2 is that in this example, paired primers with different sgRNA sequences were designed for three different target sites, and the sequences of the paired primers are shown in Table 6.

[0086] Table 6 paired primers for different target sites sgRNA on the ABCG1 gene

[0087]

[0088]

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Abstract

The invention discloses a multi-target editing vector, which comprises a Cpfl protein expression element and at least two sgRNA transcription elements. The multi-target editing vector realizes the simultaneous editing of different target sites of the genome by co-expressing Cpfl protein and multiple sgRNAs, improves the efficiency of gene editing, reduces the off-target effect of gene editing, and improves the accuracy of gene editing. The present invention also discloses a method for constructing the above-mentioned multi-target editing vector. Through two-step Golden Gate cloning, multiple sgRNAs are serially connected to the second vector with Cpfl protein expression element and fluorescent expression element to obtain the above-mentioned Multi-target editing vectors. The above-mentioned construction method has simple steps, simplifies the experimental design process, and reduces the cost of gene editing. The invention also discloses a kit including the above-mentioned multi-target site carrier, which can be used for co-editing of multi-target sites with high efficiency and low off-target.

Description

technical field [0001] The invention belongs to the technical field of gene editing, and in particular relates to a multi-target editing vector and its construction method and application. Background technique [0002] Gene editing technology is a technology that can precisely modify the genome or transcripts, and can complete gene site-directed mutation, knockout or knockin of fragments, etc. In the post-genome era, gene editing technology has become an important research content in the field of life sciences. The traditional gene editing technology is based on embryonic stem cells and gene recombination for targeted modification of biological genomes, but this technology has disadvantages such as low targeting efficiency, long experimental cycle and narrow application range. With the continuous development of gene editing technology, artificial nuclease-mediated gene editing technology has begun to be widely used. This technology specifically recognizes and cleaves the ta...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N15/66C12N15/65C12N15/113C12N15/90
CPCC12N15/113C12N15/63C12N15/65C12N15/66C12N15/902C12N2310/20C12N2310/10
Inventor 栗凤鹏曹青吴昕廖国娟
Owner GENEWIZ INC SZ
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