WO2021109302A1 - Method for preparing near-infrared light control-based crispr-cas13a system and application thereof - Google Patents
Method for preparing near-infrared light control-based crispr-cas13a system and application thereof Download PDFInfo
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- the invention belongs to the field of biotechnology, in particular to a preparation method and application of a CRISPR-Cas13a system based on near-infrared light control.
- the simple, fast and efficient near-infrared light-controlled CRISPR-Cas13a system can remotely control the release of the Cas13a-crRNA complex from the upconversion nanomaterial through near-infrared light, find the target match and activate the HEPE catalytic site of the Cas13a protein to achieve cleavage
- the purpose of target gene mRNA to inhibit gene expression is to provide new ideas and new methods for analyzing gene function. Compared with other gene expression suppression tools, this method is easier to control in terms of time, space, and efficiency. In addition, it can edit the mRNA of multiple genes at the same time.
- the near-infrared light-controlled CRISPR-Cas13a system inhibits the expression of key genes in the synthesis and metabolism pathways of food microorganisms such as yeast and mold, providing a new method for promoting the rapid development of food biotechnology and metabolic engineering.
- the present invention proposes a method for preparing a CRISPR-Cas13a system based on near-infrared light control and its application to inhibit eukaryotic gene expression, using the unique optical properties and photosensitivity of up-conversion fluorescent nanomaterials.
- the remote dynamic control of the molecularly connected Cas13a-crRNA complex adopts the near-infrared light-controlled CRISPR-Cas13a system for RNA editing; the method of the present invention has strong specificity and high stability, and can realize the target gene mRNA in eukaryotic cells. Inhibition of degradation and gene expression levels.
- a method for preparing the CRISPR-Cas13a system based on near-infrared light control According to the sequence of the target gene mRNA, the crRNA is designed and synthesized, the crRNA and the purified Cas13a protein are formed into a Cas13a-crRNA complex, and the Cas13a-crRNA complex is combined with the Cas13a-crRNA complex through a photosensitive molecule. Convert the nanomaterials together to obtain UCNPs-Cas13a; then use polyethyleneimine PEI to coat the outer layer of UCNPs-Cas13a to obtain UCNPs-Cas13a@PEI;
- the crRNA is designed into a gRNA library, and the mRNAs of multiple genes are edited to achieve suppression of the expression of multiple genes;
- sequence format of the crRNA is 5'-Cas13a protein direct repeat sequence-crRNA spacer sequence-3'; wherein the Cas13a protein direct repeat sequence depends on the source of Cas13a;
- the Cas13a protein selects LshCas13a protein, LwCas13a protein or LbuCas13a protein; the Cas13a protein specifically binds the target gene mRNA under the guidance of crRNA and cuts it, and Cas13a and crRNA are the main components to form a simple, fast and efficient CRISPR -The Cas13a system can be used as an RNA editing tool instead of RNA interference.
- the up-conversion nano-material is synthesized by a one-step solvothermal method to obtain a carboxyl-modified up-conversion nano-material (UCNP S );
- the photosensitive molecule is an ONA molecule, and the two ends of the ONA molecule are respectively connected to the Cas13a-crRNA complex and the upconversion nanomaterial;
- the Cas13a-crRNA complex connected to the photosensitive molecule is released from the upconversion nanomaterial, thereby realizing the dynamic regulation of the CRISPR-Cas13a system;
- a CRISPR-Cas13a system prepared by the above-mentioned near-infrared light-controlled CRISPR-Cas13a system preparation method is applied to the purpose of inhibiting eukaryotic gene expression in non-disease diagnosis and treatment, and the process is: the UCNPs-Cas13a@PEI and Eukaryotic cells can enter the cell through endocytosis after binding on the surface of eukaryotic cells.
- the PEI coated by UCNPs-Cas13a@PEI is used to promote endosomal escape; Cas13a-crRNA is released under NIR laser irradiation, and the target gene mRNA is searched for and crRNA
- the complementary sequence corresponding to the spacer sequence, crRNA matches the target gene mRNA to activate the HEPN catalytic site of Cas13a to cut the mRNA, thereby inhibiting the expression of the target gene in eukaryotes.
- the present invention discloses a CRISPR-Cas13a system based on near-infrared light control to inhibit eukaryotic gene expression.
- the method mainly designs and synthesizes crRNA according to the sequence of the target gene mRNA, forms a Cas13a-crRNA complex with Cas13a protein, connects it with the carboxyl modified upconversion nanomaterial through photosensitive molecules, and coats the outer layer with PEI to obtain UCNPs-Cas13a@PEI. It binds to the surface of eukaryotic cells and enters the cell through endocytosis. PEI promotes endosomal escape.
- the Cas13a-crRNA complex released under NIR laser irradiation searches for the complementary sequence of crRNA on the target gene mRNA and matches it to activate it.
- Cas13a shears mRNA to achieve the degradation of target gene mRNA and suppression of expression level.
- the invention releases the CRISPR-Cas13a system, the unique optical properties of up-converting fluorescent nanomaterials, and the remote dynamic control of the Cas13a-crRNA complex connected with photosensitive molecules, and uses the near-infrared light-controlled CRISPR-Cas13a system to carry out new ideas for RNA editing and establish eukaryotes A new method of inhibiting gene expression levels in biological cells.
- the method for inhibiting eukaryotic gene expression based on the near-infrared light-controlled CRISPR-Cas13a system disclosed in the present invention has the same effect as RNA interference, but has higher specificity and wider application range.
- crRNA is designed into a gRNA library, which can target multiple sites of mRNA at the same time to achieve the inhibition of multiple gene expression; compared with CRISPR-Cas9 editing technology, it does not change the coding gene itself, in terms of time and space.
- the efficiency is more controllable, and multiple genes can be modified at the same time, and all mRNAs of multiple copies of genes can be targeted at the same time, which is more efficient.
- the method for inhibiting eukaryotic gene expression based on the near-infrared light-controlled CRISPR-Cas13a system disclosed in the present invention can realize remote control. Inside the cell, this delivery system is activated by near-infrared light irradiation to generate visible light. The RNA editing tool is released from the up-conversion nanoparticle, and the target is combined with the target to complete the cleavage, thereby degrading the target gene mRNA and inhibiting the gene expression level.
- Figure 1 is a flow chart of the preparation of UCNPs-Cas13a@PEI
- Figure 2 is the design principle diagram of the NIR-triggered CRISPR-Cas13a delivery system for RNA editing; among them, (1) attached to the cell membrane, (2) endocytosis, (3) endosome escape, (4) up-conversion nano
- the RNA editing tool released on the particle searches for the target mRNA in the cytoplasm for editing, (5) the RNA editing tool released from the up-conversion nanoparticle enters the nucleus to find the target mRNA for editing;
- A is the map of plasmid pC013-Twinstrep-SUMO-huLwCas13a
- B is the SDS-PAGE map of purified Cas13a protein
- Figure 4 shows the fluorescence spectrum of the upconversion nanomaterial and the NaYF4:Yb/Er nanocrystal solution exhibits strong green upconversion luminescence under the excitation of a 980nm laser.
- the method for inhibiting eukaryotic gene expression based on the near-infrared light-controlled CRISPR-Cas13a system proposed by the present invention is applicable to all eukaryotes.
- only Schizosaccharomyces pombe is used for detailed description. .
- the method for preparing a CRISPR-Cas13a system based on near-infrared light control proposed by the present invention is as follows:
- LwCas13a As shown in Figure 3, the pC013-Twinstrep-SUMO-huLwCas13a plasmid and E. coli expression system were used.
- the expression and purification process of LwCas13a includes four-step purification of Cas13a protein induced by IPTG, nickel column purification, SUMO enzyme digestion to remove His, dialysis, and ion exchange. The concentration of the obtained relatively pure Cas13a protein is determined.
- Cas13a is selected as LwCas13a .
- the endogenous gene glyceraldehyde-3-phosphate dehydrogenase Tdh1 (tdh1) transcription product of Schizosaccharomyces pombe is used as the target gene mRNA for editing.
- the specific process is: preparing crRNA by chemical synthesis.
- the crRNA sequence format is 5'-Cas13a protein direct repeat sequence-crRNA spacer sequence-3', in which the designed spacer sequence is 21-28 nucleotides in length, which is complementary to a sequence in the target gene mRNA; Cas13a protein repeats directly The sequence depends on the source of Cas13a; 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 Lbu The repeat sequence is 5'-GACCACCCCAAAAATGAAGGGGACTAAAAC-3'.
- the crRNA sequence is 5'-GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACGGAUGAAUGAUCUAUACAGAAGCGAUGC-3'; a T7 promoter sequence (5'-TAATACGACTCACTATAGGG-3') is added to the 5'end of the crRNA sequence.
- tdh1-crRNA-1 5'-TAATACGACTCACTATAGGGGATTTAGACTACCCCAAAAACGAAGGGGACTAAAACGGATGAATGATCTATACAGAAGCGATGC-3'
- tdh1-crRNA-2 5'-GCATCGCTTCTGTATAGATCATTCATCCGTTTTAGTCCCCTTCTAGAAAcrRNA
- CcrRNA are produced by dhcrRNA-1: Synthesized by Industrial Biological Engineering (Shanghai) Co., Ltd.
- the two DNA strands are annealed, and then incubated with HiScribe T7 Quick High Yield RNA Synthesis kit at 37°C with T7 RNA Polymerase Mix overnight. Purify crRNA with RNA Cleanup&Concentration Kit, and measure the concentration with Nanodrop 2000.
- the purified Cas13a protein and crRNA are mixed at a molar ratio of 1:2 to form a Cas13a-crRNA complex, and the Cas13a-crRNA complex is connected with the upconversion nanomaterial through the photosensitive molecule ONA to obtain UCNPs-Cas13a;
- PEI polyethyleneimine
- PEI Polyethylenimine
- the present invention also proposes a CRISPR-Cas13a system prepared by the above-mentioned near-infrared light-controlled CRISPR-Cas13a system preparation method for non-disease diagnosis and treatment to suppress eukaryotic diseases
- UCNPs-Cas13a@PEI first attaches to the cell surface of Schizosaccharomyces pombe, and then enters the cell through endocytosis; due to the presence of many amine groups in the PEI structure, it can make internal
- the increase in osmotic pressure in the body causes ion influx and causes the membrane to burst. Therefore, the PEI coated by UCNPs-Cas13a@PEI can promote the escape of the endosome.
- NIR laser irradiates the surface of S.pombe cells.
- up-conversion nanomaterials act as "nano transducers” that can convert NIR light (980nm) into green light (as shown in Figure 4) to crack ONA molecules, thereby
- the Cas13a-crRNA complex connected with the ONA molecule is released from the upconversion nanomaterial.
- the Cas13a-crRNA complex searches for matching fragments on the target gene mRNA in the cytoplasm and nucleus.
- the combination of crRNA and target gene mRNA activates the HEPE catalytic site of the Cas13a protein Click to cut the mRNA to achieve the degradation of the target gene mRNA and the suppression of the expression level.
- the unedited S.pombe was used as the control group, and the edited S.pombe was used as the experimental group.
- the total RNA of the two cells was subjected to qRT-PCR, and the expression level of tdh1 gene was compared, and the gene expression inhibition efficiency was calculated.
- the primer sequences used were tdh1-F: 5'-TGCCTAGCATCGCTTCTGTA-3', tdh1-R: 5'-CATCAATGACGAGCTTACCAT-3'.
- Inhibition of the expression level of the gene tdh1 can cause abnormal mitotic cell cycle and lysis of vegetative cells, thereby reducing the growth rate of yeast cells.
- the implementation of the CRISPR-Cas13a system based on near-infrared light control in S.pombe can inhibit gene expression with different efficiency.
- this method provides a platform for manipulating target gene mRNA in eukaryotes.
- the expression of only a certain gene is suppressed.
- the crRNA can be designed as a gRNA library to edit the mRNA of multiple genes.
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Abstract
A method for preparing a near-infrared light control-based CRISPR-Cas13a system and an application thereof, the method comprising: CrRNA is designed and synthesized according to the sequence of a target gene mRNA; the crRNA and a purified Cas13a protein form a Cas13a-crRNA compound; the Cas13a-crRNA compound is connected to an up-conversion nanomaterial by means of a photosensitive molecule so as to obtain UCNPs-Cas13a; then the UCNPs-Cas13a is coated using PEI to obtain UCNPs-Cas13a@PEI; the prepared UCNPs-Cas13a@PEI is attached to a surface of a eukaryotic cell and enters the cell, and the PEI of the UCNPs-Cas13a@PEI is used to promote the escape of an endosome; the Cas13a-crRNA is released under the irradiation of an NIR laser; a complementary sequence corresponding to a crRNA spacer sequence is searched for on the target gene mRNA; and crRNA is matched with the target gene mRNA so as to activate a HEPN catalytic site of Cas13a to shear mRNA, and then the expression of a eukaryote target gene is inhibited.
Description
本发明属于生物技术领域，尤其是一种基于近红外光控的CRISPR-Cas13a系统制备方法及其应用。The invention belongs to the field of biotechnology, in particular to a preparation method and application of a CRISPR-Cas13a system based on near-infrared light control.
伴随着酵母菌、霉菌等食品微生物全基因组测序的完成，快速高效解析基因功能成为科研工作者予以解决的问题。针对该问题，人们运用RNA干扰、CRISPR-Cas9等技术通过抑制基因表达阐释基因功能，但这些方法存在一些不足，如操作复杂，容易产生脱靶效应，抑制效率低等，因此开发新的基因表达抑制方法迫在眉睫。With the completion of the whole genome sequencing of food microorganisms such as yeast and mold, rapid and efficient analysis of gene function has become a problem for scientific researchers to solve. In response to this problem, people use RNA interference, CRISPR-Cas9 and other technologies to interpret gene functions by inhibiting gene expression, but these methods have some shortcomings, such as complicated operations, prone to off-target effects, low inhibition efficiency, etc. Therefore, new gene expression suppression has been developed. The method is imminent.
简便、快速、高效的近红外光控的CRISPR-Cas13a系统能够通过近红外光照远程控制Cas13a-crRNA复合物从上转换纳米材料上释放，寻找靶标匹配进而激活Cas13a蛋白的HEPE催化位点，达到切割目的基因mRNA抑制基因表达的目的，为解析基因功能提供新思路和新方法。与其他基因表达抑制工具相比，该方法在时间、空间、效率上更易控制，此外，还可以同时编辑多个基因的mRNA。The simple, fast and efficient near-infrared light-controlled CRISPR-Cas13a system can remotely control the release of the Cas13a-crRNA complex from the upconversion nanomaterial through near-infrared light, find the target match and activate the HEPE catalytic site of the Cas13a protein to achieve cleavage The purpose of target gene mRNA to inhibit gene expression is to provide new ideas and new methods for analyzing gene function. Compared with other gene expression suppression tools, this method is easier to control in terms of time, space, and efficiency. In addition, it can edit the mRNA of multiple genes at the same time.
利用近红外光控的CRISPR-Cas13a系统抑制酵母菌、霉菌等食品微生物合成代谢途径中关键基因的表达，为推动食品生物技术以及代谢工程领域的快速发展提供新方法。The near-infrared light-controlled CRISPR-Cas13a system inhibits the expression of key genes in the synthesis and metabolism pathways of food microorganisms such as yeast and mold, providing a new method for promoting the rapid development of food biotechnology and metabolic engineering.
发明内容Summary of the invention
为了解决现有技术中的不足，本发明提出了一种基于近红外光控的CRISPR-Cas13a系统制备方法及其抑制真核生物基因表达的应用，利用上转换荧光纳米材料的独特光学性质以及光敏分子连接的Cas13a-crRNA复合物的远程动态调控，采用近红外光控的CRISPR-Cas13a系统进行RNA编辑；本发明的方法特异性强、稳定性高，可实现真核生物细胞中目的基因mRNA的降解和基因表达水平的抑制。In order to solve the deficiencies in the prior art, the present invention proposes a method for preparing a CRISPR-Cas13a system based on near-infrared light control and its application to inhibit eukaryotic gene expression, using the unique optical properties and photosensitivity of up-conversion fluorescent nanomaterials. The remote dynamic control of the molecularly connected Cas13a-crRNA complex adopts the near-infrared light-controlled CRISPR-Cas13a system for RNA editing; the method of the present invention has strong specificity and high stability, and can realize the target gene mRNA in eukaryotic cells. Inhibition of degradation and gene expression levels.
本发明所采用的技术方案如下：The technical scheme adopted by the present invention is as follows:
一种基于近红外光控的CRISPR-Cas13a系统制备方法，根据目的基因mRNA的序列设计合成crRNA，将crRNA和纯化的Cas13a蛋白形成Cas13a-crRNA复合物，通过光敏分子将Cas13a-crRNA复合物与上转换纳米材料连接在一起，获得UCNPs-Cas13a；再使用聚乙烯亚胺PEI涂覆UCNPs-Cas13a的外层得到UCNPs-Cas13a@PEI；A method for preparing the CRISPR-Cas13a system based on near-infrared light control. According to the sequence of the target gene mRNA, the crRNA is designed and synthesized, the crRNA and the purified Cas13a protein are formed into a Cas13a-crRNA complex, and the Cas13a-crRNA complex is combined with the Cas13a-crRNA complex through a photosensitive molecule. Convert the nanomaterials together to obtain UCNPs-Cas13a; then use polyethyleneimine PEI to coat the outer layer of UCNPs-Cas13a to obtain UCNPs-Cas13a@PEI;
进一步，将所述crRNA设计成gRNA库，编辑多个基因的mRNA，实现对多个基因 表达的抑制；Further, the crRNA is designed into a gRNA library, and the mRNAs of multiple genes are edited to achieve suppression of the expression of multiple genes;
进一步，所述crRNA的序列格式为5’-Cas13a蛋白直接重复序列-crRNA间隔序列-3’；其中，Cas13a蛋白直接重复序列依Cas13a来源而定；Further, the sequence format of the crRNA is 5'-Cas13a protein direct repeat sequence-crRNA spacer sequence-3'; wherein the Cas13a protein direct repeat sequence depends on the source of Cas13a;
进一步，所述Cas13a蛋白选择LshCas13a蛋白、LwCas13a蛋白或者LbuCas13a蛋白；Cas13a蛋白在crRNA的引导下特异性结合目的基因mRNA并将其剪切，而且以Cas13a和crRNA为主构成简单、快速、高效的CRISPR-Cas13a系统可代替RNA干扰作为RNA编辑工具使用。Further, the Cas13a protein selects LshCas13a protein, LwCas13a protein or LbuCas13a protein; the Cas13a protein specifically binds the target gene mRNA under the guidance of crRNA and cuts it, and Cas13a and crRNA are the main components to form a simple, fast and efficient CRISPR -The Cas13a system can be used as an RNA editing tool instead of RNA interference.
进一步，所述上转换纳米材料采用一步溶剂热法合成，得到羧基修饰的上转换纳米材料(UCNP
S)；
Further, the up-conversion nano-material is synthesized by a one-step solvothermal method to obtain a carboxyl-modified up-conversion nano-material (UCNP S );
进一步，所述的光敏分子为ONA分子，ONA分子的两端分别连接Cas13a-crRNA复合物和上转换纳米材料；Further, the photosensitive molecule is an ONA molecule, and the two ends of the ONA molecule are respectively connected to the Cas13a-crRNA complex and the upconversion nanomaterial;
进一步，所述光敏分子在NIR激光照射下，光敏分子所连接的Cas13a-crRNA复合物从上转换纳米材料上释放，从而实现CRISPR-Cas13a系统的动态调控；Further, when the photosensitive molecule is irradiated by the NIR laser, the Cas13a-crRNA complex connected to the photosensitive molecule is released from the upconversion nanomaterial, thereby realizing the dynamic regulation of the CRISPR-Cas13a system;
一种将上述近红外光控的CRISPR-Cas13a系统制备方法制备的CRISPR-Cas13a系统应用于非疾病诊断和治疗的抑制真核生物基因表达的用途，其过程为：所述UCNPs-Cas13a@PEI与真核生物细胞表面结合后可通过胞吞作用进入细胞，利用UCNPs-Cas13a@PEI所涂覆的PEI促进内体逃逸；在NIR激光照射下Cas13a-crRNA被释放，在目的基因mRNA上寻找与crRNA间隔序列对应的互补序列，crRNA与目的基因mRNA匹配以激活Cas13a的HEPN催化位点剪切mRNA，进而抑制真核生物目的基因表达。A CRISPR-Cas13a system prepared by the above-mentioned near-infrared light-controlled CRISPR-Cas13a system preparation method is applied to the purpose of inhibiting eukaryotic gene expression in non-disease diagnosis and treatment, and the process is: the UCNPs-Cas13a@PEI and Eukaryotic cells can enter the cell through endocytosis after binding on the surface of eukaryotic cells. The PEI coated by UCNPs-Cas13a@PEI is used to promote endosomal escape; Cas13a-crRNA is released under NIR laser irradiation, and the target gene mRNA is searched for and crRNA The complementary sequence corresponding to the spacer sequence, crRNA matches the target gene mRNA to activate the HEPN catalytic site of Cas13a to cut the mRNA, thereby inhibiting the expression of the target gene in eukaryotes.
进一步，所述PEI结构中存在许多胺基，能使内体内渗透压增加，引起离子内流，导致膜胀破，因此能够增强内体逃逸。Further, there are many amine groups in the PEI structure, which can increase the osmotic pressure of the endosome, cause ion influx, and cause the membrane to burst, thereby enhancing the escape of the endosome.
本发明的有益效果：The beneficial effects of the present invention:
1、本发明公开了一种基于近红外光控的CRISPR-Cas13a系统的抑制真核生物基因表达。该方法主要根据目的基因mRNA的序列设计合成crRNA，与Cas13a蛋白形成Cas13a-crRNA复合物，将其与羧基修饰上转换纳米材料通过光敏分子连接，PEI涂覆外层获得UCNPs-Cas13a@PEI，当其与真核生物细胞表面结合，通过胞吞作用进入细胞内，PEI促进内体逃逸，在NIR激光照射下释放的Cas13a-crRNA复合物在目的基因mRNA上寻找crRNA的互补序列，与其匹配以激活Cas13a剪切mRNA，实现目的基因mRNA的降解和表达水平的抑制。该发明将CRISPR-Cas13a系统，上转换荧光纳米材料的独特光学性质以 及远程动态调控光敏分子连接的Cas13a-crRNA复合物释放，采用近红外光控CRISPR-Cas13a系统进行RNA编辑新思路，建立真核生物细胞中基因表达水平抑制的新方法。1. The present invention discloses a CRISPR-Cas13a system based on near-infrared light control to inhibit eukaryotic gene expression. The method mainly designs and synthesizes crRNA according to the sequence of the target gene mRNA, forms a Cas13a-crRNA complex with Cas13a protein, connects it with the carboxyl modified upconversion nanomaterial through photosensitive molecules, and coats the outer layer with PEI to obtain UCNPs-Cas13a@PEI. It binds to the surface of eukaryotic cells and enters the cell through endocytosis. PEI promotes endosomal escape. The Cas13a-crRNA complex released under NIR laser irradiation searches for the complementary sequence of crRNA on the target gene mRNA and matches it to activate it. Cas13a shears mRNA to achieve the degradation of target gene mRNA and suppression of expression level. The invention releases the CRISPR-Cas13a system, the unique optical properties of up-converting fluorescent nanomaterials, and the remote dynamic control of the Cas13a-crRNA complex connected with photosensitive molecules, and uses the near-infrared light-controlled CRISPR-Cas13a system to carry out new ideas for RNA editing and establish eukaryotes A new method of inhibiting gene expression levels in biological cells.
2、本发明公开的基于近红外光控的CRISPR-Cas13a系统的抑制真核生物基因表达的方法与RNA干扰作用效果相当，但特异性更高，适用范围更广。另外，将crRNA设计成gRNA库，可同时靶向mRNA的多个位点，实现对多个基因表达的抑制；与CRISPR-Cas9编辑技术相比，其不改变编码基因本身，在时间上、空间上和效率上更加可控，而且也可同时修改多个基因、同时靶向多拷贝基因的所有mRNA，更加高效。2. The method for inhibiting eukaryotic gene expression based on the near-infrared light-controlled CRISPR-Cas13a system disclosed in the present invention has the same effect as RNA interference, but has higher specificity and wider application range. In addition, crRNA is designed into a gRNA library, which can target multiple sites of mRNA at the same time to achieve the inhibition of multiple gene expression; compared with CRISPR-Cas9 editing technology, it does not change the coding gene itself, in terms of time and space. The efficiency is more controllable, and multiple genes can be modified at the same time, and all mRNAs of multiple copies of genes can be targeted at the same time, which is more efficient.
3、本发明公开的基于近红外光控的CRISPR-Cas13a系统的抑制真核生物基因表达的方法可实现远程操控。在细胞内部，这种递送系统经近红外光照射产生可见光加以激活，RNA编辑工具从上转换纳米颗粒上释放，与靶标结合后完成切割，实现降解目的基因mRNA和抑制基因表达水平。3. The method for inhibiting eukaryotic gene expression based on the near-infrared light-controlled CRISPR-Cas13a system disclosed in the present invention can realize remote control. Inside the cell, this delivery system is activated by near-infrared light irradiation to generate visible light. The RNA editing tool is released from the up-conversion nanoparticle, and the target is combined with the target to complete the cleavage, thereby degrading the target gene mRNA and inhibiting the gene expression level.
图1为UCNPs-Cas13a@PEI的制备流程图；Figure 1 is a flow chart of the preparation of UCNPs-Cas13a@PEI;
图2为NIR触发的CRISPR-Cas13a递送系统用于RNA编辑的设计原理图；其中，(1)附着于细胞膜，(2)胞吞作用，(3)内体逃逸，(4)从上转换纳米颗粒上释放的RNA编辑工具在细胞质中寻找靶mRNA进行编辑，(5)从上转换纳米颗粒上释放的RNA编辑工具进入细胞核寻找靶mRNA进行编辑；Figure 2 is the design principle diagram of the NIR-triggered CRISPR-Cas13a delivery system for RNA editing; among them, (1) attached to the cell membrane, (2) endocytosis, (3) endosome escape, (4) up-conversion nano The RNA editing tool released on the particle searches for the target mRNA in the cytoplasm for editing, (5) the RNA editing tool released from the up-conversion nanoparticle enters the nucleus to find the target mRNA for editing;
图3中A为质粒pC013-Twinstrep-SUMO-huLwCas13a的图谱；B为纯化Cas13a蛋白的SDS-PAGE图；In Figure 3, A is the map of plasmid pC013-Twinstrep-SUMO-huLwCas13a; B is the SDS-PAGE map of purified Cas13a protein;
图4为上转换纳米材料的荧光光谱图和在980nm激光器激发下NaYF4:Yb/Er纳米晶溶液呈现较强的绿色上转换发光。Figure 4 shows the fluorescence spectrum of the upconversion nanomaterial and the NaYF4:Yb/Er nanocrystal solution exhibits strong green upconversion luminescence under the excitation of a 980nm laser.
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅用于解释本发明，并不用于限定本发明。In order to make the objectives, technical solutions, and advantages of the present invention clearer, the following further describes the present invention in detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention, but not used to limit the present invention.
本发明所提出的基于近红外光控的CRISPR-Cas13a系统的抑制真核生物基因表达的方法适用所有真核生物，在本实施例中仅以粟酒裂殖酵母(Schizosaccharomyces pombe)例作详细说明。The method for inhibiting eukaryotic gene expression based on the near-infrared light-controlled CRISPR-Cas13a system proposed by the present invention is applicable to all eukaryotes. In this embodiment, only Schizosaccharomyces pombe is used for detailed description. .
本发明所提出的一种基于近红外光控的CRISPR-Cas13a系统制备方法，具体过程如下：The method for preparing a CRISPR-Cas13a system based on near-infrared light control proposed by the present invention is as follows:
材料准备过程：Material preparation process:
1、LwCas13a蛋白的获得：1. Obtaining LwCas13a protein:
如图3，使用pC013-Twinstrep-SUMO-huLwCas13a质粒和大肠杆菌表达系统。LwCas13a的表达与纯化过程包含Cas13a蛋白经IPTG诱导表达以及镍柱纯化、SUMO酶切去除His、透析、离子交换四步纯化，测定获得的较纯Cas13a蛋白浓度，在本实施例中，Cas13a选用LwCas13a。As shown in Figure 3, the pC013-Twinstrep-SUMO-huLwCas13a plasmid and E. coli expression system were used. The expression and purification process of LwCas13a includes four-step purification of Cas13a protein induced by IPTG, nickel column purification, SUMO enzyme digestion to remove His, dialysis, and ion exchange. The concentration of the obtained relatively pure Cas13a protein is determined. In this example, Cas13a is selected as LwCas13a .
2、crRNA的制备：2. Preparation of crRNA:
在本实施例中，以粟酒裂殖酵母(Schizosaccharomyces pombe)内源基因glyceraldehyde-3-phosphate dehydrogenase Tdh1(tdh1)转录产物作为目的基因mRNA进行编辑，具体过程为：采用化学合成法制备crRNA。crRNA序列格式为5’-Cas13a蛋白直接重复序列-crRNA间隔序列-3’，其中，间隔序列设计的长度为21-28核苷酸，其与目的基因mRNA中的一段序列互补；Cas13a蛋白直接重复序列依Cas13a来源而定；当Cas13a为LshCas13a时，直接重复序列为5’-GGCCACCCCAAUAUCGAAGGGGACUAAAAC-3’；当Cas13a为LwCas13a时，直接重复序列为5’-GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAAC-3’；当Cas13a为LbuCas13a时，直接重复序列为5’-GACCACCCCAAAAATGAAGGGGACTAAAAC-3’。In this embodiment, the endogenous gene glyceraldehyde-3-phosphate dehydrogenase Tdh1 (tdh1) transcription product of Schizosaccharomyces pombe is used as the target gene mRNA for editing. The specific process is: preparing crRNA by chemical synthesis. The crRNA sequence format is 5'-Cas13a protein direct repeat sequence-crRNA spacer sequence-3', in which the designed spacer sequence is 21-28 nucleotides in length, which is complementary to a sequence in the target gene mRNA; Cas13a protein repeats directly The sequence depends on the source of Cas13a; 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 Lbu The repeat sequence is 5'-GACCACCCCAAAAATGAAGGGGACTAAAAC-3'.
由于本实施例中Cas13a选用LwCas13a，因此crRNA序列为5’-GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACGGAUGAAUGAUCUAUACAGAAGCGAUGC-3’；在crRNA序列的5’端添加T7启动子序列(5’-TAATACGACTCACTATAGGG-3’)。根据crRNA合成转录模板dsDNA，tdh1-crRNA-1：5’-TAATACGACTCACTATAGGGGATTTAGACTACCCCAAAAACGAAGGGGACTAAAACGGATGAATGATCTATACAGAAGCGATGC-3’，tdh1-crRNA-2:5’-GCATCGCTTCTGTATAGATCATTCATCCGTTTTAGTCCCCTTCGTTTTTGGGGTAGTCTAAATCCCCTATAGTGAGTCGTATTA-3’；其中，tdh1-crRNA-1和tdh1-crRNA-2由生工生物工程(上海)股份有限公司合成。将这两条DNA链进行退火，然后使用HiScribe T7 Quick High Yield RNA Synthesis试剂盒在37℃下与T7 RNA Polymerase Mix一起温育过夜。用RNA Cleanup&Concentration Kit纯化crRNA，Nanodrop 2000测定浓度。Since LwCas13a is selected for Cas13a in this embodiment, the crRNA sequence is 5'-GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACGGAUGAAUGAUCUAUACAGAAGCGAUGC-3'; a T7 promoter sequence (5'-TAATACGACTCACTATAGGG-3') is added to the 5'end of the crRNA sequence. According to the synthesis of crRNA transcription template dsDNA, tdh1-crRNA-1: 5'-TAATACGACTCACTATAGGGGATTTAGACTACCCCAAAAACGAAGGGGACTAAAACGGATGAATGATCTATACAGAAGCGATGC-3', tdh1-crRNA-2: 5'-GCATCGCTTCTGTATAGATCATTCATCCGTTTTAGTCCCCTTCTAGAAAcrRNA and CcrRNA are produced by dhcrRNA-1: Synthesized by Industrial Biological Engineering (Shanghai) Co., Ltd. The two DNA strands are annealed, and then incubated with HiScribe T7 Quick High Yield RNA Synthesis kit at 37°C with T7 RNA Polymerase Mix overnight. Purify crRNA with RNA Cleanup&Concentration Kit, and measure the concentration with Nanodrop 2000.
3、上转换纳米材料的合成：3. Synthesis of up-conversion nanomaterials:
使用一步溶剂热法合成上转换纳米粒子(UCNP
S)，将1.2mmol RECl
3(Y：Yb：Er＝80： 18：2)，2.4mmol NaCl和0.8g PAAs溶解在20mL乙二醇中并充分混匀形成透明溶液。将5mmol NH
4F溶解在15mL乙二醇中，在搅拌条件下逐滴加入到上述透明液中。所得混合物转移到反应釜中200℃保持1.5h。冷却至室温离心收集产物，用乙醇和蒸馏水洗涤3次，置于烘箱中60℃干燥，得到羧基修饰的UCNP
S。
Use one-step solvothermal method to synthesize up-conversion nanoparticles (UCNP S ), and dissolve 1.2mmol RECl 3 (Y:Yb:Er=80:18:2), 2.4mmol NaCl and 0.8g PAAs in 20mL ethylene glycol. Mix well to form a clear solution. Dissolve 5 mmol NH 4 F in 15 mL ethylene glycol, and add dropwise to the above clear liquid under stirring. The resulting mixture was transferred to the reactor at 200°C for 1.5 hours. Cooling to room temperature the product was collected by centrifugation, washed with ethanol and three times with distilled water, placed in an oven dried 60 ℃, resulting carboxyl-modified UCNP S.
4.基于上部分所准备的材料制备UCNPs-Cas13a@PEI：4. Prepare UCNPs-Cas13a@PEI based on the materials prepared in the previous section:
如图1，将纯化的Cas13a蛋白和crRNA按摩尔比1：2混合形成Cas13a-crRNA复合物，通过光敏分子ONA将Cas13a-crRNA复合物与上转换纳米材料连接在一起，获得UCNPs-Cas13a；再使用聚乙烯亚胺PEI(即Polyethylenimine)涂覆UCNPs-Cas13a的外层得到UCNPs-Cas13a@PEI。As shown in Figure 1, the purified Cas13a protein and crRNA are mixed at a molar ratio of 1:2 to form a Cas13a-crRNA complex, and the Cas13a-crRNA complex is connected with the upconversion nanomaterial through the photosensitive molecule ONA to obtain UCNPs-Cas13a; Use polyethyleneimine PEI (ie Polyethylenimine) to coat the outer layer of UCNPs-Cas13a to obtain UCNPs-Cas13a@PEI.
基于上述制备方法所制备的UCNPs-Cas13a@PEI，本发明还提出了一种将上述近红外光控的CRISPR-Cas13a系统制备方法制备的CRISPR-Cas13a系统应用于非疾病诊断和治疗的抑制真核生物基因表达的用途，具体过程如图2所示：UCNPs-Cas13a@PEI先附着于粟酒裂殖酵母细胞表面，再通过胞吞作用进入细胞；由于PEI结构中存在许多胺基，能使内体内渗透压增加，引起离子内流，导致膜胀破，因此UCNPs-Cas13a@PEI所涂覆的PEI能促进内体逃逸。Based on the UCNPs-Cas13a@PEI prepared by the above-mentioned preparation method, the present invention also proposes a CRISPR-Cas13a system prepared by the above-mentioned near-infrared light-controlled CRISPR-Cas13a system preparation method for non-disease diagnosis and treatment to suppress eukaryotic diseases The use of biological gene expression, the specific process is shown in Figure 2: UCNPs-Cas13a@PEI first attaches to the cell surface of Schizosaccharomyces pombe, and then enters the cell through endocytosis; due to the presence of many amine groups in the PEI structure, it can make internal The increase in osmotic pressure in the body causes ion influx and causes the membrane to burst. Therefore, the PEI coated by UCNPs-Cas13a@PEI can promote the escape of the endosome.
NIR激光照射S.pombe细胞表面，在NIR激光照射下，上转换纳米材料充当“纳米换能器”，能将NIR光(980nm)转变成绿光(如图4)，以裂解ONA分子，从而将ONA分子连接的Cas13a-crRNA复合物从上转换纳米材料上释放，Cas13a-crRNA复合物在细胞质和细胞核内寻找目的基因mRNA上的匹配片段，crRNA与目的基因mRNA结合激活Cas13a蛋白的HEPE催化位点以切割mRNA，实现目的基因mRNA的降解和表达水平的抑制。NIR laser irradiates the surface of S.pombe cells. Under NIR laser irradiation, up-conversion nanomaterials act as "nano transducers" that can convert NIR light (980nm) into green light (as shown in Figure 4) to crack ONA molecules, thereby The Cas13a-crRNA complex connected with the ONA molecule is released from the upconversion nanomaterial. The Cas13a-crRNA complex searches for matching fragments on the target gene mRNA in the cytoplasm and nucleus. The combination of crRNA and target gene mRNA activates the HEPE catalytic site of the Cas13a protein Click to cut the mRNA to achieve the degradation of the target gene mRNA and the suppression of the expression level.
为了验证本发明所提出的基于近红外光控的CRISPR-Cas13a系统的抑制真核生物基因表达的方法的效果，以未编辑的S.pombe作为对照组，编辑的S.pombe为实验组，提取两者细胞总RNA进行qRT-PCR，比较tdh1基因的表达水平，计算基因表达抑制效率。所用引物序列tdh1-F：5’-TGCCTAGCATCGCTTCTGTA-3’，tdh1-R：5’-CATCAATGACGAGCTTACCAT-3’。In order to verify the effect of the method for inhibiting eukaryotic gene expression based on the near-infrared light-controlled CRISPR-Cas13a system proposed by the present invention, the unedited S.pombe was used as the control group, and the edited S.pombe was used as the experimental group. The total RNA of the two cells was subjected to qRT-PCR, and the expression level of tdh1 gene was compared, and the gene expression inhibition efficiency was calculated. The primer sequences used were tdh1-F: 5'-TGCCTAGCATCGCTTCTGTA-3', tdh1-R: 5'-CATCAATGACGAGCTTACCAT-3'.
基因tdh1表达水平的抑制会使有丝分裂细胞周期异常和营养细胞裂解，从而降低酵母细胞生长速率。在S.pombe中实施基于近红外光控的CRISPR-Cas13a系统能以不同的效率抑制基因表达，该方法作为一种新的RNA编辑工具为在真核生物中操纵目的基因mRNA提供了平台。Inhibition of the expression level of the gene tdh1 can cause abnormal mitotic cell cycle and lysis of vegetative cells, thereby reducing the growth rate of yeast cells. The implementation of the CRISPR-Cas13a system based on near-infrared light control in S.pombe can inhibit gene expression with different efficiency. As a new RNA editing tool, this method provides a platform for manipulating target gene mRNA in eukaryotes.
另外，在本实施例中仅对某一个基因进行抑制表达，如当需要同时抑制多个基因的表达，可以将crRNA设计成gRNA库，编辑多个基因的mRNA。In addition, in this embodiment, the expression of only a certain gene is suppressed. For example, when the expression of multiple genes needs to be suppressed at the same time, the crRNA can be designed as a gRNA library to edit the mRNA of multiple genes.
以上实施例仅用于说明本发明的设计思想和特点，其目的在于使本领域内的技术人员能够了解本发明的内容并据以实施，本发明的保护范围不限于上述实施例。所以，凡依据本发明所揭示的原理、设计思路所作的等同变化或修饰，均在本发明的保护范围之内。The above embodiments are only used to illustrate the design ideas and features of the present invention, and their purpose is to enable those skilled in the art to understand the content of the present invention and implement them accordingly. The protection scope of the present invention is not limited to the above embodiments. Therefore, all equivalent changes or modifications made according to the principles and design ideas disclosed in the present invention fall within the protection scope of the present invention.
Claims (9)
- 一种基于近红外光控的CRISPR-Cas13a系统制备方法，其特征在于，根据目的基因mRNA的序列设计合成crRNA，将crRNA和纯化的Cas13a蛋白形成Cas13a-crRNA复合物，通过光敏分子将Cas13a-crRNA复合物与上转换纳米材料连接在一起，获得UCNPs-Cas13a；再使用聚乙烯亚胺PEI涂覆UCNPs-Cas13a的外层得到UCNPs-Cas13a@PEI。A method for preparing a CRISPR-Cas13a system based on near-infrared light control, which is characterized in that crRNA is designed and synthesized according to the sequence of the target gene mRNA, the crRNA and the purified Cas13a protein are formed into a Cas13a-crRNA complex, and the Cas13a-crRNA is combined by a photosensitive molecule. The composite is connected with the up-conversion nanomaterial to obtain UCNPs-Cas13a; and then polyethyleneimine PEI is used to coat the outer layer of UCNPs-Cas13a to obtain UCNPs-Cas13a@PEI.
- 根据权利要求1所述的一种基于近红外光控的CRISPR-Cas13a系统制备方法，其特征在于，所述crRNA的序列格式为5’-Cas13a蛋白直接重复序列-crRNA间隔序列-3’；其中，Cas13a蛋白直接重复序列依Cas13a来源而定。The method for preparing a CRISPR-Cas13a system based on near-infrared light control according to claim 1, wherein the sequence format of the crRNA is 5'-Cas13a protein direct repeat sequence-crRNA spacer sequence-3'; wherein , The direct repeat sequence of Cas13a protein depends on the source of Cas13a.
- 根据权利要求2所述的一种基于近红外光控的CRISPR-Cas13a系统制备方法，其特征在于，所述Cas13a蛋白选择LshCas13a蛋白、LwCas13a蛋白或者LbuCas13a蛋白。The method for preparing a CRISPR-Cas13a system based on near-infrared light control according to claim 2, wherein the Cas13a protein is selected from LshCas13a protein, LwCas13a protein or LbuCas13a protein.
- 根据权利要求1、2或3所述的一种基于近红外光控的CRISPR-Cas13a系统制备方法，其特征在于，所述上转换纳米材料采用一步溶剂热法合成，得到羧基修饰的上转换纳米材料。The method for preparing a CRISPR-Cas13a system based on near-infrared light control according to claim 1, 2 or 3, wherein the upconversion nanomaterial is synthesized by a one-step solvothermal method to obtain a carboxyl-modified upconversion nanomaterial. material.
- 根据权利要求4所述的一种基于近红外光控的CRISPR-Cas13a系统制备方法，其特征在于，所述光敏分子在NIR激光照射下，光敏分子所连接的Cas13a-crRNA复合物从上转换纳米材料上释放，从而实现CRISPR-Cas13a系统的动态调控。The method for preparing a CRISPR-Cas13a system based on near-infrared light control according to claim 4, wherein the photosensitive molecule is irradiated by the NIR laser, and the Cas13a-crRNA complex connected to the photosensitive molecule is upconverted from the nanometer Released on the material, thereby realizing the dynamic regulation of the CRISPR-Cas13a system.
- 根据权利要求5所述的一种基于近红外光控的CRISPR-Cas13a系统制备方法，其特征在于，所述的光敏分子为ONA分子，ONA分子的两端分别连接Cas13a-crRNA复合物和上转换纳米材料。The method for preparing a CRISPR-Cas13a system based on near-infrared light control according to claim 5, wherein the photosensitive molecule is an ONA molecule, and both ends of the ONA molecule are respectively connected to the Cas13a-crRNA complex and upconverter nanomaterials.
- 根据权利要求6所述的一种基于近红外光控的CRISPR-Cas13a系统制备方法，其特征在于，将所述crRNA设计成gRNA库，编辑多个基因的mRNA，实现对多个基因表达的抑制。The method for preparing a CRISPR-Cas13a system based on near-infrared light control according to claim 6, wherein the crRNA is designed as a gRNA library, and the mRNAs of multiple genes are edited to inhibit the expression of multiple genes. .
- 一种将权利要求7所述近红外光控的CRISPR-Cas13a系统制备方法制备的CRISPR-Cas13a系统应用于非疾病诊断和治疗的抑制真核生物基因表达的用途，其特征在于，将UCNPs-Cas13a@PEI与真核生物细胞表面结合后通过胞吞作用进入细胞，利用UCNPs-Cas13a@PEI所涂覆的PEI促进内体逃逸；在NIR激光照射下Cas13a-crRNA被释放，在目的基因mRNA上寻找与crRNA间隔序列对应的互补序列，crRNA与目的基因mRNA匹配以激活Cas13a的HEPN催化位点剪切mRNA，进而抑制真核生物目的基因表 达。A CRISPR-Cas13a system prepared by the preparation method of the near-infrared light-controlled CRISPR-Cas13a system according to claim 7 is applied to non-disease diagnosis and treatment for inhibiting eukaryotic gene expression, characterized in that UCNPs-Cas13a @PEI binds to the surface of eukaryotic cells and enters the cell through endocytosis. The PEI coated with UCNPs-Cas13a@PEI is used to promote endosomal escape; Cas13a-crRNA is released under NIR laser irradiation, and the target gene mRNA is searched Complementary sequence corresponding to the crRNA spacer sequence. The crRNA matches the target gene mRNA to activate the HEPN catalytic site of Cas13a to cut the mRNA, thereby inhibiting the expression of the target gene in eukaryotes.
- 根据权利要求8所述的用途，其特征在于，所述PEI结构中存在许多胺基，能使内体内渗透压增加，引起离子内流，导致膜胀破，因此能够增强内体逃逸。The use according to claim 8, characterized in that there are many amine groups in the PEI structure, which can increase the osmotic pressure in the endosome, cause ion influx, cause the membrane to burst, and thus can enhance the escape of the endosome.
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