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Construction and application of high-flux detection system for nuclease induced insertion and deletions (Indels)

A nuclease, a technology to be detected, applied in the field of genetic engineering, which can solve problems such as destroying the function of proteins

Pending Publication Date: 2019-10-08
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The introduction of Indels mediated by NHEJ may show extremely high diversity and uncertainty due to differences in artificial nuclease systems, cell types, and gene site selection. When the number of bases deleted or inserted is a multiple of 3, No frame-shifted mutations disrupt protein function

Method used

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  • Construction and application of high-flux detection system for nuclease induced insertion and deletions (Indels)
  • Construction and application of high-flux detection system for nuclease induced insertion and deletions (Indels)
  • Construction and application of high-flux detection system for nuclease induced insertion and deletions (Indels)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: Detection of Indels mutations on the genome of 293T cells after CRISPR / Cas9 action --- different target gene loci (Indels mutation efficiency > 20%)

[0052] 1. Construct a CRISPR / Cas9 system specifically targeting EMX1, HPRT1, and CCR5 genes and transfect 293T cells (human embryonic kidney cells). The sequences of each target gene are shown in Table 2.

[0053] Table 2 Comparison table of target gene sequences

[0054]

[0055] Specifically include the following steps:

[0056] (1) The sgRNA sequence and gene target sequence amplified by Annealing PCR were cloned into the sgRNA-Cas9 expression vector and the NHEJ-RPG reporter vector (the carrier owned by the applicant's laboratory, The specific construction process will not be described in detail), and the sgRNA.EMX1-Cas9 expression vector, NHEJ-RPG.EMX1 reporter vector, sgRNA.HPRT1-Cas9 expression vector, NHEJ-RPG.HPRT1 reporter vector, sgRNA.CCR5-Cas9 expression vector and NHEJ -RPG.CCR5 reporter vec...

Embodiment 2

[0086] Example 2: Indels mutation detection for different target gene loci of unscreened 293T cells

[0087] 1. Construct and obtain 293T cell lines specifically targeting EMX1, HPRT1, and CCR5 genes, and only use a single sgRNA.EMX1-Cas9, sgRNA.HPRT1-Cas9, and sgRNA.CCR5-Cas9 expression vectors to achieve different target gene positions For point genome editing, the cells were harvested 5 to 7 days later and the cell genome was extracted. The subsequent detection methods and steps were the same as in Example 1.

[0088] 2. Indels frequency statistical results are shown in Table 10. Figure 7 shown.

[0089] Table 10 CRISPResso2 analysis and ara-FabI system detection Indels frequency statistics

[0090] Indels frequency (%) CCR5 EMX1 HPRT1 NGS 4.51 15.73 17.96 ara-mFabI 9.11 10.94 25.63

[0091] It can be seen from the above results that the system constructed by the present invention can not only effectively reveal the higher frequency (...

Embodiment 3

[0092] Example 3: Detection of Indels mutations at the CCR5 gene locus on different cell lines

[0093] 1. Construct 293T, A375 (human melanoma cells) and U2OS (human osteosarcoma cells) specifically targeting the CCR5 gene, and transfect the sgRNA.CCR5-Cas9 expression vector to achieve the same target in different cell lines For genome editing of gene loci, the cells were harvested 5 to 7 days later and the cell genome was extracted. The subsequent detection methods and steps were the same as in Example 1.

[0094] 2. Indels frequency statistical results are shown in Table 11. Figure 8 shown.

[0095] Table 11 CRISPResso2 analysis and ara-FabI system detection Indels frequency statistics

[0096] Indels frequency (%) 293T A375 U2OS NGS 4.51 1.29 0.62 ara-mFabI 9.11 1.08 0.4

[0097] It can be seen from the results that the constructed system has a certain revealing effect even for <1% of target gene Indels mutations, and maintains a high ...

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Abstract

The invention belongs to the field of gene engineering and particularly relates to construction and application of a mutation detection system for detecting artificial nuclease induced insertion and deletions (Indels). According to the adopted specific technical scheme, the ara-mFabI detection system is provided and comprises three detection carriers, and each carrier is separately responsible forquantitative and qualitative reveal of one type of insert fragment. The three carriers are distinguished in that the types of open reading frames (ORFs) of mFabI genes are different; and nine basic groups, ten basic groups and eleven basic groups are introduced through upstream primers of amplification primers of the mFabI genes correspondingly so as to change the ORFs of the mFabI genes. By using the detection system, quantitative reveal of the mutation frequency of all the types of nuclease induced Indels can be realized; and the mutation frequency of the Indels close to amplicon sequencingand type distribution can be realized.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a system for detecting mutations of Indels induced by artificial nucleases, a construction method and an application. Background technique [0002] Nuclease-dependent gene editing tools are rapidly affecting and changing the genomes of cells and organisms with extremely high precision and high throughput, and have broad application prospects in the field of genome editing. In particular, the editing system based on type II sgRNA-guided CRISPR / Cas9 nuclease (clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated protein-9 nuclease (Cas9)) has been favored for its simple design and efficient function. Used a lot. Artificial nucleases ZFNs, TALENs, CRISPR / Cas, etc. can induce DNA double-strand breaks (double strand breaks, DSBs) at specific target gene loci, and are usually repaired by non-homologous end joining (non-homologous end-joinin...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/66C12Q1/10C12Q1/06C12R1/19
CPCC12N15/70C12N15/66C12Q1/10C12Q1/06G01N2333/245
Inventor 张智英程心珍邢佳妮闫娜娜马锦荣
Owner NORTHWEST A & F UNIV
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