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CRISPR/Cpf1 gene editing method applicable to marine crustacean mitochondrion genome

A mitochondrial genome and gene editing technology, applied in the field of molecular biology, can solve unseen problems and achieve the effect of simple operation and good editing effect

Inactive Publication Date: 2018-08-17
HUAIHAI INST OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But apart from this (human), there are no other species, especially research reports on gene editing technology related to the mitochondrial genome of crustaceans

Method used

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Examples

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Embodiment Construction

[0017] The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those skilled in the art.

[0018] The implementation of the present invention will be further described in detail below in conjunction with specific examples of application in Portunus trituberculatus. It should be understood that the examples are only for illustrating the present invention and should not and will not be used to limit the scope of the present invention. Various procedures and methods not described in detail in the following examples are conventional methods well known in the art.

[0019] 1. Construction of plasmid vector

[0020] Since there is no suitable restriction endonuclease available in the NLS sequence region of pY094 plasmid Cpf1, it is necessary to introduce two enzyme cutting site regions, XcmI (position: 4870bp) and BamHI (position 5201), specifically as follows, manually 合成“5'CACCATGGTGGCCCTGATCCGCAGCGTGCTGCAGATGCGGAACT...

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Abstract

The invention discloses a CRISPR / Cpf1 gene editing method applicable to a marine crustacean mitochondrion genome. The method includes the steps: introducing MLS signals positioned by a marine crustacean mitochondrion into a CRISPR / Cpf1 expression plasmid; designing gRNA; micro-injecting fertilized eggs; culturing the fertilized eggs in an in-vitro manner; detecting editing effect to complete editing of the marine crustacean mitochondrion genome. By the method, the marine crustacean mitochondrion genome can be directionally edited, and the method has significant value to a condition that artificial proliferation releasing markers are introduced into the marine crustacean mitochondrion genome.

Description

technical field [0001] The invention belongs to the technical field of molecular biology and relates to a CRISPR / Cpf1 gene editing method suitable for mitochondrial genomes of marine crustaceans. Background technique [0002] Gene editing technology is a technology that modifies specific DNA sequences at the DNA level by means of deletion, insertion, etc. Transcription activator-like effector (TALENs), a qualitative leap has taken place since the establishment of CRISPR / Cas9 technology. On the basis of the CRISPR / Cas9 system, Zhang Feng's research group at the Massachusetts Institute of Technology further improved CRISPR / Cas9 and established the CRISPR / Cpf1 gene editing system. The main difference between it and the CRISPR / Cas9 system is that it uses Cpf1 nuclease instead of Cas9 , but the advantages are more obvious: first, compared with Cas9 nuclease, the Cpf1 enzyme expressed in this system is smaller in size, so it is easier to enter small organelles and tissues such as...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/90
CPCC12N15/8509C12N15/902C12N2800/80C12N2810/10
Inventor 高焕马杭柯阎斌伦孙金秋徐莞媛李光光王玉
Owner HUAIHAI INST OF TECH
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