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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
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  • 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 the target gene is low, generally between 5% and 30%, and the application will be limited to a certain extent

Method used

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  • 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

Embodiment 1

[0042] The principle of embodiment 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 The expression level of the luciferase gene was then checked. 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 about 50% higher than that of gRNA.

Embodiment 2

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

Embodiment 3

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

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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

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Application Information

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IPC IPC(8): C12N15/11C12N15/10
Inventor 席建忠叶延桢孙常宏
Owner PEKING UNIV
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