Method used for construction of CRISPR/Cas9 genome knockout library with enzymatically digested genome

A genome and library technology, applied in the field of genome knockout library construction, can solve the problems of time-consuming and labor-intensive, low coverage of manual design, imperfect genetic information of species, etc., to reduce production costs, improve coverage, and maintain stability Effect

Active Publication Date: 2017-08-29
NORTHEAST AGRICULTURAL UNIVERSITY
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Problems solved by technology

[0005] The present invention aims to solve the problems of incomplete species gene information, low artificial design coverage and time-consuming and labor-intensive prob

Method used

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  • Method used for construction of CRISPR/Cas9 genome knockout library with enzymatically digested genome
  • Method used for construction of CRISPR/Cas9 genome knockout library with enzymatically digested genome
  • Method used for construction of CRISPR/Cas9 genome knockout library with enzymatically digested genome

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Embodiment 1

[0047] In this embodiment, the method for constructing a CRISPR / Cas9 genome knockout library by enzymatically cutting the genome comprises the following steps:

[0048] 1. Screening of cells stably expressing green fluorescent protein

[0049] The commercial pEGFP-C1 plasmid was transfected into the PK-15 cell line by liposome transfection, and G418 was used to screen, and the clones stably expressing green fluorescent protein were picked and continued to be cultured, and finally the stable expression green fluorescent protein was obtained. The PK-15 cell line was named PKpG-Pi.

[0050] A clone of PK-15 cells expressing green fluorescent protein observed under a fluorescent microscope and an optical microscope is as follows: figure 2 as shown, figure 2 The upper part is the photo under the fluorescence microscope, and the lower part is the photo under the light microscope. A PKpG-Pi cell line stably expressing green fluorescent protein observed under a fluorescent micros...

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Abstract

The invention relates to a construction method of a genome knockout library, and more specifically discloses a method used for construction of a CRISPR/Cas9 genome knockout library with an enzymatically digested genome. The method is used for solving problems in the prior art that species gene information is incomplete, artificial design coverage is low, and both time and labor are consumed. The method comprises following steps: 1, construction of a Mspl.f-library library; 2, obtaining of 20bp gRNA target sequence fragments; 3, construction of a PAM-f.library library; and 4, construction of a Lenti-gRNA-library library. The method used for obtaining gRNA libraries is independent of any known species genome sequence information, so that the problem in the prior art that species gene information is incomplete, artificial design coverage is low, and both time and labor are consumed are solved, and the knockout library coverage rate is increased greatly. The method is used for construction of a CRISPR/Cas9 genome knockout library.

Description

technical field [0001] The invention relates to a method for constructing a genome knockout library. Background technique [0002] Gene knockout refers to a genetic engineering technique that uses genetic engineering techniques to modify specific genes or loci in chromosomes so as to silence their expression. The CRISPR / Cas9 system is derived from the immune-related mechanism in bacteria. Since 2013, many articles have described its gene editing effect in eukaryotic cells. The CRISPR / Cas9 system consists of two parts: the Cas9 protein with nuclease properties and the guideRNA containing the target site sequence fragment and the guideRNA secondary structure. The principle is: the sequence fragment of the target site in the guideRNA specifically binds to the genomic target site and guides the Cas9 protein to cut the genomic target site, causing double-stranded DNA breaks, and there is a probability that bases will be lost or added. After non-homologous end joining occurs, th...

Claims

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Application Information

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IPC IPC(8): C40B50/06C12N15/113
CPCC12N15/1082C12N15/113C12N2310/10C40B50/06
Inventor 吕嘉伟赵艳华刘忠华
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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