Method for targeted knockout of gene ALK6 by using CRISPR-Cas9

A gene and targeting technology, applied in the field of molecular biology, can solve the problems of low efficiency, low applicability, and inability to accurately find target sequence detection methods, and achieve the effect of simple detection methods and improved universality

Inactive Publication Date: 2016-09-07
GUANGXI ZHUANG AUTONOMOUS REGION BUFFALO INST
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  • Abstract
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  • Claims
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Problems solved by technology

Therefore, the present invention needs to solve the following problems: (1) construct an ALK6 gene editing method that can target multiple species; Low and low applicability, the entire platform still needs to be optimized and conditionally explored; (3) The existing technology does not have a detection method that can quickly and accurately find the target sequence, thereby further improving the success rate of gene editing

Method used

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  • Method for targeted knockout of gene ALK6 by using CRISPR-Cas9
  • Method for targeted knockout of gene ALK6 by using CRISPR-Cas9
  • Method for targeted knockout of gene ALK6 by using CRISPR-Cas9

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Embodiment

[0061] 1. Preparation of pPDNA330 vector:

[0062] (1) Optimizing the puro gene, removing the restriction site in its coding region, artificially synthesizing it, and connecting it into the pcDNA3.1(-) vector to prepare a pPDNA vector. See the sequence listing for the sequence optimized by puro. Methods as below:

[0063] 1. Optimize the puro gene, remove the enzyme cleavage site in its coding region, but do not change its amino acid composition, artificially synthesize the puro gene at Sangon Bioengineering (Shanghai) Co., Ltd., and the synthetic puro gene is located in the puc57-puro vector .

[0064] 2. Digest puc57-puro with StuI (Fermentas) and ClaI (Fermentas), and recover the puro gene from the gel. The enzyme digestion system is: 3 μg of puc57-puro plasmid, 5 μL of 10×FastDigest, 3 μL of StuI, 3 μL of ClaI, and water Add to 50 μL, incubate at 37°C for 3 hours; perform agarose gel electrophoresis on the digested product, recover the target fragment of about 1638 bp, ...

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Abstract

The invention discloses a method for targeted knockout of a gene ALK6 by using CRISPR-Cas9. The method comprises the steps of carrying out homologous comparison on a plurality of species so as to select two pairs of gene ALK6 target sequences, synthesizing DNA double strands, of which two pairs of target sequences are different and expressed proteins are the same and which have the same BsmBI cohesive ends, by using PAM design principles of gRNA, connecting the double strands with plasmid pPDNA330 so as to obtain recombinant vectors carrying ALK6 homologous genes of a plurality of species, detecting gene knockout efficiency of the recombinant vectors by using a fluorescin expression method, carrying out transfection on a vector with higher knockout efficiency with recipient cells of a plurality of species, and carrying out gene knockout and verification, thereby completing simple, efficient and accurate gene knockout of genes ALK6 of a plurality of species.

Description

【Technical field】 [0001] The present invention relates to the field of molecular biology, in particular to a method for targeted knockout of ALK6 gene by using CRISPR-Cas9. 【Background technique】 [0002] ALK6 is another name for bone morphogenetic protein receptor 1B (bone morphogenetic protein receptor 1B, BMPR-1B), which is a type 1 receptor of the transforming growth factor-β (transforming growth factor-β) superfamily. Bone morphogenetic protein 2 (BMP2), bone morphogenetic protein 4 (BMP4), bone morphogenetic protein 6 (bone morphogenetic protein 6, BMP6) and bone morphogenetic protein 15 (bone morphogenetic protein 15, BMP15 ) and other ligand signals play a very important role in the reproductive process of mammals. Under natural circumstances, the 109th base A in the coding region of the ALK6 gene of Burra Merino and other sheep breeds is mutated to G, and the ovulation rate and multiple births of mutant individuals significantly higher than wild-type individuals. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/65
CPCC12N15/8509A01K2267/02C07K14/71C12N15/65C12N2800/107C12N2800/80C12N2810/10
Inventor 朱鹏梁贤威庞春英邓廷贤段安琴陆杏蓉
Owner GUANGXI ZHUANG AUTONOMOUS REGION BUFFALO INST
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