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A nucleic acid molecule safely encoding cas9 protein and its expression vector

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: 2018-04-13
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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  • A nucleic acid molecule safely encoding cas9 protein and its expression vector
  • A nucleic acid molecule safely encoding cas9 protein and its expression vector
  • A nucleic acid molecule safely encoding cas9 protein and its expression vector

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, that is, the 229th to the 246th 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, b...

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Abstract

The invention relates to the field of biotechnology, and provides a safer nucleic acid molecule encoding Cas9 protein and its recombinant expression vector, as well as its application in CRISPR-Cas9 technology, and a method for weakening the combination of Cas9 nucleic acid molecule and microRNA. Experiments have found that although the currently widely used Cas9 expression vector can successfully express Cas9 protein and play a role, the mRNA sequence transcribed by the vector itself can act as an adsorbent of the microRNA let-7 family to inhibit the function of let-7, and let-7 can inhibit the function of let-7. The expression of multiple oncogenes, so the currently applied Cas9 expression vector can increase the expression of some oncogenes and thus increase the risk of cancer. In the M-mir-Cas9 expression vector provided by the present invention, the transcribed mRNA cannot adsorb let-7, and can still be translated into a complete Cas9 protein to function, increasing the safety of the CRISPR-Cas9 gene editing tool.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a nucleic acid molecule safely encoding a Cas9 protein and an expression vector thereof. 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 the target DNA sequence at a precise location, including mutation, modification, insertion and other changes, so that the genetic code of life can be changed according to human wishes, either at the cell level or in life individual level. This technology has also been widely used in recent years, such as gene activation, interference, gene labeling of living cells, RNA cutting and so on. 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 Patents(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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