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Method for remarkably improving fish genome editing efficiency

A genome editing and genome technology, applied in the biological field, can solve the problem of unequal distribution of injected nucleic acid and other problems, and achieve the effect of reducing invalid F1 generation individuals and reducing time.

Active Publication Date: 2014-12-10
NANJING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, researchers in this field have not yet recognized the unequal distribution of injected nucleic acids in embryos and taken corresponding measures to improve the passage efficiency of genome editing

Method used

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  • Method for remarkably improving fish genome editing efficiency
  • Method for remarkably improving fish genome editing efficiency
  • Method for remarkably improving fish genome editing efficiency

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Directed knock-in of mloxP in the intron of zebrafish aldh1a2 gene

[0037] 1. Construction of a CRISPR / Cas9 system targeting the third and fourth introns of the zebrafish aldh1a2 gene

[0038] Table 1. Primer and template sequences used to construct sgRNA targeting aldh1a2

[0039]

[0040] Use primers ali3gRF11 (Seq ID No: 1) and sgRNAR (Seq ID No: 3) to amplify the plasmid template containing the sgRNA backbone, and the PCR conditions are: 95°C for 2 minutes, 35 cycles (94°C for 30 seconds, 56°C for 30 seconds , 72°C for 30 seconds), and a final 72°C extension for 5 minutes. The same method was used to amplify the plasmid template containing the sgRNA backbone (Seq ID No: 4) with primers ali4gRF5 (Seq ID No: 2) and sgRNAR (Seq ID No: 3). The above PCR products were transcribed into RNA with MEGAscript Kit (Ambion, USA), and the products were ali3gR11 and ali4gR5, respectively. The total RNA of zebrafish 24hpf embryos was extracted with TRIzol reagent,...

Embodiment 2

[0060] Example 2 Introducing an insertion-deletion mutation in the intron of the zebrafish aldh1a2 gene

[0061] 1. Construction of a CRISPR / Cas9 system targeting the third and fourth introns of the zebrafish aldh1a2 gene

[0062] Table 1. Primer and template sequences used to construct sgRNA targeting aldh1a2

[0063]

[0064] Use primers ali3gRF11 (Seq ID No: 1) and sgRNAR (Seq ID No: 3) to amplify the plasmid template containing the sgRNA backbone, and the PCR conditions are: 95°C for 2 minutes, 35 cycles (94°C for 30 seconds, 56°C for 30 seconds , 72°C for 30 seconds), and a final 72°C extension for 5 minutes. The same method was used to amplify the plasmid template containing the sgRNA backbone (Seq ID No: 4) with primers ali4gRF5 (Seq ID No: 2) and sgRNAR (Seq ID No: 3). The above PCR products were transcribed into RNA with MEGAscript Kit (Ambion, USA), and the products were ali3gR11 and ali4gR5, respectively. The total RNA of zebrafish 24hpf embryos was extracted ...

Embodiment 3

[0079] Embodiment 3 introduces insertion-deletion mutations in the yellow catfish mstna gene and mstnb gene

[0080]As two independent genome editing tools, zinc finger nuclease and transcription activator-like effector nuclease can respectively complete the recognition and cutting of specific sequences. In the experiment, you can choose to edit the genome separately or together according to the needs of the experiment. In order to make the present invention concise, in this embodiment, the zinc finger nuclease mRNA that specifically recognizes and cleaves the specified site sequence of the mstna gene, and the transcription activator-like effector nuclease mRNA that specifically recognizes and cleaves the specified site sequence of the mstnb gene Co-injection into yellow catfish embryos to carry out the present invention, but use the zinc finger nuclease mRNA that specifically recognizes and cuts the sequence of the designated site of the mstna gene or the transcription activa...

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Abstract

The invention discloses a method for remarkably improving fish genome editing efficiency. The method for remarkably improving fish genome editing efficiency comprises the following steps: designing a genome editing tool for specifically identifying and cutting a specified site sequence of the fish genome, designing a homologous donor corresponding to the specified site sequence and containing a knock-in exogenous gene fragment, introducing the genome editing tool, the homologous donor and mRNA for specifically and stably expressing fluorescent protein in primordial germ cells into fish animal embryos by using a codominant microinjection method, and selecting the embryos by detecting fluorescent protein expressed by the fluorescent protein mRNA and obtaining stable inheritable characters. The efficiency of the method for knocking the exogenous gene fragment into the specified site of the fish genome so as to obtain a first filial generation of fish with the knock-in exogenous gene fragment is remarkably higher than that of the prior art.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to a method for significantly improving the editing efficiency of fish genomes. Background technique [0002] Gene targeting is an important genetic technique. Fish are genetic model animals for vertebrates. Traditional gene targeting relies on embryonic stem cell culture and homologous recombination. It is difficult to apply gene targeting based on homologous recombination in many model animals and many economic animals because there is no establishment of embryonic stem cell lines. In the past, the methods of reverse genetics in fish such as zebrafish were relatively limited, and when it was necessary to artificially inhibit gene expression, morpholino oligonucleotides or siRNA were usually used for gene knockdown. Artificial endonucleases, including zinc finger endonuclease (ZFN) technology, transcription activator-like effector endonuclease (TALEN) technology, and CRISPR / Cas9 endonu...

Claims

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Application Information

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IPC IPC(8): C12N15/89
Inventor 赵庆顺董张及
Owner NANJING UNIV
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