CN111041049A - Preparation method and application of CRISPR-Cas13a system based on near-infrared light control - Google Patents
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Abstract
The invention discloses a preparation method and application of a CRISPR-Cas13a system based on near-infrared light control, wherein crRNA is synthesized according to the sequence design of target gene mRNA, a Cas13a-crRNA compound is formed by the crRNA and purified Cas13a protein, and UCNPs-Cas13a is obtained by connecting the Cas13a-crRNA compound and an up-conversion nano material through photosensitive molecules; coating UCNPs-Cas13a with PEI to obtain UCNPs-Cas13a @ PEI; the prepared UCNPs-Cas13a @ PEI is attached to the surface of a eukaryotic cell and enters the eukaryotic cell, and the escape of endosomes is promoted by the PEI of the UCNPs-Cas13a @ PEI; cas13a-crRNA is released under NIR laser irradiation, a complementary sequence corresponding to a crRNA spacer sequence is searched on target gene mRNA, the crRNA is matched with the target gene mRNA to activate HEPN catalytic sites of Cas13a to shear the mRNA, and further target gene expression of eukaryotes is inhibited.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a preparation method and application of a CRISPR-Cas13a system based on near infrared light control.
Background
Along with the completion of sequencing of the whole genome of food microorganisms such as saccharomycetes, mycete and the like, the rapid and efficient analysis of gene functions becomes a problem to be solved by researchers. In order to solve the problem, the gene function is explained by inhibiting gene expression by using technologies such as RNA interference, CRISPR-Cas9 and the like, but the methods have some defects, such as complex operation, easy off-target effect generation, low inhibition efficiency and the like, so that the development of a novel gene expression inhibition method is urgent.
The simple, rapid and efficient near-infrared light-controlled CRISPR-Cas13a system can remotely control the release of a Cas13a-crRNA compound from an up-conversion nano material through near-infrared light irradiation, search target matching and further activate a HEPE catalytic site of a Cas13a protein, achieve the purpose of cutting target gene mRNA to inhibit gene expression, and provide a new thought and a new method for analyzing gene functions. Compared with other gene expression inhibiting tools, the method is easier to control in time, space and efficiency, and can simultaneously edit mRNA of a plurality of genes.
The CRISPR-Cas13a system controlled by near infrared light is used for inhibiting the expression of key genes in the metabolic pathway of synthesizing food microorganisms such as yeast, mould and the like, and a new method is provided for promoting the rapid development of the food biotechnology and the metabolic engineering field.
Disclosure of Invention
In order to solve the defects in the prior art, the invention provides a preparation method of a near-infrared light-controlled CRISPR-Cas13a system and application thereof in inhibiting eukaryotic gene expression, wherein the unique optical property of an upconversion fluorescent nano material and the remote dynamic regulation and control of a Cas13a-crRNA compound connected with photosensitive molecules are utilized, and the near-infrared light-controlled CRISPR-Cas13a system is adopted for RNA editing; the method has strong specificity and high stability, and can realize the degradation of target gene mRNA and the inhibition of gene expression level in eukaryotic cells.
The technical scheme adopted by the invention is as follows:
a CRISPR-Cas13a system preparation method based on near-infrared light control comprises the steps of synthesizing crRNA according to the sequence design of target gene mRNA, forming a Cas13a-crRNA compound by the crRNA and purified Cas13a protein, and connecting the Cas13a-crRNA compound and an up-conversion nano material together through photosensitive molecules to obtain UCNPs-Cas13 a; coating the outer layer of UCNPs-Cas13a with polyethyleneimine PEI to obtain UCNPs-Cas13a @ PEI;
further, designing the crRNA into a gRNA library, editing mRNA of a plurality of genes, and realizing the inhibition of the expression of the plurality of genes;
further, the sequence format of the crRNA is 5 '-Cas 13a protein direct repeat-crRNA spacer-3'; wherein, the direct repeat sequence of the Cas13a protein depends on the source of Cas13 a;
further, the Cas13a protein is selected from LshCas13a protein, LwCas13a protein or LbuCas13a protein; the Cas13a protein specifically binds and cleaves target gene mRNA under the guidance of crRNA, and a simple, rapid and efficient CRISPR-Cas13a system which takes Cas13a and crRNA as main components can replace RNA interference to be used as an RNA editing tool.
Further, the upconversion nanometer material is synthesized by adopting a one-step solvothermal method to obtain the carboxyl modified upconversion nanometer material (UCNP)S)；
Further, the photosensitive molecule is an ONA molecule, and two ends of the ONA molecule are respectively connected with the Cas13a-crRNA compound and the up-conversion nano material;
further, under the irradiation of NIR laser, the Cas13a-crRNA compound connected with the photosensitive molecule is released from the up-conversion nano material, so that the dynamic regulation and control of a CRISPR-Cas13a system are realized;
an application of the CRISPR-Cas13a system prepared by the preparation method of the near-infrared light-controlled CRISPR-Cas13a system in inhibiting eukaryotic gene expression in non-disease diagnosis and treatment is as follows: the UCNPs-Cas13a @ PEI can enter cells through endocytosis after being combined with the surfaces of eukaryotic cells, and the escape of endosomes is promoted by the PEI coated with the UCNPs-Cas13a @ PEI; cas13a-crRNA is released under NIR laser irradiation, a complementary sequence corresponding to a crRNA spacer sequence is searched on target gene mRNA, the crRNA is matched with the target gene mRNA to activate HEPN catalytic sites of Cas13a to shear the mRNA, and further target gene expression of eukaryotes is inhibited.
Further, the presence of a plurality of amine groups in the PEI structure can increase endosomal osmotic pressure, cause ion influx, cause membrane rupture, and thus can enhance endosomal escape.
The invention has the beneficial effects that:
1. the invention discloses a near-infrared light control-based CRISPR-Cas13a system for inhibiting eukaryotic gene expression. The method mainly comprises the steps of designing and synthesizing crRNA according to a sequence of target gene mRNA, forming a Cas13a-crRNA compound with Cas13a protein, connecting the crRNA compound with a carboxyl modified up-conversion nano material through photosensitive molecules, coating an outer layer with PEI to obtain UCNPs-Cas13a @ PEI, enabling the UCNPs-Cas13a @ PEI to enter cells through endocytosis when the UCNPs-Cas 13-Cas 14-PEI is combined with surfaces of eukaryotic cells, enabling PEI to promote endosome escape, searching a complementary sequence of the crRNA on the target gene mRNA by the Cas13a-crRNA compound released under NIR laser irradiation, matching the complementary sequence with the CRRNA to activate Cas13a to cut the mRNA, and achieving degradation and. The invention releases the unique optical properties of a CRISPR-Cas13a system and an upconversion fluorescent nano material as well as a Cas13a-crRNA compound connected with a remotely and dynamically regulated photosensitive molecule, adopts a near-infrared light-controlled CRISPR-Cas13a system to carry out a new idea of RNA editing, and establishes a new method for inhibiting the gene expression level in eukaryotic cells.
2. The method for inhibiting the eukaryotic gene expression based on the near-infrared light-controlled CRISPR-Cas13a system has the same effect as the RNA interference effect, but has higher specificity and wider application range. In addition, the crRNA is designed into a gRNA library, so that multiple sites of mRNA can be targeted simultaneously, and the inhibition of multiple gene expressions can be realized; compared with the CRISPR-Cas9 editing technology, the encoding gene is not changed, the encoding gene is more controllable in time, space and efficiency, and multiple genes can be modified simultaneously, all mRNA of multiple copies of the genes can be targeted simultaneously, and the method is more efficient.
3. The method for inhibiting the eukaryotic gene expression based on the near-infrared light-controlled CRISPR-Cas13a system can realize remote control. Inside the cell, the delivery system is activated by visible light generated by near infrared light irradiation, and the RNA editing tool is released from the upconversion nanoparticles and is cut after being combined with a target, so that the target gene mRNA is degraded and the gene expression level is inhibited.
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FIG. 1 is a flow chart of the preparation of UCNPs-Cas13a @ PEI;
fig. 2 is a schematic design diagram of a NIR triggered CRISPR-Cas13a delivery system for RNA editing; wherein, (1) attaching to a cell membrane, (2) performing endocytosis, (3) escaping endosome, (4) searching target mRNA in cytoplasm for editing by an RNA editing tool released from the up-conversion nanoparticle, and (5) searching target mRNA in nucleus for editing by the RNA editing tool released from the up-conversion nanoparticle;
FIG. 3A is a map of plasmid pC013-Twinstrep-SUMO-huLwCas13 a; b is an SDS-PAGE map of purified Cas13a protein;
FIG. 4 is a fluorescence spectrum of the upconversion nanomaterial and the solution of NaYF4: Yb/Er nanocrystals shows strong green upconversion luminescence under the excitation of a 980nm laser.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The method for inhibiting the eukaryotic gene expression based on the near-infrared light-controlled CRISPR-Cas13a system is applicable to all eukaryotic organisms, and only Schizosaccharomyces pombe (Schizosaccharomyces pombe) is taken as an example for detailed description in the embodiment.
The invention provides a preparation method of a CRISPR-Cas13a system based on near-infrared light control, which comprises the following specific steps:
the preparation process of the material comprises the following steps:
1. acquisition of LwCas13a protein:
as shown in FIG. 3, the pC013-Twinstrep-SUMO-huLwCas13a plasmid and E.coli expression system were used. The expression and purification process of the LwCas13a comprises the steps of IPTG-induced expression of Cas13a protein, nickel column purification, SUMO enzyme digestion to remove His, dialysis and ion exchange purification, and the concentration of the obtained purer Cas13a protein is determined, wherein in the embodiment, the LwCas13a is selected as the Cas13 a.
2. Preparation of crRNA:
in this example, the transcription product of the endogenous gene, namely, glyceraldehyde-3-phosphate dehydrogenase Tdh1(Tdh1), of Schizosaccharomyces pombe (Schizosaccharomyces pombe) was used as the target gene mRNA for editing, and the specific process was as follows: crRNA is prepared by chemical synthesis. The format of the crRNA sequence is 5 '-Cas 13a protein direct repeat-crRNA spacer-3', wherein the length of the spacer is designed to be 21-28 nucleotides, and the spacer is complementary with a sequence in target gene mRNA; the Cas13a protein direct repeat sequence is dependent on the Cas13a source; when Cas13a is LshCas13a, the direct repeat sequence is 5'-GGCCACCCCAAUAUCGAAGGGGACUAAAAC-3'; when Cas13a is LwCas13a, the direct repeat sequence is 5'-GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAAC-3'; when Cas13a is LbuCas13a, the direct repeat sequence is 5'-GACCACCCCAAAAATGAAGGGGACTAAAAC-3'.
Since the Cas13a in this embodiment is selected from LwCas13a, the crRNA sequence is 5'-GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACGGAUGAAUGAUCUAUACAGAAGCGAUGC-3'; the T7 promoter sequence (5'-TAATACGACTCACTATAGGG-3') was added to the 5 ' end of the crRNA sequence. Transcription template dsDNA was synthesized from crRNA, tdh 1-crRNA-1: 5'-TAATACGACTCACTATAGGGGATTTAGACTACCCCAAAAACGAAGGGGACTAAAACGGATGAATGATCTATACAGAAGCGATGC-3', tdh1-crRNA-2: 5'-GCATCGCTTCTGTATAGATCATTCATCCGTTTTAGTCCCCTTCGTTTTTGGGGTAGTCTAAATCCCCTATAGTGAGTCGTATTA-3'; among them, tdh1-crRNA-1 and tdh1-crRNA-2 were synthesized by Biotechnology engineering (Shanghai) Ltd. The two DNA strands were annealed and then incubated with T7 RNA Polymerase Mix overnight at 37 ℃ using the HiScribe T7Quick High Yield RNA Synthesis kit. The crRNA was purified using the RNA clean & Concentration Kit and the Concentration was determined by Nanodrop 2000.
3. Synthesis of up-conversion nano material:
synthesis of upconversion nanoparticles (UCNPs) using one-step solvothermal methodS) 1.2mmol of RECl3(Y: Yb: Er ═ 80: 18: 2), 2.4mmol of NaCl and 0.8g of PAAs were dissolved in 20mL of ethylene glycol and mixed well to form a clear solution. Adding 5mmol NH4F was dissolved in 15mL of ethylene glycol and added dropwise to the above clear solution with stirring. The resulting mixture was transferred to a reaction kettle and held at 200 ℃ for 1.5 h. Cooling to room temperature, centrifuging, collecting the product, washing with ethanol and distilled water for 3 times, and drying in an oven at 60 deg.C to obtain carboxyl-modified UCNPS。
4. Preparation of UCNPs-Cas13a @ PEI based on the material prepared in the upper section:
as in fig. 1, the purified Cas13a protein and crRNA were mixed in a molar ratio of 1: 2, mixing to form a Cas13a-crRNA compound, and connecting the Cas13a-crRNA compound with an up-conversion nano material through a photosensitive molecule ONA to obtain UCNPs-Cas13 a; and coating the outer layer of the UCNPs-Cas13a with polyethyleneimine PEI (namely polyethyleneimine) to obtain the UCNPs-Cas13a @ PEI.
Based on UCNPs-Cas13a @ PEI prepared by the preparation method, the invention also provides an application of the CRISPR-Cas13a system prepared by the preparation method of the near-infrared light-controlled CRISPR-Cas13a system in inhibiting eukaryotic gene expression in non-disease diagnosis and treatment, and the specific process is shown in figure 2: UCNPs-Cas13a @ PEI is firstly attached to the cell surface of the Schizosaccharomyces pombe and then enters the cell through endocytosis; since the presence of many amine groups in the PEI structure increases endosomal endosmosis pressure, causing ion influx, leading to membrane rupture, the UCNPs-Cas13a @ PEI coated PEI can facilitate endosomal escape.
The NIR laser irradiates the surface of s.pommbe cells, under the irradiation of the NIR laser, the upconversion nanomaterial serves as a 'nano transducer', NIR light (980nm) can be converted into green light (as shown in fig. 4) to cleave the ONA molecules, so that Cas13a-crRNA complexes connected with the ONA molecules are released from the upconversion nanomaterial, the Cas13a-crRNA complexes search matching fragments on target gene mRNA in cytoplasm and nucleus, the binding of the crRNA and the target gene mRNA activates HEPE catalytic sites of the Cas13a protein to cleave the mRNA, and the degradation and the inhibition of the expression level of the target gene mRNA are realized.
In order to verify the effect of the method for inhibiting the eukaryotic gene expression based on the near-infrared light-controlled CRISPR-Cas13a system, unedited S.pombe is taken as a control group, edited S.pombe is taken as an experimental group, total RNA of cells of the unedited S.pombe and the edited S.pombe is extracted to carry out qRT-PCR, the expression level of tdh1 gene is compared, and the gene expression inhibition efficiency is calculated. The primer sequence used was tdh 1-F: 5'-TGCCTAGCATCGCTTCTGTA-3', tdh 1-R: 5'-CATCAATGACGAGCTTACCAT-3' are provided.
Inhibition of the expression level of gene tdh1 results in mitotic cell cycle abnormalities and lysis of vegetative cells, thereby reducing the yeast cell growth rate. The near-infrared light control-based CRISPR-Cas13a system implemented in S.pombe can inhibit gene expression with different efficiencies, and the method serves as a novel RNA editing tool and provides a platform for manipulating target gene mRNA in eukaryotes.
In this example, if it is necessary to inhibit the expression of only one gene, the crRNA may be designed as a gRNA library to edit mrnas of a plurality of genes.
The above embodiments are only used for illustrating the design idea and features of the present invention, and the purpose of the present invention is to enable those skilled in the art to understand the content of the present invention and implement the present invention accordingly, and the protection scope of the present invention is not limited to the above embodiments. Therefore, all equivalent changes and modifications made in accordance with the principles and concepts disclosed herein are intended to be included within the scope of the present invention.
Claims (9)
1. A preparation method of a CRISPR-Cas13a system based on near-infrared light control is characterized in that crRNA is synthesized according to the sequence design of target gene mRNA, the crRNA and purified Cas13a protein form a Cas13a-crRNA compound, and the Cas13a-crRNA compound is connected with an up-conversion nano material through photosensitive molecules to obtain UCNPs-Cas13 a; and coating the outer layer of UCNPs-Cas13a with polyethyleneimine PEI to obtain UCNPs-Cas13a @ PEI.
2. The method for preparing the near-infrared light control-based CRISPR-Cas13a system according to claim 1, wherein the sequence format of the crRNA is 5 '-Cas 13a protein direct repeat-crRNA spacer-3'; the direct repeat sequence of the Cas13a protein depends on the source of Cas13 a.
3. The method for preparing the near-infrared light control-based CRISPR-Cas13a system according to claim 2, wherein the Cas13a protein is selected from LshCas13a protein, LwCas13a protein or LbuCas13a protein.
4. The preparation method of the near-infrared light control-based CRISPR-Cas13a system according to claim 1, 2 or 3, wherein the up-conversion nanomaterial is synthesized by a one-step solvothermal method to obtain the carboxyl-modified up-conversion nanomaterial.
5. The preparation method of the near-infrared light control-based CRISPR-Cas13a system according to claim 4, wherein the photosensitive molecule is irradiated by NIR laser, and the Cas13a-crRNA complex connected with the photosensitive molecule is released from the up-conversion nanomaterial, so that the dynamic regulation of the CRISPR-Cas13a system is realized.
6. The preparation method of the near-infrared light control-based CRISPR-Cas13a system according to claim 5, wherein the photosensitive molecule is an ONA molecule, and the two ends of the ONA molecule are respectively connected with the Cas13a-crRNA complex and the upconversion nanomaterial.
7. The preparation method of the near-infrared light control-based CRISPR-Cas13a system according to claim 6, wherein the crRNA is designed into a gRNA library, and the mRNA of multiple genes is edited to realize the inhibition of the expression of the multiple genes.
8. An application of the CRISPR-Cas13a system prepared by the near-infrared light-controlled CRISPR-Cas13a system preparation method of claim 7 in inhibiting eukaryotic gene expression in non-disease diagnosis and treatment is characterized in that UCNPs-Cas13a @ PEI is combined with the eukaryotic cell surface and then enters the cell through endocytosis, and the escape of endosomes is promoted by the PEI coated with the UCNPs-Cas13a @ PEI; cas13a-crRNA is released under NIR laser irradiation, a complementary sequence corresponding to a crRNA spacer sequence is searched on target gene mRNA, the crRNA is matched with the target gene mRNA to activate HEPN catalytic sites of Cas13a to shear the mRNA, and further target gene expression of eukaryotes is inhibited.
9. The use according to claim 8, wherein the presence of a plurality of amine groups in the PEI structure increases endosomal osmotic pressure, causing ion influx, resulting in membrane burst, and thus enhanced endosomal escape.
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| CN114774389B (en) * | 2022-04-11 | 2023-09-29 | 天津市天津医院 | Gene editing method, composition and application based on light-operated CRISPR/Cas13d gene editing system |
| CN114703258A (en) * | 2022-06-06 | 2022-07-05 | 深圳大学 | One-pot type RPA-CRISPR nucleic acid detection method and system based on light activation |
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