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Eukaryote CRISPR/Cas whole genome editing vector library and construction method

A technology of eukaryotic organisms and construction methods, which is applied in the field of gene editing, can solve problems such as time-consuming, labor-intensive, powerless, etc., and achieve good results

Inactive Publication Date: 2020-08-18
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the face of massive functional genomic data, traditional research methods are time-consuming and labor-intensive, and seem powerless

Method used

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  • Eukaryote CRISPR/Cas whole genome editing vector library and construction method
  • Eukaryote CRISPR/Cas whole genome editing vector library and construction method
  • Eukaryote CRISPR/Cas whole genome editing vector library and construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0039] The Bombyx mori embryonic cell line (BmE) used in this example is a commonly used cell line in biological experiments (PMID: 17570024).

[0040] Construction of genome-wide marker vector library of silkworm CRISPR / Cas9 mediated by piggyBac transposon system

[0041] 1. Taking piggyBacModify (its nucleotide sequence as SEQ ID NO.11), the basic vector of the piggyBac transposon system, as the initial vector, construct a piggyBac transposon system-mediated CRISPR / Cas9 gene knockout vector skeleton, mainly including piggyBac Transposable arm (including two piggyBac transposon terminal inverted repeats, invertedterminal repeat, ITR), screening marker Zeocin resistance gene expression cassette, Cas9 protein expression cassette, E. coli lethal gene ccDB expression cassette, named pB-CRISPRv2 , its nucleotide sequence is shown in SEQ ID NO.1, and the vector map is shown in figure 1 shown.

[0042] 2. Construct two vectors for subsequent library construction experiments. One ...

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Abstract

The invention relates to a eukaryote CRISPR / Cas9 whole genome editing vector library and a construction method. The method comprises the following steps: firstly, constructing a piggyBac transposon system mediated eukaryote CRISPR / Cas9 knockout skeleton vector, and then constructing an eukaryote whole genome knockout mutant library by comprehensively applying a bridging PCR (Polymerase Chain Reaction) method and enzyme digestion connection method. The method is characterized in that a piggyBac transposon system with wide biological applicability and super-large exogenous gene bearing capacityis selected as a delivery system, and a bridging PCR and enzyme digestion connection method is selected as a construction method. The constructed eukaryote whole genome knockout vector library has a good effect.

Description

technical field [0001] The invention belongs to the technical field of gene editing, and relates to a eukaryotic CRISPR / Cas whole genome editing carrier library and a construction method. Background technique [0002] With the development of sequencing technology, more and more organisms have completed whole genome sequencing, and functional genome research has increasingly become an important field of life science research. In the face of massive functional genomic data, traditional research methods are time-consuming and labor-intensive, and seem powerless. How to efficiently and quickly study the functional genome has increasingly become a concern of life scientists. Gene editing technology is a genetic manipulation technology developed in recent years, and CRISPR / Cas9 is the most efficient and economical technology among gene editing technologies. In addition to realizing the editing of a single gene, CRISRP / Cas9 can also be designed to construct a simple sgRNA library...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/79C40B50/06C40B40/04C12N15/113C12N15/90
CPCC12N15/79C40B50/06C40B40/04C12N15/113C12N15/902C12N2310/20
Inventor 马三垣常珈菘夏庆友
Owner SOUTHWEST UNIV