Design, synthesis and use of RNA molecule for high-efficiency genome editing

A base and base number technology, applied in the field of genetic engineering and nucleic acid detection, can solve the problems of application limitations and low cutting efficiency

Inactive Publication Date: 2015-07-29
PEKING UNIV
View PDF0 Cites 45 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Problems existing in the existing technology: the cutting efficiency of the existing Cas9/gRNA to th

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
  • Design, synthesis and use of RNA molecule for high-efficiency genome editing
  • Design, synthesis and use of RNA molecule for high-efficiency genome editing
  • Design, synthesis and use of RNA molecule for high-efficiency genome editing

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0042] The principle of Example 1 is as follows figure 2 with image 3 As shown, the experimental results are as Figure 18 Shown. First, we constructed the gRNA recognition sequence into the SSA vector, and then constructed the gRNA expression vector on the eukaryotic expression vector; secondly, we co-transfected the gRNA expression vector, cas9 expression vector, and SSA expression vector into HEK293T cells for 24 hours Then check the expression level of the luciferase gene. At the same time, we co-transfected the gRNA expression vector and the cas9 expression vector into HEK293T cells. After 48 hours, we extracted the genomic DNA and performed the SURVEYOR experiment to reflect the gRNA / cas9 activity. From Figure 18 It can be seen that the activity of gRNA1 is increased by about 50% relative to the activity of gRNA.

Example Embodiment

[0043] The experimental process of Example 2 is similar to that of Example 1, except that the gRNA1 sequence is replaced with gRNA2. See the results of the experiment Figure 19 . by Figure 19 It can be seen that the activity of gRNA2 is increased by about 30% relative to the activity of gRNA.

Example Embodiment

[0044] The experimental process of Example 3 is similar to that of Example 1, except that the gRNA1 sequence is replaced with gRNA3. See the results of the experiment Figure 20 . by Figure 20 It can be seen that the activity of gRNA3 is increased by about 30% relative to the activity of gRNA.

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

In prokaryote, a cas9 protein of a clustered regularly interspaced short palindromic repeals and CRISPR associated (CRISPR/Cas) system and two small RNAs (of crRNA and tracrRNA) can be combined and used for recognition and direct shearing of exogenous nucleic acid so that self-protection is realized. Scientists fully utilizes the above characteristic and utilizes cas9 and gRNA (which is a long RNA obtained by fusion of crRNA and tracrRNA) to shear a DNA sequence so that gene fixed point modification is realized. The existing Cas9/gRNA has low target gene cutting efficiency generally of 5-30% and is limited in use. Through a high efficiency screening system, a series of gRNAs for improving Cas9 activity are selected.

Description

Technical field: [0001] The invention relates to the fields of genetic engineering and nucleic acid detection. Specifically, the present invention relates to genome editing methods and uses of biological nucleic acid sequences of viruses, microorganisms, animals and plants. Background technique: [0002] Gene site-directed modification has always been one of the important means to study gene function, and it is also used in the treatment of human genetic diseases, so this kind of technology has become a research hotspot in modern molecular biology. The early gene targeting technology based only on homologous recombination was extremely inefficient and its application was limited. [0003] The emergence of zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENS) allows scientists to efficiently carry out gene-directed modification, and has been applied to animal models such as zebrafish, mice, rats, and pigs , experiments have proved that th...

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): C12N15/11C12N15/10
Inventor 席建忠叶延桢孙常宏
Owner PEKING UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap