CN110305863B - sgRNA, recombinant plasmid and cell strain for up-regulating expression of non-coding RNA of human DLK1-DIO3 imprinting domain - Google Patents
sgRNA, recombinant plasmid and cell strain for up-regulating expression of non-coding RNA of human DLK1-DIO3 imprinting domain Download PDFInfo
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Abstract
The invention discloses sgRNA, recombinant plasmid and cell strain for up-regulating non-coding RNA expression of human DLK1-DIO3 imprinting domain. The sequence of sgRNA is shown as SEQ ID NO.1, specifically 5'-TTTATATGGAGGCGCAGAAG-3'; synthesizing, synthesizing a nucleic acid fragment of a sgRNA sequence, inserting the nucleic acid fragment into a multiple cloning site of an MS2-P65-HSF1 expression plasmid vector, transforming, selecting a monoclonal strain, extracting an MS2-P65-HSF1-sgRNA-DLK1-DIO3 recombinant plasmid, transfecting the recombinant plasmid to a target cell strain which is transfected with a dCAS9-VP64 plasmid in advance, and obtaining a cell strain which can up-regulate the expression of non-coding RNA of a DLK1-DIO3 imprinting domain. The invention can quickly, simply, conveniently and accurately up-regulate the expression of the non-coding RNA on the DLK1-DIO3 imprinting domain in the cell strain.
Description
Technical Field
The invention relates to gene editing and application thereof, in particular to a special sgRNA primer sequence for up-regulating DLK1-DIO3 imprinting domain non-coding RNA expression and application thereof.
Technical Field
Gene editing technology is a genome modification technology developed in recent years, and operations such as InDel mutation, knock-in, multi-site mutation, deletion of small fragments, and fragment replacement can be performed at specific sites of a genome. In the field of scientific research, gene editing technology can be used for rapid construction of model organisms, such as construction of specific gene knockout mice; in the agricultural field, the technology can be used for optimizing and modifying animal and plant varieties; in the field of medical health, the technology also has the aim of treating diseases from the source by modifying human self genes. Therefore, the gene editing technology has extremely wide application value and development prospect.
CRISPR/Cas belongs to the third generation gene editing technology, which is derived from the naturally occurring prokaryotic adaptive immune system. 8 month 2012, USA JiaThe working principle of this system, which was first reported by researchers at the Burkeley university of State and the Hannover medical college, Germany in the journal Science, was primarily directed to the guide RNA sequence (i.e., sgRNA) to effect the editing of a particular gene. The CRISPR/Cas system can realize efficient and reliable RNA guide genome modification in a plurality of mammal systems after years of development, thereby greatly improving the convenience of genome editing and genome regulation. The first published articles on CRISPR systems all describe how to cut DNA using CRISPR systems. Qi and Jaenisch et al have recently constructed a nuclease-free active Cas9 protein (dCas9-VP64) fused with activator proteins (VP48, VP64, VP96, p65), and the expression of downstream target genes can be enhanced by targeting the gene promoter sequence with sgRNA and recruiting transcription factors by the activator proteins. Jaenisch et al named this technique "CRISPR-on" (www.crispr- on.org)。
The development and progression of DLK1-DIO3 imprinted domain non-coding RNAs and tumors was associated, in particular, with maternal expression of imprinted genes (mainly non-coding RNAs: e.g., miRNAs and a few lncRNAs). These non-coding RNAs are transcribed in the same direction and share a common imprinted regulatory region, intergenic DMR (IG-DMR). In the existing research, most reports suggest that the expression of the non-coding RNA of the DLK1-DIO3 imprinting domain in the tumor is inhibited, and suggest that the RNA may play a role in inhibiting cancer. Their transcription and expression can thus be globally upregulated using the CRISPR-on system, the key to sgRNA design and effectiveness.
Disclosure of Invention
In order to solve the problems in the background art, the invention aims to provide a specific sgRNA sequence aiming at a DLK1-DIO3 imprinting domain by utilizing a CRISPR-on technology and considering the characteristics of clustered distribution and simultaneous transcription of non-coding RNA of the DLK1-DIO3 imprinting domain, so that the expression of the non-coding RNA on the DLK1-DIO3 imprinting domain in a cell strain can be quickly, simply, conveniently and accurately up-regulated, and the transcription and the expression of the non-coding RNA can be further comprehensively up-regulated by utilizing a CRISPR-on system. Another objective of the invention is to provide a recombinant plasmid MS2-P65-HSF1-sgRNA-DLK1-DIO 3. The invention also provides cell lines that up-regulate the expression of non-coding RNAs on the imprinting domain of DLK1-DIO 3.
The technical scheme of the invention is as follows:
a sgRNA sequence designed for DLK1-DIO3 imprinting domain promoter region: the sequence is shown in SEQ ID NO.1, specifically 5'-TTTATATGGAGGCGCAGAAG-3', and the sgRNA is named as sgRNA (DLK1-DIO 3). Located at the upstream of the transcription start site of the non-coding RNA on the DLK1-DIO3 imprinting domain from-32 to-13 and has the length of 20 nt.
The first base at the 5' end of the sgRNA sequence needs to be G, if the first base is not G, a base G needs to be added in front of the first base, a CACC needs to be added as a joint for a forward primer, an AAAC needs to be added as a joint for a reverse primer, and after the sgRNA sequence of a target region is determined, a strand complementary with the sgRNA sequence is designed by taking the sgRNA sequence as a template.
The sgRNA (DLK1-DIO3) specifically targets a DLK1-DIO3 imprinting domain promoter region, and the expression level of DLK1-DIO3 imprinting domain non-coding RNA is up-regulated in a target cell strain pre-transfected with dCAS9-VP64 plasmid.
The invention comprises application of the sgRNA sequence in up-regulating the expression quantity of DLK1-DIO3 imprinting domain non-coding RNA.
The invention includes MS2-P65-HSF1-sgRNA-DLK1-DIO3 recombinant plasmids comprising the sgRNA sequence of claim 1.
The invention comprises a construction method of the MS2-P65-HSF1-sgRNA-DLK1-DIO3 recombinant plasmid, wherein the sgRNA is inserted into a multiple cloning site of an expression plasmid vector of MS2-P65-HSF1 and is transformed, and the transformation refers to clone amplification, so that the MS2-P65-HSF1-sgRNA-DLK1-DIO3 recombinant plasmid is obtained.
The invention comprises application of the MS2-P65-HSF1-sgRNA-DLK1-DIO3 recombinant plasmid in preparation of a cell strain for up-regulating expression of DLK1-DIO3 imprinted domain non-coding RNA.
Secondly, a method for constructing a cell strain for up-regulating the expression of DLK1-DIO3 imprinted domain non-coding RNA, which comprises the following steps:
(1) synthesizing the sgRNA sequence of claim 1;
(2) inserting the nucleic acid fragment synthesized in the step (1) into a multiple cloning site of an MS2-P65-HSF1 expression plasmid vector, transforming, selecting a monoclonal strain, extracting an MS2-P65-HSF1-sgRNA-DLK1-DIO3 recombinant plasmid, and sequencing and identifying to obtain a correctly sequenced MS2-P65-HSF1-sgRNA-DLK1-DIO3 recombinant plasmid;
(3) transfecting the MS2-P65-HSF1-sgRNA-DLK1-DIO3 recombinant plasmid obtained in the step (2) into a target cell strain which is transfected with a dCAS9-VP64 plasmid in advance to obtain a cell strain which can up-regulate the expression of non-coding RNA of the DLK1-DIO3 imprinting domain.
In addition, the invention also comprises a cell strain which is constructed by the method and used for up-regulating the expression of the DLK1-DIO3 imprinting domain non-coding RNA.
The cell strain is preferably a human bladder cancer cell strain.
The invention has the beneficial effects that:
the specific sgRNA sequence designed and constructed by the invention can target a DLK1-DIO3 imprinting domain promoter region, and can quickly, simply, conveniently and accurately up-regulate the expression of non-coding RNA on a DLK1-DIO3 imprinting domain in a cell strain based on a CRISPR-on system.
Drawings
FIG. 1 is a schematic diagram of the construction process of MS2-P65-HSF1-sgRNA-DLK1-DIO3 recombinant plasmid;
FIG. 2 is a graph showing the determination of the expression level of non-coding RNA in the imprinting domain of DLK1-DIO3 in UM-UC-3 cells of bladder cancer. As the non-coding RNA on the DLK1-DIO3 imprinted domain contains more than 50 miRNAs, part of miRNAs are randomly selected for quantitative detection in the experiment and comprise miR-300, miR-323B, miR-381, miR-409, miR-433, miR-487A and miR-539. Statistical tests determined that the expression level of these non-coding RNAs, located on the imprinting domain of DLK1-DIO3, was significantly up-regulated.
Detailed Description
The invention is further illustrated below in connection with specific embodiments, it being understood that the following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention.
The specific embodiment of the invention is as follows:
and culturing the UM-UC-3 cells with good growth state, plating the UM-UC-3 cells one day before virus infection, and adding dCAS9-VP64 lentiviral particles according to the group of experimental design on the day of infection to carry out the infection experiment of the target cells. Puromycin screening is carried out 3 days after infection, sgRNA (DLK1-DIO3) lentivirus is infected, G418 screening is carried out, and cell collection detection or related functional study is carried out.
Experimental Material
i. Reagent
Culture medium (DMEM, Hyclone), Fetal Bovine Serum (FBS), PBS, pancreatin, DMSO, RNAiosofor Small RNA, One Step
miRNA cDNA Synthesis Kit、
Premix Ex TaqTM II, primer, chloroform, ethanol, isopropanol.
An apparatus:
pipette (1000. mu.l, 200. mu.l, Gilson; 100ml, BIOHIT), CO2An incubator, an inverted microscope, a biological safety cabinet, a-80 ℃ low-temperature refrigerator, a common refrigerator, a thermometer (0-100 ℃), a centrifuge, a constant-temperature circulating water bath box, a liquid nitrogen tank and a fluorescent quantitative PCR amplification instrument.
Experimental procedure
i. Preparing the cells of interest
1.1 cell Resuscitation
1) Taking out the cell freezing tube from the liquid nitrogen tank;
2) quickly putting into 37 ℃ water bath to quickly thaw;
3) after complete thawing, centrifuging at 1000rpm for 2 min;
4) wiping the freezing tube with 70% alcohol for disinfection, and moving to a super clean bench;
5) removing supernatant of the frozen stock solution by suction, adding 1ml of fresh complete culture medium to suspend cells, inoculating the cell suspension into a 6-hole plate containing 3ml of complete culture medium, gently shaking uniformly, and culturing in a 5% CO2 incubator at 37 ℃;
6) the culture solution is replaced once the next day and then the culture is continued.
1.2 cell passages
1) Passaging cells grown to 90% confluence;
2) discarding the old culture solution, adding 2ml of sterilized D-Hank's solution, washing the cell growth surface, and then discarding the solution;
3) adding 1ml pancreatin digestive juice, and digesting at 37 deg.C for about 1-2min until the cells are completely digested;
4) adding 2ml of complete culture medium, blowing and beating for a plurality of times by using a graduated pipette, and washing off cells on the wall;
5) after mixing the cells evenly, dividing the cells into two new 6-cm dish, supplementing the complete culture medium to 4ml, and continuing to culture.
um-UC-3 cell lentivirus infection
1) Carrying out pancreatin digestion on target cells in a logarithmic growth phase to prepare a cell suspension;
2) suspending the cells (cell number about 5X 10)4) Inoculating in 6-well plates, 5% CO at 37 ℃2Culturing in an incubator until the cell fusion degree reaches about 30%;
3) adding a proper amount of virus according to the MOI value of the cells;
4) cell status was observed after 12 h: if no obvious cytotoxicity exists, the culture medium is replaced after the culture is continued for 24 hours; if the cytotoxicity is obvious, the culture medium is immediately replaced;
5) 3 days after infection, the appropriate concentration of puromycin is added for screening for 3 days, then culture is continued with the maintenance of low concentration of puromycin, and sgRNA (DLK1-DIO3) lentivirus is further infected, and G418 screening is carried out.
Quantitative PCR assay
1) Small RNA extraction
After collecting cells, 200ul of chloroform was added to an EP tube, vortexed for 20 seconds, the solution was thoroughly mixed and then allowed to stand on ice for 5min, followed by centrifugation at 4 ℃ for 15 min.
After centrifugation, the supernatant was divided into three layers, RNA was in the supernatant, so that approximately 200. sup. & 250. mu.l of supernatant was gently aspirated and transferred to a new 1.5ml EP tube.
600ul of isopropanol was added to the EP tube of the previous step, vortexed for 15 seconds to mix thoroughly, and then placed in a low temperature freezer at-20 ℃ for 10 min. 12000g at 4 ℃ for 10 min. The isopropanol was carefully discarded and a white precipitate was visible at the bottom of the tube, i.e., Small RNA.
The precipitate was washed with 1ml of 75% ethanol to remove residual organic reagent. 12000g were centrifuged at 4 ℃ for 5min and the alcohol carefully discarded.
Reverse-covering the EP tube on a paper towel at normal temperature, drying RNA precipitate for about 10-15min, and adding appropriate amount of DEPC water (40-60ul) to dissolve precipitate.
The concentration of sample RNA and OD260/280 values (optimally 1.8-2.0) were determined using NanoDrop 2000.
2) Reverse transcription reaction
Small RNA was reverse-transcribed into cDNA using the One Step PrimeScript miRNA cDNA Synthesis Kit. The reaction system is as follows:
TABLE 1 One Step PrimeScript miRNA eDNA Synthesis Kit reaction System
Reaction conditions are as follows: 60min at 37 ℃ (tailing and reverse transcription)
5s at 85 ℃ (enzyme activity removed)
Adding RNase Free dH into the reaction product2Diluting O to 100ul for further quantitative PCR detection, or freezing in a low-temperature refrigerator at-20 ℃ for later use.
3) Quantitative PCR
The kit adopts
Premix Ex TaqTMII (perfect Real Time), done on ABI 7500 Fastclean-Time PCR amplificator. The internal reference is U6small RNA, and the relative expression quantity of miRNA in a treatment group compared with a control group is 2-ΔΔCtThe method was calculated and expressed in fold format. The calculation formula is as follows:ΔΔct ═ (Ct (mirna) -Ct (U6)) cancer tissue- (Ct (mirna) -Ct (U6)) paracancerous tissue; folds ═ 2-ΔΔCt。
The Real Time PCR reaction is as follows:
TABLE 2 quantitative PCR reaction System
The cycle conditions are 95 ℃ for 5s and 60 ℃ for 34s, and the results are analyzed after 40 cycles of amplification.
EXAMPLES expression levels of non-coding RNAs on the DLK1-DIO3 imprinting domain in UM-UC-3 cells of bladder cancer are shown in FIG. 2, which shows that 7 non-coding RNAs on the DLK1-DIO3 imprinting domain are randomly selected, and include miR-300, miR-323B, miR-381, miR-409, miR-433, miR-487A and miR-539. It can be seen that quantitative PCR analysis and statistical tests determined that the expression level of non-coding RNA on the DLK1-DIO3 imprinting domain was significantly up-regulated.
Sequence listing
<110> Zhejiang university
<120> sgRNA, recombinant plasmid and cell strain for up-regulating expression of non-coding RNA of human DLK1-DIO3 imprinting domain
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
tttatatgga ggcgcagaag 20
Claims (7)
1. A sgRNA sequence designed for DLK1-DIO3 imprinted domain promoter region, characterized in that: the sequence is shown as SEQ ID NO.1, specifically 5'-TTTATATGGAGGCGCAGAAG-3', the sgRNA is named as sgRNA (DLK1-DIO3), is positioned at the upstream of a non-coding RNA transcription start site on a DLK1-DIO3 imprinting domain from-32 to-13, and has the length of 20 nt.
2. Use of the sgRNA sequence of claim 1 to up-regulate the expression level of DLK1-DIO3 imprinted domain non-coding RNA.
3. An MS2-P65-HSF1-sgRNA-DLK1-DIO3 recombinant plasmid, which is characterized in that: the recombinant plasmid comprises the sgRNA sequence of claim 1.
4. A method for constructing the recombinant plasmid MS2-P65-HSF1-sgRNA-DLK1-DIO3 as claimed in claim 3, wherein the method comprises the following steps: the sgRNA is inserted into the multiple cloning site of an MS2-P65-HSF1 expression plasmid vector to obtain an MS2-P65-HSF1-sgRNA-DLK1-DIO3 recombinant plasmid.
5. The use of the MS2-P65-HSF1-sgRNA-DLK1-DIO3 recombinant plasmid of claim 3 in the preparation of a cell strain that upregulates the expression of DLK1-DIO3 imprinted domain non-coding RNA.
6. A method for constructing a cell strain for up-regulating the expression of DLK1-DIO3 imprinted domain non-coding RNA, which is characterized by comprising the following steps:
(1) synthesizing the sgRNA sequence of claim 1;
(2) inserting the nucleic acid fragment synthesized in the step (1) into a multiple cloning site of an MS2-P65-HSF1 expression plasmid vector, transforming, selecting a monoclonal strain, extracting an MS2-P65-HSF1-sgRNA-DLK1-DIO3 recombinant plasmid, and sequencing and identifying to obtain a correctly sequenced MS2-P65-HSF1-sgRNA-DLK1-DIO3 recombinant plasmid;
(3) transfecting the MS2-P65-HSF1-sgRNA-DLK1-DIO3 recombinant plasmid obtained in the step (2) into a target cell strain which is transfected with a dCAS9-VP64 plasmid in advance to obtain a cell strain which can up-regulate the expression of non-coding RNA of the DLK1-DIO3 imprinting domain.
7. A cell line that up-regulates the expression of DLK1-DIO3 imprinted domain non-coding RNA, characterized by:
the method according to claim 6.
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