阅读说明：本技术 基于CRISPR-Cas13a系统特异性靶向F3-T3融合基因的crRNA及应用 (CRRNA (crribonucleic acid) of specific targeting F3-T3 fusion gene based on CRISPR-Cas13a system and application ) 是由 康春生 武烨 王琦雪 于 2021-07-09 设计创作，主要内容包括：本发明创造提供了基于CRISPR-Cas13a系统特异性靶向F3-T3融合基因的crRNA及应用,所述crRNA的序列如SEQID NO：1所示。本发明创造所述的crRNA介导的CRISPR-Cas13a基因编辑系统能够明显抑制胶质瘤细胞增殖。(The invention provides CRRNA based on CRISPR-Cas13a system specificity targeting F3-T3 fusion gene and application thereof, wherein the sequence of the CRRNA is shown as SEQID NO: 1 is shown. The CRRNA-mediated CRISPR-Cas13a gene editing system can obviously inhibit glioma cell proliferation.)

1. A CRRNA specifically targeting F3-T3 fusion gene based on CRISPR-Cas13a system, characterized in that: the sequence of the crRNA is shown as SEQ ID NO: 1 is shown.

2. The crRNA-mediated CRISPR-Cas13a gene editing system of claim 1 is applied.

3. The use of the crRNA-mediated CRISPR-Cas13a gene editing system of claim 2 to inhibit tumor cell proliferation or kill tumor cells.

4. Use of a crRNA-mediated CRISPR-Cas13a gene editing system according to claim 3, characterized in that: the crRNA-mediated CRISPR-Cas13a gene editing system inhibits tumor cell proliferation or kills tumor cells by triggering a side effect to cause RNA degradation.

5. Use of a crRNA-mediated CRISPR-Cas13a gene editing system according to claim 3, characterized in that: the tumor cells are glioma cells.

6. Use of a crRNA-mediated CRISPR-Cas13a gene editing system according to claim 3, characterized in that: the glioma cells are glioblastoma cells.

7. Use of a crRNA-mediated CRISPR-Cas13a gene editing system according to claim 3, characterized in that: the glioma cells are human glioma cells U87 cells or TBD0220 cells.

8. The use of the crRNA-mediated CRISPR-Cas13a gene editing system of claim 2 to specifically target FGFR3-TACC3 fusion genes.

9. The use of a crRNA-mediated CRISPR-Cas13a gene editing system according to claim 8, wherein: the expression vector of the Cas13a gene in tumor cells is a lentivirus expression vector.

10. Use of the crRNA-mediated CRISPR-Cas13a gene editing system according to claim 9, characterized in that: the lentiviral expression vector was p GV 341.

Technical Field

The invention belongs to the field of biology, and particularly relates to CRRNA of a CRISPR-Cas13a system-based specific targeting F3-T3 fusion gene and application thereof.

Background

Cas13a is an RNA-guided CRISPR effector that targets and cleaves single-stranded RNA (ssrna) and has attracted considerable attention as a therapeutic and diagnostic tool for RNA virus infectious diseases and malignancies. The CRISPR-Cas13a system consists of two parts, including RNA-targeting CRISPR-RNA (crRNA) and the rnase Cas13a guided by the crRNA. To date, the CRISPR-Cas13a system is considered to be a method of directly inhibiting gene expression at the transcriptional level. Cas13a provides a potentially safer alternative to Cas9, as it results in loss of functional phenotype, but does not disrupt the genome. More importantly, Cas13a has a unique "side-cleavage" effect: upon recognition of its target RNA, activated Cas13a exhibits cleavage not only of the target RNA, but also of non-target RNAs.

The FGFR3-TACC3(F3-T3) fusion gene is reported in a glioblastoma sample for the first time and has become an oncogenic driver of various cancers, including urothelial cancer, non-small cell lung cancer, cervical cancer, head and neck cancer, gastrointestinal cancer malignant tumors and malignant melanoma. It is expected that about 1.2-8.3% of GBM will carry this chromosomal translocation. The genes encoding FGFR3 and TACC3 are located 48kb apart in human chromosome 4p16 and represent the most common FGFR-TACC chromosomal rearrangement in glioblastomas. FGFR-TACC fusion is an effective oncogene, and has a growth promoting effect and induces aneuploidy. However, its intracellular signaling pathway is not yet clear. The prognosis of the tumor patients carrying FGFR-TACC fusion is poor, and the mortality rate is high. Currently, fusion proteins against FGFR3-TACC3 are limited to kinase inhibitors against FGFRs, and these small molecule compounds are typical multi-kinase inhibitors, targeting FGFR and other tyrosine kinases. Therefore, the FGFR3-TACC3 is precisely targeted, so that adverse reactions are reduced, and the method has important significance for targeted therapy of tumors.

Disclosure of Invention

In view of the above, the invention provides crRNA specifically targeting F3-T3 fusion gene based on CRISPR-Cas13a system and application thereof, aiming at overcoming the defects in the prior art.

In order to achieve the purpose, the technical scheme of the invention is realized as follows:

CRRNA specifically targeting F3-T3 fusion gene based on CRISPR-Cas13a system, and the sequence is shown as SEQ ID NO: 1 is shown.

An application of the CRISPR-Cas13a gene editing system mediated by crRNA.

An application of the CRISPR-Cas13a gene editing system mediated by crRNA in inhibiting cancer cell proliferation or killing tumor cells.

Preferably, the crRNA-mediated CRISPR-Cas13a gene editing system inhibits tumor cell proliferation or kills tumor cells by triggering a side effect to cause RNA degradation.

Preferably, the tumor cell is a glioma cell.

More preferably, the glioma cell is a glioblastoma cell.

Preferably, the glioma cell is a human glioma cell U87 cell or TBD0220 cell.

The CRRNA-mediated CRISPR-Cas13a gene editing system is applied to the specific targeting of FGFR3-TACC3 fusion genes.

Preferably, the expression vector of the Cas13a gene in tumor cells is a lentiviral expression vector.

More preferably, the lentiviral expression vector is p GV 341.

Compared with the prior art, the invention has the following advantages:

(1) the CRRNA-mediated CRISPR-Cas13a gene editing system has high target gene knocking efficiency;

(2) the CRRNA-mediated CRISPR-Cas13a gene editing system can trigger side effect;

(3) the CRRNA-mediated CRISPR-Cas13a gene editing system can obviously inhibit glioma cell proliferation;

(4) the CRRNA-mediated CRISPR-Cas13a gene editing system can inhibit the formation of mouse intracranial tumors.

Drawings

FIG. 1 is a graph showing the efficiency of the crRNA1 for knocking down target genes, which is created by the present invention (FIG. 1A shows the efficiency of four crRNAs in knocking down F3-T3 mRNA in U87-F3-T3-Cas13a cells, and FIG. 1B shows the efficiency of four crRNAs in knocking down F3-T3 mRNA in TBD0220-F3-T3-Cas13a cells);

FIG. 2 is a Cas13a-crRNA1 trigger side effect graph (FIG. 2A is an electrophoretogram of total RNA isolated from U87 and TBD0220 cell populations, FIG. 2B is the RNA integrity value determined on Agilent 2100; FIG. 2C is the 28S:18S ratio determined on Agilent 2100) created by the present invention;

fig. 3 is a graph of inhibition of glioma cell proliferation by Cas13A-crRNA1 as invented by the present invention (fig. 3A is a graph of colony formation analysis in U87 and TBD0220 cells, fig. 3B is a graph of quantification of colony numbers in fig. 3A, fig. 3C is a growth curve of U87 cells measured with CCK8, and fig. 3D is a growth curve of TBD0220 cells measured with CCK 8);

FIG. 4 shows the formation of Cas13a-crRNA1 inhibiting intracranial tumors in mice (FIG. 4A is a representative bioluminescent image of mice implanted with intracranial tumors taken at days 7, 14 and 21 after implantation; FIG. 4B is a quantitative signal intensity graph of bioluminescence of mice implanted with intracranial tumors taken at days 7, 14 and 21 after implantation; and FIG. 4C is a survival curve of mice).

Detailed Description

Unless defined otherwise, technical terms used in the following examples have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods are conventional methods unless otherwise specified.

The invention will be described in detail with reference to the following examples.

Example 1 design of crRNA

Following the imbricated design principle, a crRNA library targeting specific fragments was designed, containing a total of 4 crrnas:

crRNA1：

5’-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACCCUUUACGUCGGUG GACGUCACGGUAAG-3’

crRNA2：

5’-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACUCGCCUUUACGUCG GUGGACGUCACGGU-3’

crRNA3：

5’-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACGUGUCGCCUUUACG UCGGUGGACGUCAC-3’

crRNA4：

5’-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACCCUGUGUCGCCUUU ACGUCGGUGGACGU-3’

example 2 screening for optimal crRNA sequences

The computer simulates the butt joint of the crRNA, the Cas13a protein and the target RNA, calculates the binding energy of the Cas13a-crRNA and Cas13a-crRNA-RNA compound, and performs screening according to the size of the binding energy, wherein the smaller the binding energy is, the more stable the compound is, and the crRNA1 with the minimum binding energy is the potential optimal crRNA sequence screened by the computer.

Example 3 packaging of Cas13a lentivirus

The Cas13a lentiviral expression vector was chosen to be p GV 341. Transient co-transfection method is adopted, transfection systems containing Cas13a expression plasmid and packaging plasmid are transferred into HEK293T cells, and slow virus packaging is carried out in the cells. The packaged lentivirus particles are secreted into extracellular culture supernatant, and the supernatant is collected to obtain lentivirus.

EXAMPLE 4 Synthesis of crRNA

(1) DNA templates for crRNA were generated by PCR using T7 flanking primers. Primer extension was performed by denaturing the reaction mixture by heating at 95 ℃ for 5 minutes, followed by quenching on ice for 10 minutes and incubating at 72 ℃ for 30 minutes.

(2) The T7 PCR product was transcribed in vitro using the T7 sgRNA MICscript TM kit (Biomics Biotech, Jiangsu, China).

(3) The transcribed crRNA was purified using the EzOmicsTM RNA Quick Clear kit (Biomics Biotech, Jiangsu, China).

Example 5 cell culture and transfection

Human glioma cells U87 cells and TBD0220 cells were cultured in DMEM and DMEM-F12 medium at 10% FBS, and 1% penicillin-streptomycin. Placing at 37 ℃ and 5% CO₂The medium was changed 1 time every 1 day. With Lipofectamine R3000 (I)nvitrogen, california, usa) to transfect crRNA into cells.

Example 6 efficiency of detecting crRNA knockdown of Gene of interest at cellular level Using qRT-PCR

Cells were lysed in TRIzol reagent (Invitrogen, california, usa). According to TRIzol: volume of chloroform 5: chloroform was added at a ratio of 1, shaken for 10 seconds, and allowed to stand for 2 minutes. Centrifuge (4 ℃) for 15min at 12,000g and take the supernatant. Adding appropriate amount of isopropanol, mixing, and standing for 10 min. Centrifuge (4 ℃ C.) at 12,000g for 10min and discard the supernatant. Adding appropriate amount of 75% ethanol, gently washing the precipitate, centrifuging at 7500g (4 deg.C) for 5min, and discarding the supernatant. Adding appropriate amount of DEPC H after drying₂O dissolves the RNA. Then, cDNA synthesis was performed using a reverse transcription kit (RR047A, TaKaRa, Japan). The qRT-PCR experiments were performed on a DNA Engine Opticon 2 two-color qRT-PCR detection system using SYBR Green Master Mix reagents (Life Technologies). The PCR primers used included:

FGFR3-TACC3-F:5’-CCAACTGCACACACGACCT-3’；

FGFR3-TACC3-R:5’-TCCTCCTGTGTCGCCTTTAC-3’；

GAPDH-F:5’-TGCACCACCAACTGCTTAGC-3’；

GAPDH-R:5’-GGCATGGACTGTGGTCATGAG-3’；

the detection result is shown in FIG. 1, and as can be seen from FIG. 1A and FIG. 1B, the crRNA1 of the present invention has the highest efficiency for knocking down the mRNA of the target gene F3-T3 in U87-F3-T3-Cas13a and TBD0220-F3-T3-Cas13a cells.

Example 7 Agilent 2100 Mass control verification side Effect

RNA was extracted by the method of example 6 and tested on the Agilent 2100 quality control platform. The results are shown in fig. 2, and it can be seen from fig. 2 that Cas13a-crRNA1 of the present invention can trigger side effects and cause extensive RNA degradation.

Example 8 cell level validation that Cas13a-cr1 can inhibit glioma cell proliferation

(1) Plate cloning: taking tumor cells in good growth state, digesting the tumor cells by using 0.25% trypsin, centrifuging the tumor cells, then resuspending the cells by using a culture medium and blowing the cells into a single cell suspension for later use. Diluting the cell suspension by multiple times, respectively inoculating the cells of each group according to the number of 50, 100 and 200 cells per six-hole plate, wherein the holes contain 2mL of culture medium, and then shaking the cells until the cells are uniformly dispersed. Then cultured in an incubator for about 2 weeks. During which time the solution is changed. It is often observed that the culture is terminated when macroscopic clumps of cells appear in the wells. The supernatant was aspirated off, washed 2 times with PBS and the medium was removed. Cells were washed with 4% paraformaldehyde and fixed for 15 minutes at room temperature. The fixative was then aspirated off, a small amount of crystal violet stain was added to submerge the cells, room temperature 15 minutes, then the stain was washed indirectly with a slow flow to see good clones, and air dried. The six well plate was inverted and a piece of film with a grid was placed underneath, the number of clones counted and photographed. The detection results are shown in FIGS. 3A-3B, and it can be seen from FIGS. 3A-3B that Cas13A-crRNA1 of the present invention can significantly inhibit the proliferation of glioma.

(2) CCK-8 method cell activity test: in 96 well plates, 100ul of cell suspension containing 2000-3000 tumor cells in logarithmic growth phase was added per well. The plates were cultured in an incubator for 24 hours (37 ℃ C., 5% CO)₂Incubation conditions of (a). Plates were grouped as required with 3-5 replicate wells per group. 10ul of different treatments were added to each well of the plate. The plates were incubated in the incubator for the appropriate time (experiment time 48 hours). After incubation, 10ul of CCK-8 solution was added to each well suspension (care was taken not to generate air bubbles in the wells, affect the O.D. value, and contact the cells at the bottom of the plate, to avoid cell death). Incubation of the 96-well plates in the incubator was continued for 1-4 hours as required. The absorbance at 450nm was measured with a microplate reader and the data was recorded. The growth curve test results are shown in FIGS. 3C-3D, and it can be seen from FIGS. 3C-3D that the Cas13a-crRNA1 of the present invention can significantly inhibit the proliferation of U87 and TBD0220 cells.

Example 9 animal level validation that Cas13a-cr1 can inhibit intracranial tumor formation

Five-week old female nude mice (institute for tumor, national academy of medical sciences) were used to establish intracranial in situ tumor models. U87 and U87-FGFR3-TACC3 cells were used as cellular models. The cultured tissue is implanted into the intracranial area of a nude mouse in a stereotactic manner by using a stereotactic instrument of a small animal. The operation is performed in a sterile environment, comprising the steps of: mice were anesthetized with gas, the skin of the head was then incised, a hole was drilled 2mm to the right of the midline of the skull, 50 ten thousand cells were then injected 2mm into the cranium with the aid of a stereotaxic apparatus, and the skin was finally sutured. On days 7, 14, 21, mice were tested for intracranial tumor growth status using bioluminescent imaging. The test results are shown in figures 4A-4C, and the test results show that the Cas13a-crRNA1 group can obviously inhibit the formation of intracranial tumors of mice and improve the survival rate and the survival time of the mice.

In conclusion, the CRISPR-Cas13a guided by crRNA can improve the efficiency of knocking down target genes and trigger side effects, thereby causing extensive RNA degradation, inhibiting the proliferation of glioma cells and inhibiting the formation of intracranial tumors.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and should not be taken as limiting the invention, so that any modifications, equivalents, improvements and the like, which are within the spirit and principle of the present invention, should be included in the scope of the present invention.

Sequence listing

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