Method for knocking out MSTN (myostatin) genes in targeted manner by utilizing CRISPR-Cas9

Abstract

The invention discloses a method for knocking out MSTN (myostatin) genes in a targeted manner by utilizing CRISPR-Cas9. According to the method, multiple species are subjected to homology comparison, two pairs of MSTN gene target sequences are selected, two pairs of DNA double strands which have the sequences different from the target sequences, the same expression protein and the same BsmBi sticky ends are synthesized according to the design principle of PAM of gRNA, the double strands are connected with pPDNA330 plasmids, a recombinant vector carrying MSTN homologous genes of multiple species is obtained and is used for detecting the gene knockout efficiency with a fluorescent protein expression method, carrier-transfected receptor cells, having higher knockout efficiency, of multiple species are selected, gene knockout and verification are performed, and simple, efficient and accurate gene knockout on the MSTN genes of the multiple species is completed.

Description

【Technical field】 [0001] The invention relates to the field of molecular biology, in particular to a method for targeted knockout of MSTN gene by using CRISPR-Cas9. 【Background technique】 [0002] MSTN is a myostatin protein (myostatin, MSTN), a member of the transforming growth factor-β (transforming growth factor-β) superfamily. Since its discovery in 1997, a large number of research results have shown that it is a skeletal muscle growth inhibitory gene. Compared with the wild-type mice, the body weight of MSTN- / - mice increased by about 30%, the weight of skeletal muscle increased by about 1-2 times, and the number of skeletal muscle fibers increased by about 86%. Under natural circumstances, the muscles of cattle with mutations in the MSTN gene grow significantly, and the phenomenon of "double muscle buttocks" appears. This double muscle phenomenon occurs in many animals in nature. A bimuscular phenotype appears in signs. [0003] Changing the gene structure is usuall...

AI Technical Summary

Problems solved by technology

[0006] At present, some studies have begun to use the CRISPR / Cas9 system for gene knockout experiments. Experiments have proved that using the CRISPR / Cas9 system for gene knockout has the advantages of higher efficiency and easier operation, but a method that can be used simultaneously for knockout has not yet been developed. In addition to the recombinant vector of the MSTN gene of multiple species, find out a detection method that can quickly and accurately find the target sequence

For example, PCT patent CN 105142669 A discloses genome modification and regulation based on CRISPR, which illustrates that the CRISPR system can be used for genome modification and regulation, and that the CRISPR system is simpler and more efficient than ZFN and TALEN. The specific restriction site and base sequence of the knockout, the detection and selection method for the preferred fragment of the MSTN gene has not been published, and the construction of the recombinant plasmid vector used for the knockout of the MSTN gene in the present invention has not been published; Chinese patent CN 104232669 A discloses a vector and its construction method based on the fish CRISPR / Cas9 system using gene knockout method, but this technical solution can only knock out the MSTN gene of fish, not for other species, and the universality rate is low. At the same time, this technical solution does not disclose a detection method that can quickly and accurately find the target sequence

Therefore, the present invention needs to solve the following problems: (1) construction of MSTN gene editing method capable of targeted knockout of multiple species; (2) existing technology in the gene editing process, especially the technology platform of human and buffalo genome editing Low and low applicability, the entire platform still needs to be optimized and conditionally explored; (3) There is no detection method that can quickly and accurately find the target sequence in the existing technology, thereby further improving the success rate of gene editing

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