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CRISPR/Cas9 random library, and its construction and application

A library and random body technology, applied in libraries, chemical libraries, nucleotide libraries, etc., can solve unsatisfactory problems and achieve far-reaching social and economic value

Inactive Publication Date: 2018-06-01
SHANGHAI TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Obviously, it is far from satisfying the human 3x10 9 BP's Genome-Wide Requirements

Method used

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  • CRISPR/Cas9 random library, and its construction and application
  • CRISPR/Cas9 random library, and its construction and application
  • CRISPR/Cas9 random library, and its construction and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0075] 1. Design and preparation of sgRNA random library

[0076] A strategy of setting the first 20 nucleotides of the sgRNA recognition sequence as G, and 2 to 20 as random nucleotides N, driven by a 17bp T7 promoter (taatacgactcactata, SEQ ID NO: 2) sgRNA expression. The sgRNA consists of a 83bp backbone and a 20bp recognition region. In order to improve the transcription efficiency of the T7 promoter, the first nucleotide of the recognition region is set as G, and the 2nd to 20th are random nucleotides N, with a total length of 120bp, (SEQ ID NO: 3,

[0077] taatacgactcactatagnnnnnnnnnnnnnnnnnngttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcttttttt). sgRNA templates are synthesized from random upstream and downstream primers. The synthesized sgRNA primers were annealed and completed in vitro as random transcription templates, and then in vitro transcription was performed after sequencing verification. The specific steps are as follows:

[00...

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Abstract

The invention provides a CRISPR / Cas9 random library, and its construction and application. The CRISPR / Cas9 random library is characterized by comprising sgRNA, wherein the sgRNA contains a recognitionsequence, the first nucleotide in the 20 nucleotides of the sequence is set as G, and the second to twentieth nucleotides are set as random nucleotides. The sgRNA random library of the invention hashigh-throughput, is universal and free of preference, and provides an efficient tool for high-throughput function-annotation genomic functional elements used in functional genomics.

Description

technical field [0001] The invention relates to a CRISPR / Cas9 random library and its application. Background technique [0002] After the genome project is completed, one of the challenges scientists face in functional genomics research is high-throughput functional annotation of functional gene elements. RNA interference is an effective strategy for high-throughput functional studies [Kaelin, 2012]. However, there are factors such as off-target effects, knocking down rather than knocking out genes, only affecting expressed genes but not targeting non-coding functional elements, which limit its application. The CRISPR / Cas9 system is an ideal high-throughput gene knockout tool, but the throughput of existing sgRNA libraries is very limited. This invention intends to develop a strategy for constructing a high-throughput, universal, and unbiased sgRNA random library. [0003] One of the challenges in genomics research is to functionally annotate different genome functional el...

Claims

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Application Information

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IPC IPC(8): C40B40/06C40B50/06C12N15/10C12N15/113
CPCC12N15/102C12N15/1093C12N15/113C12N2310/10C40B40/06C40B50/06
Inventor 周建奎黄行许
Owner SHANGHAI TECH UNIV
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