Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Cas9 (CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) associated protein 9) nuclease Q920P and application thereof

A nuclease and polynucleotide technology, applied in the field of Cas9 nuclease Q920P, to achieve the effect of precise editing

Active Publication Date: 2017-07-14
SHANGHAI JIAO TONG UNIV
View PDF1 Cites 31 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the editing of DNA fragments can be realized through the CRISPR / Cas9 system, but for the in-depth study of the precise function of specific DNA segments, the Cas9 nuclease that can effectively realize the precise genetic editing of DNA fragments has yet to be discovered

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Cas9 (CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) associated protein 9) nuclease Q920P and application thereof
  • Cas9 (CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) associated protein 9) nuclease Q920P and application thereof
  • Cas9 (CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) associated protein 9) nuclease Q920P and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1 Study on the connection of adapters for DNA fragment editing and discovery of a new mechanism for Cas9 cutting

[0077] For the HS51 site, construct the sgRNAs plasmid targeting the HS51 site:

[0078] (1) Purchase primers

[0079] Purchase forward and reverse deoxy oligonucleotides with 5' hanging ends "ACCG" and "AAAC" that can be complementary paired for the HS51 site and the sgRNAs targeting sequence respectively from Shanghai Sunny Biotechnology Co., Ltd.;

[0080] Targeting sequences of sgRNAs targeting the above HS51 site:

[0081] HS51 RE1 sgRNA1: GCCACACATCCAAGGCTGAC (SEQ ID NO.1)

[0082] HS51 RE1 sgRNA2: GAGATTTGGGGCGTCAGGAAG (SEQ ID NO.2)

[0083] (2) Obtain complementary paired double-stranded DNA with hanging ends

[0084] 1) use ddH 2 O Dissolve deoxyoligonucleotides to 100 μM and dilute to 20 μM;

[0085] 2) Add positive and negative deoxy oligonucleotides to the following reaction system:

[0086]

[0087] Reaction conditions: 95°C w...

Embodiment 2

[0152] Example 2 Mutation of SpCas9 to obtain a specific Cas9 with a changed cutting method to achieve precise DNA fragment editing

[0153] 1. Construction of Cas9 mutants

[0154] 1) Use NEB Mutagenesis Kit (Q5Site-Directed Mutagenesis Kit, #E0554S) to construct Cas9 mutants, first perform PCR amplification, and the reaction is as follows:

[0155]

[0156]

[0157] 2) KLD (Kinase, Ligase&DpnI) treatment, the reaction is as follows:

[0158]

[0159] Reaction conditions: 10 minutes at room temperature

[0160] 3) All the reaction products in 2) were used for the transformation of competent bacteria Stbl3 (50 μl), and cultured on LB plates containing ampicillin antibiotic (Amp, 100 mg / L) overnight at 37° C. Single clones were picked, plasmids were extracted and sent for sequencing.

[0161] The amino acid sequence of SpCas9 (Cas9WT) is shown in SEQ ID NO.7, specifically:

[0162]

[0163]

[0164] The coding nucleotide sequence of SpCas9 (Cas9WT) is shown i...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention belongs to the technical field of biology and in particular relates to Cas9 (CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) associated protein 9) nuclease and application thereof. The Cas9 nuclease (Cas9-Q920P) has Cas9 nuclease activity and is applicable to a CRISPR / Cas9 system; the Cas9 nuclease (Cas9-Q920P) is obtained by mutating a 920th glutamine of wild type Cas9 nuclease into proline. The Cas9 nuclease (Cas9-Q920P) is used for cutting DNA (Deoxyribonucleic Acid) double strand to generate an protruded broken terminal and an alkaline group, which is complementary with the protruded broken terminal, can be added in a filling-in connecting manner, so that accurate editing of a specific position of a DNA segment of a genome can be realized.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a Cas9 nuclease Q920P and its application. Background technique [0002] Since the completion of the Human Genome Project and the Encyclopedia of DNA Elements, scientists have analyzed and identified a large number of genes and DNA regulatory elements in the genome [1,2]. DNA regulatory elements that play an important role in the regulation of gene expression include promoters, enhancers, silencers, and insulators. However, the functions of most regulatory elements have not been experimentally verified and elucidated [2-8]. Exploring the function of genes and DNA regulatory elements can be studied through genetic DNA segment editing. [0003] Early gene editing and gene function modification were achieved through gene transposition and transgenesis [9-14]. With the development of sequencing technology, reverse genetics has been applied to make specific mutations in the...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12N9/22C12N15/55C12N15/63C12N15/10
CPCC12N9/22C12N15/102C12N15/63C12N2310/10
Inventor 吴强李金环寿佳
Owner SHANGHAI JIAO TONG UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com