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Nucleic acid molecule for encoding Cas9 (CRISPR (clustered regularly interspaced short palindromic repeat)-associated endonuclease 9) protein securely and expression vector of nucleic acid molecule

A technology for nucleic acid molecules and expression vectors, which is applied in the field of nucleic acid molecules and their expression vectors that safely encode Cas9 protein. It can solve the problems of interaction between mRNA and microRNA that have not been reported in the literature, and achieve the effect of overcoming cancer risks and increasing safety.

Active Publication Date: 2015-07-29
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, there is no literature reporting the interaction between Cas9 mRNA and microRNA, and improving CRISPR-Cas9 technology from this aspect

Method used

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  • Nucleic acid molecule for encoding Cas9 (CRISPR (clustered regularly interspaced short palindromic repeat)-associated endonuclease 9) protein securely and expression vector of nucleic acid molecule
  • Nucleic acid molecule for encoding Cas9 (CRISPR (clustered regularly interspaced short palindromic repeat)-associated endonuclease 9) protein securely and expression vector of nucleic acid molecule
  • Nucleic acid molecule for encoding Cas9 (CRISPR (clustered regularly interspaced short palindromic repeat)-associated endonuclease 9) protein securely and expression vector of nucleic acid molecule

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Bioinformatics analysis of the site where the widely used Cas9 expression vector binds to microRNA.

[0045] MicroRNA and RNA interaction analysis software miRanda can be downloaded from the following website:

[0046] http: / / www.microrna.org / microrna / getDownloads.do

[0047] After downloading, use it according to the software instructions provided by the website. Specifically, we will use the widely used Cas9 coding sequence (SEQ ID NO: 2, Addgene ID is Plasmid#41815, URL: http: / / www.addgene.org / search / advanced / ?q=41815) into the analysis software to analyze the combination of microRNA with all the people included in the software, we use Score Threshold:

[0048] The condition control index of 140 has obtained the situation of all microRNAs that are mutually combined with SEQ ID NO: 2, and the corresponding positions in SEQ ID NO: 2, such as figure 1 A special case shown in B.

[0049] After analysis, we found that there are 3 binding sites on SEQ ID NO:...

Embodiment 2

[0051] Example 2: Experimental analysis of the effect of the mRNA transcribed by the existing Cas9 expression vector on the genes inhibited by let-7.

[0052] 1. Cell culture and transfection

[0053] Human embryonic kidney epithelial cells (HEK293, purchased from the Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences) were divided into two groups and cultured in high-glucose DMEM medium containing 10% fetal bovine serum, placed in a 5% carbon dioxide incubator at 37°C Set up culture. When the cell density of the two groups reached about 50%, Lipofectamine2000 reagent (Invitrogene, USA) was used to transfect the existing widely used Cas9 expression vector (purchased from AddgenePlasmid ID number: 41815, Its protein coding sequence reference sequence 2) and an empty plasmid without Cas9 coding sequence were used as controls. Cells were harvested 72 hours after transfection for Real-time PCR experiments.

[0054] 2. Real-time PCR detection ...

Embodiment 3

[0075] Example 3: Synonymously mutate the binding site between existing Cas9 and microRNA let-7, and construct an M-mir-Cas9 expression vector.

[0076] The currently widely used Cas9 expression vector is purchased from Addgene, and its Plasmid ID number is: 41815. Synonymous mutations are based on this plasmid.

[0077] The rapid site-directed mutagenesis kit was purchased from TIANGEN BIOTECH (BEIJING) CO., LTD, and its catalog number is KM101.

[0078] Since there are three binding sites with Let-7 on the original Cas9, we mutated the Cas9 plasmid in three steps.

[0079] 1. Mutation Cas9 expression plasmid site 1

[0080] The goal of the mutation is to mutate the sequence 5'-AATCGGATCTGCTACCTG-3' (SEQ ID NO: 15, 229th to 246th in SEQ ID NO: 2) to 5'-AA C CGGATCTG T TA TT TG-3' (SEQ ID NO: 16, i.e. the 229th to 246th positions in SEQ ID NO: 1). There is no frame shift mutation before and after the mutation, and the amino acid sequence after translation is consistent,...

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Abstract

The invention relates to the technical field of biology, and provides a nucleic acid molecule for encoding Cas9 (clustered regularly interspaced short palindromic repeat-associated endonuclease 9) protein securely, a recombinant expression vector of the nucleic acid molecule, an application of the nucleic acid molecule in the CRISPR-Cas9 technology and a method for reducing combination of Cas9 nucleic acid molecule and microRNA (ribonucleic acid). Experiments find that although the extensively applied Cas9 expression vector can express the Cas9 protein successfully and play a role, an mRNA sequence transcribed by the vector can serve as an adsorber of the microRNA let-7 family to inhibit functions of let-7 which can inhibit expression of multiple oncogenes, accordingly, the Cas9 expression vector applied at present can increase expression of the oncogenes, and the risk of cancer is increased. According to the provided M-mir-Cas9 expression vector, the transcribed mRNA cannot adsorb let-7 and can be still translated into complete Cas9 protein to play a role, and the safety of CRISPR-Cas9 gene editing tool is improved.

Description

Technical field [0001] The present invention relates to the field of biotechnology, and in particular to a nucleic acid molecule that safely encodes Cas9 protein and its expression vector. Background technique [0002] CRISPR-Cas9 (Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated endonuclease (Cas9)) technology is a revolutionary gene editing technology that has emerged in recent years. This technology can quickly and easily edit target DNA sequences at precise sites, including mutations, modifications, insertions and other changes, so that the genetic code of life can be changed according to human wishes, either at the cellular level or in life. individual level. This technology has also been widely used in recent years, such as gene activation, interference, living cell gene labeling, RNA cleavage, etc. It has broad application prospects for agriculture, industry, medicine, and human health (see literature: Hsu, P.D., Lander, E.S., and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12N15/63C12N15/10
Inventor 刘厚奇蒋俊锋王越张莉仵敏娟
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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