MiR-205 gene knockout kit based on CRISPR-Cas9 gene knockout technology
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A mir-205, mir-205-cas9 technology, applied in the field of genetic engineering, can solve the problem of ineffective inhibition of oncogenes
Active Publication Date: 2015-12-02
广州辉园苑医药科技有限公司
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When miRNA is overexpressed, it inhibits the expression of tumor suppressor genes...
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Embodiment 1
[0020] Embodiment 1: vector construction
[0021] (1) Optimized design of miR-205 target
[0022] For the miR-205 gene (gene name MIR205, gene ID number: 406988, http: / / www.ncbi.nlm.nih.gov / gene / 406988), retrieve and download the miR-205 genome sequence (SEQ ID NO: 1):
[0024]Use the online software FengZhanglab'sTargetFinder (http: / / crispr.mit.edu / ) and DNA2.0gRNA design tool software (https: / / www.dna20.com / eCommerce / cas9 / input) to design sgRNA, and input the above miR-205 Sequence, set and search to obtain the optimal target sequence. Select the target sequences recommended by both software, and select the optimal 4 target sequences, as shown in the following table:
[0025] Table 1
[0026] serial number target sequence Location SEQ ID NO:2 TCTCTTGTCCTTCATTCCAC 27-46 SEQ ID NO:3 CATACCCAACCAGATTTCAG 59-78 SEQ...
Embodiment 2
[0081] Example 2: Kit use and Knockout efficiency identification
[0082] (1) Plasmid transformation and amplification
[0083] 1) Take competent cells from a -80°C refrigerator and thaw in an ice box.
[0084] 2) Add 5uL of the ligation product to 50μL of DH5α competent cells in an ice bath, swirl gently to mix well, and bathe in ice for 30min.
[0085] 3) Heat shock in 42°C water bath for 90s (do not let the sample float).
[0086] 4) Quickly transfer the tube to an ice bath for 3 minutes.
[0087] 5) Add 500 μL SOC medium, mix well, put in a shaker, and activate at 200 rpm at 37° C. for 1 hour.
[0088] 6) Spread all on the LB plate containing Amp antibiotics, and place it at room temperature for 10 minutes until the liquid is absorbed. At the same time, add an appropriate amount of glass beads and shake back and forth to make the bacteria solution evenly coated. Invert the plate and transfer to a 37°C biochemical incubator for overnight culture.
[0089] 7) Pour 6 mL ...
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Abstract
The invention discloses a miR-205 gene knockout kit based on CRISPR-Cas9 gene knockout technology. According to the invention, an optimal CRISPR-Cas9 target sequence of a certain amount of miR-205 is obtained through target design software and an sgRNA single chain is synthesized in vitro; an insertion fragment is obtained through processing; then sgRNA is inoculated into a plasmid vector by using T4 ligase; and a miR-205 gene knockout cell strain is obtained through transfection of an LNCap cell strain, continuous drug screening and fluorescence detection. Heterogenous hybridization double strands are obtained by extracting DNAs of a cell, PCR amplification of miR-205, purification, denaturation of a PCR product and annealing; T7E1 enzyme digestion test is employed to determining shearing efficiency of a CRISPR-Cas9 system on miR-205; a verified optimized a miR-205 gene knockout CRISPR-Cas9 target sequence is obtained; and the kit is constructed on the basis of the target sequence and can be used for directional knockout of miR-205 genes. The kit has the characteristics of high gene knockout efficiency, fast speed, easiness and economic performance and has wide prospects in the aspects of construction of animal models and clinical research of medical science.
Description
technical field [0001] The invention belongs to the field of genetic engineering and relates to a miR-205 gene knockout kit, in particular to a miR-205 gene knockout kit based on CRISPR-Cas9 gene knockout technology. Background technique [0002] In 1987, when Osaka University (Osaka University) was studying a bacterial-encoded alkaline phosphatase (alkalinephosphatase) gene, it was found that there was a tandem spaced repeat DNA sequence near the coding region of the gene. In 2002, it was officially called "Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)" by scientists. [0003] CRISPR is a special family of DNA repeat sequences widely distributed in bacterial and archaeal genomes. CRISPR sites are usually composed of short highly conserved repeats (repeats), the length of the repeats is usually 21-48bp, and the repeats are separated by 26-72bp spacers. CRISPR recognizes target genes through these spacers. Cas (CRISPR associated) exists near the CRISPR...
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