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A method to dramatically increase the efficiency of genome editing in fish

A genome editing and genome technology, applied in the biological field, can solve problems such as unaware of the unequal distribution of injected nucleic acids, and achieve the effect of reducing invalid F1 generation individuals and reducing time

Active Publication Date: 2017-06-09
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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  • A method to dramatically increase the efficiency of genome editing in fish
  • A method to dramatically increase the efficiency of genome editing in fish
  • A method to dramatically increase the efficiency of genome editing in fish

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0037] 1. Construct a CRISPR / Cas9 system that targets the third and fourth introns of zebrafish aldh1a2 gene

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

[0039]

[0040] The primers ali3gRF11 (Seq ID No: 1) and sgRNAR (Seq ID No: 3) were used to amplify the plasmid template containing the sgRNA backbone. The PCR conditions were: 95°C for 2 minutes, 35 cycles (94°C for 30 seconds, 56°C for 30 seconds) , 72°C for 30 seconds), and finally extended at 72°C for 5 minutes. In the same way, the primers ali4gRF5 (Seq ID No: 2) and sgRNAR (Seq ID No: 3) were used to amplify the plasmid template containing the sgRNA backbone (Seq ID No: 4). 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, and the...

Embodiment 2

[0060] Example 2 Introducing an indel mutation in the zebrafish aldh1a2 gene intron

[0061] 1. Construct a CRISPR / Cas9 system that targets the third and fourth introns of zebrafish aldh1a2 gene

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

[0063]

[0064] The primers ali3gRF11 (Seq ID No: 1) and sgRNAR (Seq ID No: 3) were used to amplify the plasmid template containing the sgRNA backbone. The PCR conditions were: 95°C for 2 minutes, 35 cycles (94°C for 30 seconds, 56°C for 30 seconds) , 72°C for 30 seconds), and finally extended at 72°C for 5 minutes. In the same way, the primers ali4gRF5 (Seq ID No: 2) and sgRNAR (Seq ID No: 3) were used to amplify the plasmid template containing the sgRNA backbone (Seq ID No: 4). 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, and ...

Embodiment 3

[0079] Example 3 Introducing indel mutations in the mstna gene and mstnb gene of yellow catfish

[0080] As two independent genome editing tools, zinc finger nuclease and transcription activator-like effector nuclease can respectively complete the identification and cutting of specific sequences. In the experiment, you can choose whether to edit the genome separately or together according to the needs of the experiment. In order to make the present invention concise, in this example, 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 implement the present invention, but using zinc finger nuclease mRNA that specifically recognizes and cleaves the specified site sequence of the mstna gene or transcription activator-like effects ...

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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 fish genome editing efficiency. Background technique [0002] Gene targeting is an important genetic technique. Fish are the genetic model animals of 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 multiple model animals and many economic animals because embryonic stem cell lines have not been established. In the past, reverse genetics methods 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, transcriptional activator-like effector endonuclease (TALEN) technology, and CRISPR / Cas9 endonuclease technolo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/89
Inventor 赵庆顺董张及
Owner NANJING UNIV
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