WO2023137926A1 - 一种基于光控的CRISPR-Cas核酸检测试剂盒及检测方法 - Google Patents

一种基于光控的CRISPR-Cas核酸检测试剂盒及检测方法 Download PDF

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WO2023137926A1
WO2023137926A1 PCT/CN2022/091068 CN2022091068W WO2023137926A1 WO 2023137926 A1 WO2023137926 A1 WO 2023137926A1 CN 2022091068 W CN2022091068 W CN 2022091068W WO 2023137926 A1 WO2023137926 A1 WO 2023137926A1
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guide rna
silencing
nucleic acid
cas
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周小明
胡梦露
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华南师范大学
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    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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  • the invention belongs to the field of biological detection, and in particular relates to a light-controlled CRISPR-Cas nucleic acid detection kit and detection method.
  • CRISPR-Cas systems such as CRISPR-Cas12 and CRISPR-Cas13 systems
  • CRISPR-Cas12 and CRISPR-Cas13 systems have been widely used in the field of nucleic acid diagnosis, but the sensitivity of a single CRISPR-Cas detection system is still difficult to reach the level of PCR, so most of the current nucleic acid detection methods based on CRISPR-Cas are combined with nucleic acid amplification.
  • the CRISPR-Cas detection system In the detection method that combines nucleic acid amplification and CRISPR-Cas recognition system into a single test tube, because the CRISPR-Cas detection system will recognize and cut the target nucleic acid, it will cause the fragmentation of the amplification template, thereby reducing the amplification efficiency. On the other hand, during the amplification process, the initial amplification product will also be recognized and cut by the CRISPR-Cas detection system, resulting in the fragmentation of the template that can be recycled, resulting in a decrease in amplification efficiency.
  • the object of the present invention is to provide a CRISPR-Cas nucleic acid detection kit and detection method based on light control, which can separate nucleic acid amplification and CRISPR-Cas detection in time, and can be sealed in one test tube, avoiding the reagent transfer process of opening the cap, and ensuring high detection sensitivity while ensuring that the detection is not affected by aerosol pollution.
  • the kit also includes more than one of fluorescent reporter probes, amplification primers, enzymes or buffers;
  • the silencing guide RNA is formed by annealing hybridization of silencing nucleotides and guide RNA;
  • the guide RNA is designed according to the target nucleic acid sequence and includes two regions, a repetitive sequence and a spacer sequence.
  • the guide RNA combines with the Cas protein to form a protein-nucleic acid complex, and the guide protein-nucleic acid complex recognizes and cuts the target nucleic acid;
  • the bases of the silent nucleotides are connected by a PC linker that can be activated and degraded by light;
  • the silent nucleotide is completely complementary to the spacer sequence of the guide RNA, or is completely paired with the repeat region sequence of the guide RNA;
  • the silent nucleotide in addition to completely matching with the guide RNA spacer sequence, also pairs several (1-21) more bases to the repeat region, preferably more than 5 bases;
  • the silent nucleotide may contain 1-6 PC linkers, preferably contains 3 PC linkers;
  • a PC linker is embedded every 5-10 bases; preferably a PC linker is embedded every 6 bases;
  • the molar concentration ratio of the silent nucleotide to the guide RNA is (1-2):1, preferably 2:1;
  • the base length of the guide RNA is 30-60, preferably 41 or 51;
  • the base length of the silent nucleotide is 20-40, preferably 25 or 30;
  • the CAS protein is CAS12 protein or CAS13 protein. These proteins not only have cashmere cutting activity, but also transliterate activity. This is a place where CAS9 protein is different (CAS9 protein has only cashmere cutting activity).
  • CAS12 protein is a DNA (DSDNA or SSDNA) as the. Active nucleic acid intrase enzyme, its activated transliteration activity can cut SSDNA; CAS13 protein is a nucleic acid enzyme with RNA as a target.
  • the isothermal amplification primers are designed according to the target nucleic acid sequence
  • the isothermal amplification specifically refers to recombinase polymerase isothermal amplification (RPA), loop-mediated isothermal amplification (LAMP), helicase-dependent constant temperature amplification (HDA), strand displacement amplification (SDA), rolling circle amplification (RCA), exponential isothermal amplification (EXPAR), nucleic acid sequence-dependent amplification (NASBA), single primer isothermal amplification (SPIA), chimeric primer-induced nucleic acid isothermal amplification (ICAN), strand exchange amplification (SEA), etc., preferably recombinase polymerase, etc. warm amplification (RPA).
  • RPA recombinase polymerase isothermal amplification
  • LAMP loop-mediated isothermal amplification
  • HDA helicase-dependent constant temperature amplification
  • SDA strand displacement amplification
  • RCA rolling circle amplification
  • EXPAR exponential isothermal amplification
  • the final concentration of the Cas protein can be 10-500nM
  • the final concentration of the silencing nucleotide-guide RNA complex is 10-500nM to ensure that the cleavage reaction between the Cas protein and the target nucleic acid can occur with high efficiency
  • the silencing nucleotide-guide RNA complex is combined with the Cas protein to form a protein-nucleic acid complex
  • the concentration of the silencing nucleotide-guide RNA complex is in a corresponding relationship with the concentration of the Cas protein.
  • the final concentration of the fluorescent reporter probe is 200nM-1 ⁇ M, so as to ensure that there is sufficient fluorescent reporter probe near the target nucleic acid to be cleaved.
  • reaction temperature of the isothermal amplification in step (2) is 25-65° C. to ensure that the nucleic acid amplification reaction proceeds normally and the Cas protein is not inactivated.
  • reaction time of the isothermal amplification in step (2) is at least 5 minutes
  • the UV light time is at least 5 seconds
  • the CRISPR-Cas nucleic acid detection time is at least 5 minutes.
  • the Cas protein combines with the guide RNA to form a protein-nucleic acid complex.
  • the protein-nucleic acid complex recognizes and cuts the target nucleic acid.
  • the trans-cleavage activity of the Cas protein is stimulated to cut the surrounding fluorescent reporter probe in a non-specific manner.
  • One end of the fluorescent reporter probe is connected to fluorescein, and the other end is connected to a quencher group, which can be excited to generate a fluorescent signal after being cut off.
  • the silent nucleotide is designed to be complementary to the guide RNA, thereby blocking the recognition of the target nucleic acid by the guide RNA.
  • the silent nucleotides are designed to be connected by light-activated linkers, which can be easily excited by UV light to cause the cleavage of the silent nucleotides, thereby reviving the CRISPR-Cas detection system.
  • CRISPR-Cas detection system When this inactivated CRISPR-Cas detection system is combined with nucleic acid amplification technology into one tube reaction, the template for nucleic acid amplification will not be cut by CRISPR-Cas, so the amplification efficiency will not be affected. After nucleic acid amplification is complete, the CRISPR-Cas detection system is activated with light.
  • this detection method separates nucleic acid amplification and CRISPR-Cas detection in time, it can be sealed in one test tube, avoiding the reagent transfer process of opening the cap, and ensuring high detection sensitivity while ensuring that the detection is not affected by aerosol pollution.
  • the present invention has the following advantages and effects:
  • the kit and method of the present invention avoid the generation of aerosol pollution and simplify the experimental steps.
  • the kit and method of the present invention simplify the reaction system and experimental steps.
  • the kit and method of the present invention solve the problem of instability and do not need to rely on centrifuge equipment.
  • the kit and method of the present invention can realize highly sensitive nucleic acid detection with only one guide RNA, which simplifies the reaction system and experimental steps.
  • Figure 1 shows the fluorescence signal intensity caused by different silencing guide DNA.
  • Figure 2 shows the fluorescence signal intensity caused by silencing guide RNA.
  • Figure 3 shows the fluorescence signal intensity caused by different silencing guide RNAs.
  • Fig. 4 is the fluorescence intensity curve when using the kit and method of the present invention to detect the target.
  • Fig. 5 is the fluorescence intensity curve when using the kit and method of the present invention to detect the target.
  • Fig. 6 is the fluorescence intensity when different targets are detected by using the kit and method of the present invention.
  • Fig. 7 is the fluorescence intensity curve when using the kit and method of the present invention to detect different target amounts.
  • Fig. 8 is the fluorescence intensity curve when using the traditional CRISPR-Cas12 method to detect the target.
  • Fig. 9 is an electrophoresis diagram when the target is detected by agarose gel electrophoresis.
  • Figure 10 shows the fluorescence signal intensity caused by different silencing guide DNA.
  • Figure 11 shows the fluorescence signal intensity caused by silencing guide DNA.
  • the guide RNA is inactivated by annealing hybridization.
  • the annealing premix reaction solution includes guide RNA, silencing DNA, and Cas protein reaction buffer (NEBuffer 2.1, purchased from New England Biotechnology (Beijing) Co., Ltd., the same below).
  • the final concentrations of each component in the annealing premixed reaction solution are: the final concentration of guide RNA is 10 ⁇ M, the final concentration of silencing DNA is 20 ⁇ M, and the final concentration of Cas protein reaction buffer is 1 ⁇ .
  • RNA uaauuucuacuaaguguaagauggggguuugagguccauuaca (SEQ.ID.NO.1), bases in italics are repeat regions, and the rest are spacers;
  • the sequence of the guide RNA is divided into two parts, a repetitive sequence and a spacer sequence. Therefore, for the complementary pairing between silencing DNA and guide RNA, the abbreviation corresponding to the repeat region is called R, and the abbreviation corresponding to the spacer region is called S. Different numbers represent the base lengths of complementary pairings.
  • the target used is double-stranded DNA formed by annealing, and the target sequence is:
  • Target F ctgatagtatttaggggtttgaggtccattacagctgtaatgaacattacgtcttatgt (SEQ.ID.NO.31)
  • Target R acataagacgtaatgttcattacagctgtaatggacctcaaacccctaaatactatcag (SEQ.ID.NO.32)
  • the annealing premix reaction solution includes target F, target R, and Cas protein reaction buffer.
  • the final concentrations of the components in the annealing premixed reaction solution are respectively: the final concentration of target F is 10 ⁇ M, the final concentration of target R is 10 ⁇ M, and the final concentration of Cas protein reaction buffer is 1 ⁇ .
  • the Cas protein premix reaction solution includes Cas protein reaction buffer, Cas12 protein (purchased from Guangzhou Bolaisi Biotechnology Co., Ltd., the same below), silencing guide RNA, FQ probe, and target DNA.
  • the final concentrations of each component in the reaction solution were: the final concentration of the Cas protein reaction buffer was 1 ⁇ , the final concentration of the Cas12 protein was 100 nM, the final concentration of the silent guide RNA was 10 nM, the final concentration of the FQ probe was 400 nM, and the final concentration of the target was 1 nM.
  • the FQ probe used in this example is FQC6 (purchased from Huzhou Hippo Biotechnology Co., Ltd.), and its sequence is FAM-CCCCCC-BHQ1.
  • the Cas protein premixed reaction solution was incubated at 37° C. for 30 minutes. Measure the fluorescence signal intensity per minute of the Cas protein premixed reaction solution.
  • the target DNA was added to the positive group (P), the target DNA was replaced by RNase-free water in the negative group (N), and the CG group did not contain silencing DNA.
  • the guide RNA is inactivated by annealing hybridization.
  • the annealing premix reaction solution includes guide RNA, silencing RNA, and Cas protein reaction buffer (NEBuffer 2.1, purchased from New England Biotechnology (Beijing) Co., Ltd., the same below).
  • the final concentrations of each component in the annealing premixed reaction solution are: the final concentration of guide RNA is 10 ⁇ M, the final concentration of silencing RNA is 20 ⁇ M, and the final concentration of Cas protein reaction buffer is 1 ⁇ .
  • silencing RNA The sequence of the silencing RNA is: uguaauggaccucaaacccc (SEQ.ID.NO.33), and the guide RNA (that is, the "paired crRNA” in Table 2) is SEQ.ID.NO.1.
  • the main formulations of each experimental group in Figure 2 are shown in Table 2.
  • the "mismatched crRNA” in Table 2 is a guide RNA that is not completely complementary to the silencing RNA (SEQ.ID.NO.33), and the sequence is uaauuucuacuaaguguagauaaaaauuacagaagagguug (SEQ.ID.NO.34).
  • the experimental group without target was replaced by RNase-free water.
  • silencing RNA that is completely complementary to the spacer sequence of the guide RNA can completely block the activity of the guide RNA, and the addition of silencing RNA to the Cas12a cleavage system that does not pair with the guide RNA will not affect the system.
  • the guide RNA is inactivated by hybridization, and the molar concentration ratio of silencing RNA (SEQ.ID.NO.33) and guide RNA (SEQ.ID.NO.1) is 1:1, 1.5:1, and 2:1, respectively.
  • the modified PC-linker sequence used in the present invention was purchased from Huzhou Hippo Biotechnology Co., Ltd.
  • the sequence of the silencing RNA 6PC, 3PC, and 2PC used in this embodiment is the same as SEQ.ID.NO.33, and different numbers of PC-linkers are connected in its bases.
  • R5-3PC also paired 5 more bases to the repeat region.
  • + represents that the premixed reaction solution was irradiated with UV light for 30 s before step 3
  • - represents that no light treatment was performed before step 3.
  • the target DNA is a plasmid DNA (SEQ.ID.NO.35) containing the partial sequence of the B646L gene of African swine fever virus (ASFV))
  • a guide RNA (SEQ.ID.NO.1) is designed for the B646L gene of African swine fever virus (ASFV).
  • the silencing RNA sequence used was R5-3PC.
  • the Cas protein premixed reaction solution which includes the Cas protein reaction buffer, Cas12 protein, silencing guide RNA, and FQ probe.
  • the final concentration of the reaction solution is as follows: the final concentration of the Cas protein reaction buffer is 1 ⁇ , the final concentration of the Cas protein is 100 nM, the final concentration of the silent guide RNA is 100 nM, and the final concentration of the FQ probe is 400 nM.
  • the FQ probe used in this example is FQC6 with the sequence FAM-CCCCCC-BHQ1.
  • the recombinant enzyme polymerase isothermal amplification reaction solution includes primers, amplification buffer, amplification enzyme, plasmid DNA (synthesized by Nanjing Qingke Biotechnology Co., Ltd.) containing African swine fever virus (ASFV) B646L gene partial sequence, and magnesium acetate.
  • the final concentration of the reaction solution is as follows: the final concentration of the primer is 480nM, the final concentration of the amplification buffer is 1 ⁇ , the final concentration of the target is 100ag/ ⁇ L, and the final concentration of magnesium acetate is 14mM.
  • Front primer sequence gccgaagggaatggatactgagggaatagcaa (SEQ.ID.NO.36);
  • the target is added to the positive group (P), and RNase-free water is added to the negative group (N).
  • the target RNA is the O gene of the new coronavirus (SARS-CoV-2) (genome coordinates: 13201-15600, GenBank No.NC_045512)
  • the sequence of the designed guide RNA is uaauuucuacuaaguguagauggugauuucauacaaaccac (SEQ.ID.NO.38), the bases in italics are repeat regions, and the rest are spacers.
  • the silencing RNA sequence used was gugguu(PC-linker)uguauug(PC-linker)aaauca(PC-linker)ccaucua.
  • the reverse transcription recombinase polymerase isothermal amplification reaction solution includes primers, amplification buffer, amplification enzyme, reverse transcriptase, target, and magnesium acetate.
  • the final concentration of the reaction solution is: the final concentration of the primer is 480nM, the final concentration of the amplification buffer is 1 ⁇ , the final concentration of the reverse transcriptase is 5U/ ⁇ L, the final concentration of the target is 100aM, and the final concentration of magnesium acetate is 14mM.
  • the sequence of the front primer is: tgatgccatgcgaaatgctggtattgttgg (SEQ.ID.NO.39);
  • the rear primer sequence is: ctgcagttaaagccctggtcaaggttaata (SEQ.ID.NO.40);
  • the target used in this example is an RNA standard (the same below) that contains part of the novel coronavirus (SARS-CoV-2) O gene sequence (genome coordinates: 13201-15600, GenBank No. NC_045512) purchased from the China Institute of Metrology.
  • SARS-CoV-2 novel coronavirus
  • the target is added to the positive group, and RNase-free water is added to the negative group.
  • SARS-CoV-2 novel coronavirus
  • the sequence of the designed guide RNA is uaauuucuacuaaguguaagaucccccagcgcuucagcguuc (SEQ.ID.NO.41), the bases in italics are repeat regions, and the rest are spacer regions.
  • the silencing RNA sequence used was gaacgc(PC-linker)ugaagc(PC-linker)gcuggg(PC-linker)ggaucua.
  • the sequence of the front primer is: agacgtggtccagaacaaacccaaggaaatt (SEQ.ID.NO.42);
  • the rear primer sequence is: tgtgtaggtcaaccacgttcccgaaggtgt (SEQ.ID.NO.43);
  • the added targets are: novel coronavirus (SARS-CoV-2) RNA standard (purchased from China Institute of Metrology), coronavirus (SARS-CoV) RNA (SEQ.ID.NO.66), bat SARS-like coronavirus (bat-SL-CoVZC45) RNA (SEQ.ID.NO.67).
  • SARS-CoV-2 novel coronavirus
  • SARS-CoV coronavirus
  • bat SARS-like coronavirus bat SARS-like coronavirus
  • bat-SL-CoVZC45 bat SARS-like coronavirus
  • Coronavirus (SARS-CoV) RNA and bat SARS-like coronavirus (bat-SL-CoVZC45) RNA are plasmids with N-gene sequences synthesized by biological companies, and corresponding RNAs are obtained through transcription.
  • SARS-CoV-2 novel coronavirus
  • the amounts of targets added were: 10 4 copies, 10 3 copies, 10 2 copies, 10 1 copies, 10 0 copies, 0 copies (NTC).
  • This comparative example is a conventional single-tube CRISPR-Cas12-based DNA detection method, using the target DNA, Cas12a protein, guide RNA, fluorescent reporter probe and buffer of Example 1.
  • Cas protein premixed reaction liquid is prepared, and described reaction liquid comprises Cas protein reaction buffer, Cas protein, guide RNA, FQ probe.
  • the final concentration of the reaction solution is as follows: the final concentration of the Cas protein reaction buffer is 1 ⁇ , the final concentration of the Cas protein is 100 nM, the final concentration of the guide RNA is 100 nM, and the final concentration of the FQ probe is 400 nM.
  • the FQ probe used in this example is FQC6, and its sequence is FAM-CCCCCC-BHQ1.
  • RNA sequence is R5-3PC.
  • the target is added to the positive group, and RNase-free water is added to the negative group.
  • This comparative example uses agarose gel electrophoresis to verify the specificity of RT-RPA amplification novel coronavirus (SARS-CoV-2) RNA standard, coronavirus (SARS-CoV) RNA, bat SARS-like coronavirus (bat-SL-CoVZC45) RNA.
  • SARS-CoV-2 novel coronavirus
  • SARS-CoV coronavirus
  • the RT-RPA amplification reaction solution was the same as in Example 6, and the above RT-RPA amplification reaction solution was incubated at 37° C. for 30 min. The amplified products were then verified by agarose gel electrophoresis.
  • Example 6 when using the method proposed in this patent for the specific analysis of these three viruses, except that the amplified target is the new coronavirus (SARS-CoV-2) RNA standard, no other fluorescent signals are generated ( Figure 6), indicating that the method proposed in this patent has better specificity.
  • SARS-CoV-2 new coronavirus
  • the guide RNA is inactivated by annealing hybridization.
  • the annealing premixed reaction solution includes guide RNA, silencing DNA, and Cas protein reaction buffer (PCR buffer, purchased from Baoriji Biotechnology (Beijing) Co., Ltd., the same below).
  • the final concentrations of each component in the annealing premixed reaction solution are: the final concentration of guide RNA is 10 ⁇ M, the final concentration of silencing DNA is 20 ⁇ M, and the final concentration of Cas protein reaction buffer is 1 ⁇ .
  • RNA gaccaccccaaaaaugaaggggacuaaaaccaacaucagucugauaagcua (SEQ.ID.NO.44), bases in italics are repeat regions, and the rest are spacers;
  • the sequence of the guide RNA is divided into two parts, a repetitive sequence and a spacer sequence. Therefore, for the complementary pairing between silencing DNA and guide RNA, the abbreviation corresponding to the repeat region is called R, and the abbreviation corresponding to the spacer region is called S. Different numbers represent the base lengths of complementary pairings.
  • Cas protein premixed reaction solution which includes Cas protein reaction buffer, Cas13 protein (purchased from Guangzhou Bolaisi Biotechnology Co., Ltd., the same below), silencing guide RNA, FQ probe, and target RNA (same as Example 1).
  • the final concentrations of each component in the reaction solution are respectively: the final concentration of the Cas protein reaction buffer is 1 ⁇ , the final concentration of the Cas13 protein is 100 nM, the final concentration of the silent guide RNA is 10 nM, the final concentration of the FQ probe is 400 nM, and the final concentration of the target is 1 nM.
  • the FQ probe used in this example is FQU5 (purchased from Huzhou Hippo Biotechnology Co., Ltd.), and its sequence is FAM-UUUUU-BHQ1.
  • the target used is single-stranded RNA, and the target sequence is: uagcuuaucagacugauguuga (SEQ.ID.NO.65).
  • the target RNA was added to the positive group (P), the target DNA was replaced by RNase-free water in the negative group (N), and the CG group did not contain silencing DNA.
  • silencing guide RNA Preparation of silencing guide RNA.
  • the guide RNA was inactivated by hybridization, and the molar concentration ratios of silencing RNA and guide RNA were 1:1, 1.5:1, and 2:1, respectively.
  • Steps (2)-(3) are the same as in Example 7.
  • the target RNA was added to the positive group (P), the target DNA was replaced by RNase-free water in the negative group (N), and the CG group did not contain silencing DNA.
  • the fluorescence intensity shown in the figure is the fluorescence signal intensity when the Cas protein premixed reaction solution was reacted for 60 minutes.
  • + represents that the premixed reaction solution was irradiated with UV light for 30 s before step 3
  • - represents that no light treatment was performed before step 3.

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Abstract

本发明公开了一种基于光控的CRISPR-Cas核酸检测试剂盒及检测方法，该试剂盒包括沉默引导RNA和Cas蛋白；沉默引导RNA是由沉默核苷酸和引导RNA通过退火杂交形成；引导RNA是根据靶标核酸序列设计的，包含重复区和间隔区两个区域；沉默核苷酸与引导RNA的间隔区序列完全互补配对，或者与引导RNA的重复区序列完全配对；沉默核苷酸的碱基之间采用PC linker连接；Cas蛋白为Cas12蛋白或Cas13蛋白。本申请将核酸扩增和CRISPR-Cas检测在时间上分开，但是可以实现在一个试管里面封闭完成，避免了开盖试剂转移过程，在确保高的检测灵敏度的同时，又保证了检测不受气溶胶污染影响。

Description

一种基于光控的CRISPR-Cas核酸检测试剂盒及检测方法 技术领域
本发明属于生物检测领域，具体涉及一种基于光控的CRISPR-Cas核酸检测试剂盒及检测方法。
背景技术
目前，CRISPR-Cas系统，如CRISPR-Cas12和CRISPR-Cas13系统，已广泛地应用于核酸诊断领域，但是单独的CRISPR-Cas检测体系灵敏度仍然难以达到PCR的水平，所以当前基于CRISPR-Cas发展起来的核酸检测方法绝大多数要结合核酸扩增。
在将核酸扩增和CRISPR-Cas识别体系结合到单个试管的检测方法中，因为CRISPR-Cas检测体系将识别并切割靶核酸，所以会导致扩增模板的断裂，从而降低扩增效率。另一方面，在扩增过程当中，初期扩增产物也会被CRISPR-Cas检测体系识别并切割，从而导致可被循环使用的模板的断裂，导致扩增效率下降。
这些因素导致现有的基于CRISPR-Cas的单管核酸检测技术仍然效率较低，满足不了高灵敏度的核酸检测要求。
由于这些原因，很多报道的基于CRISPR-Cas的核酸检测技术将核酸扩增过程和CRISPR-Cas检测过程分步进行。但是分步过程会涉及到反应试管的开盖和液体转移，这些过程将容易引起气溶胶污染，从而导致假阳性。因此，发展能提高CRISPR-Cas系统灵敏度的单管检测方法是本领域重要的科学课题。
发明内容
本发明的目的在于提供一种基于光控的CRISPR-Cas核酸检测试剂盒及检测方法，将核酸扩增和CRISPR-Cas检测在时间上分开，又可以在一个试管中封闭完成，避免了开盖试剂转移过程，在确保高检测灵敏度的同时，保证了检测不受气溶胶污染影响。
本发明的目的通过下述技术方案实现：
一种基于光控的CRISPR-Cas核酸检测试剂盒，包括沉默引导RNA和Cas蛋白；
所述的试剂盒还包括荧光报告探针、扩增引物、酶或缓冲液中的一种以上；
所述的沉默引导RNA，是由沉默核苷酸和引导RNA通过退火杂交形成；
所述引导RNA是根据靶标核酸序列设计的，包含重复区(repetitive sequence)和间隔区(spacer sequence)两个区域，引导RNA与Cas蛋白结合形成蛋白核酸复合物，引导蛋白核酸复合物识别并切割靶标核酸；
所述沉默核苷酸的碱基之间采用可被光激活降解的PC linker连接；
所述沉默核苷酸与引导RNA的间隔区序列完全互补配对，或者与引导RNA的重复区序列完全配对；
优选地，所述沉默核苷酸除了与引导RNA间隔区序列完全互配之外，还往重复区多配对若干个(1-21个)碱基，优选多配对5个碱基；
所述沉默核苷酸中可以含有1-6个PC linker，优选含有3个PC linker；
所述沉默核苷酸中，每隔5-10个碱基嵌入一个PC linker；优选每隔6个碱基嵌入一个PC linker；
所述沉默核苷酸与引导RNA的摩尔浓度比为(1～2):1，优选2:1；
所述引导RNA的碱基长度为30-60个，优选41或51个；
所述沉默核苷酸的碱基长度为20-40个，优选25个或30个；
所述的Cas蛋白为Cas12蛋白或Cas13蛋白，这些蛋白不仅具有顺式切割活性，还具有反式切割活性，这是其不同于Cas9蛋白的地方(Cas9蛋白仅具有顺式切割活性)；同时，Cas12蛋白是一种以DNA(dsDNA或ssDNA)为靶标的核酸内切酶，其激活的反式切割活性可切割ssDNA；Cas13蛋白是一种以RNA为靶标的核酸内切酶，其激活的反式切割活性可切割ssRNA。
所述的等温扩增引物是根据靶标核酸序列设计的；
所述的等温扩增具体指重组酶聚合酶等温扩增(RPA)、环介导等温扩增(LAMP)、依赖解旋酶恒温扩增(HDA)、链置换扩增(SDA)、滚环扩增(RCA)、指数等温扩增(EXPAR)、依赖核酸序列扩增(NASBA)、单引物等温扩增技术(SPIA)、嵌合引物引发的核酸等温扩增(ICAN)、链交换扩增技术(SEA)等，优选重组酶聚合酶等温扩增(RPA)。
一种基于上述试剂盒的光控CRISPR-Cas核酸检测方法，包括以下步骤：
(1)将沉默引导RNA、Cas蛋白、荧光报告探针、扩增引物、酶与缓冲液混合配制成预混反应液，加入包含靶标核酸的待测物形成单管内的混合体系；
(2)将上述混合体系进行等温扩增，待等温扩增完成，启动UV光照，连接子断裂，沉默核苷酸从引导RNA上脱离，CRISPR-Cas核酸切割反应启动，最后检测所述混合体系中的荧光信号；
进一步地，步骤(1)所述混合体系中，Cas蛋白终浓度可为10-500nM，所述沉默核苷酸-引导RNA复合物终浓度为10-500nM，确保Cas蛋白与靶标核酸的切割反应能够高效率发生，所述沉默核苷酸-引导RNA复合物与Cas蛋白结合形成蛋白核酸复合物，沉默核苷酸-引导RNA复合物的浓度与Cas蛋白的浓度成对应关系。
进一步地，步骤(1)所述混合体系中，所述荧光报告探针终浓度为200nM-1μM，以 保证靶标核酸附近存在充足的荧光报告探针可被切割。
进一步地，步骤(2)所述等温扩增的反应温度为25-65℃，以保证核酸扩增反应正常进行和Cas蛋白不失活。
进一步地，步骤(2)所述等温扩增的反应时间至少5min以上，所述UV光照时间至少5s以上，所述CRISPR-Cas核酸检测的时间至少5min以上。
本发明的原理如下：
Cas蛋白与引导RNA结合形成蛋白核酸复合物，当存在靶标核酸时，在引导RNA介导下，蛋白核酸复合物识别并切割靶标核酸，同时Cas蛋白的反式切割活性被激发，以非特异性的方式切割周围的荧光报告探针。荧光报告探针一端连接荧光素，一端连接淬灭基团，其被切断后则可被激发产生荧光信号。
本发明设计沉默核苷酸与引导RNA互补，从而阻断引导RNA对靶标核酸的识别。同时，设计沉默核苷酸由光激活的连接子连接，可以很便捷地用UV光激发导致沉默核苷酸断裂，从而对CRISPR-Cas检测体系进行复活。
当将这种失活的CRISPR-Cas检测体系与核酸扩增技术结合到一管反应时，核酸扩增的模板不会被CRISPR-Cas所切割，所以扩增效率不受影响。当核酸扩增完成以后，用光激活CRISPR-Cas检测体系。
这种检测方式虽然将核酸扩增和CRISPR-Cas检测在时间上分开，但是可以实现在一个试管里面封闭完成，避免了开盖试剂转移过程，在确保高的检测灵敏度的同时，又保证了检测不受气溶胶污染影响。
本发明相对于现有技术具有如下的优点及效果：
1、相对于分步反应体系，本发明的试剂盒和方法避免了气溶胶污染的产生，简化了实验步骤。
2、相对基于相分离的一管反应体系，本发明的试剂盒和方法简化了反应体系和实验步骤。
3、相对于在管内不同位置发生Cas反应和扩增反应的一管反应体系，本发明的试剂盒和方法解决了其不稳定的问题，并且不需要依赖离心设备。
4、相对于优化Cas反应体系(例如：需要两条及以上的引导RNA)，本发明的试剂盒和方法仅用一条引导RNA即可实现高灵敏度的核酸检测，简化了反应体系和实验步骤。
附图说明
图1是不同沉默引导DNA所致的荧光信号强度。
图2是沉默引导RNA所致的荧光信号强度。
图3是不同沉默引导RNA所致的荧光信号强度。
图4是采用本发明试剂盒和方法检测靶标时的荧光强度曲线。
图5是采用本发明试剂盒和方法检测靶标时的荧光强度曲线。
图6是采用本发明试剂盒和方法检测不同靶标时的荧光强度。
图7是采用本发明试剂盒和方法对不同靶标量检测时的荧光强度曲线。
图8是采用传统的CRISPR-Cas12方法检测靶标时的荧光强度曲线。
图9是采用琼脂糖凝胶电泳检测靶标时的电泳图。
图10是不同沉默引导DNA所致的荧光信号强度。
图11是沉默引导DNA所致的荧光信号强度。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。
实施例1
沉默DNA(未修饰PC-linker)的筛选
(1)制备沉默引导RNA
采用退火杂交的方式将引导RNA失活。退火预混反应液包括引导RNA、沉默DNA、Cas蛋白反应缓冲液(NEBuffer 2.1，购买于纽英伦生物技术(北京)有限公司，下同)。所述退火预混反应液中各组分的终浓度分别为：引导RNA终浓度为10μM、沉默DNA终浓度为20μM、Cas蛋白反应缓冲液的终浓度为1×。将上述预混反应液置于核酸扩增仪上。在70℃条件下温育5min，并逐步降温直至温度降至室温，得到沉默引导RNA；
引导RNA：uaauuucuacuaaguguagaugggguuugagguccauuaca(SEQ.ID.NO.1)，斜体的碱基为重复区，其余为间隔区；
沉默DNA的代号、序列、序号如表1所示：
表1
代号 序列 序号
S5R20 accccatctacacttagtagaaatt SEQ.ID.NO.2
S5R19 accccatctacacttagtagaaat SEQ.ID.NO.3
S5R18 accccatctacacttagtagaaa SEQ.ID.NO.4
S5R17 accccatctacacttagtagaa SEQ.ID.NO.5
S5R16 accccatctacacttagtaga SEQ.ID.NO.6
S5R14 accccatctacacttagta SEQ.ID.NO.7
S5R13 accccatctacacttagt SEQ.ID.NO.8
S5R12 accccatctacacttag SEQ.ID.NO.9
S5R11 accccatctacactta SEQ.ID.NO.10
S5R10 accccatctacactt SEQ.ID.NO.11
S10R15 ctcaaaccccatctacacttagtag SEQ.ID.NO.12
S9R15 tcaaaccccatctacacttagtag SEQ.ID.NO.13
S8R15 caaaccccatctacacttagtag SEQ.ID.NO.14
S7R15 aaaccccatctacacttagtag SEQ.ID.NO.15
S6R15 aaccccatctacacttagtag SEQ.ID.NO.16
S5R15 accccatctacacttagtag SEQ.ID.NO.17
S4R15 ccccatctacacttagtag SEQ.ID.NO.18
S3R15 cccatctacacttagtag SEQ.ID.NO.19
S2R15 ccatctacacttagtag SEQ.ID.NO.20
S1R15 catctacacttagtag SEQ.ID.NO.21
R15 atctacacttagtag SEQ.ID.NO.22
R21 atctacacttagtagaaatta SEQ.ID.NO.23
S-17 tgtaatggacctcaaac SEQ.ID.NO.24
S-16 tgtaatggacctcaaa SEQ.ID.NO.25
S-15 tgtaatggacctcaa SEQ.ID.NO.26
S-14 tgtaatggacctca SEQ.ID.NO.27
S-13 tgtaatggacctc SEQ.ID.NO.28
S-12 tgtaatggacct SEQ.ID.NO.29
S-11 tgtaatggacc SEQ.ID.NO.30
关于沉默DNA代号的含义，引导RNA的序列分为两个部分，重复区(repetitive sequence)和间隔区(spacer sequence)。因此，沉默DNA与引导RNA之间的互补配对，对应重复区的简称为R，对应间隔区的简称为S。不同的数字代表互补配对的碱基长度。
(2)制备靶标DNA
所用靶标为通过退火形成的双链DNA，靶标序列为：
靶标F：ctgatagtatttaggggtttgaggtccattacagctgtaatgaacattacgtcttatgt(SEQ.ID.NO.31)
靶标R：acataagacgtaatgttcattacagctgtaatggacctcaaacccctaaatactatcag(SEQ.ID.NO.32)
退火预混反应液包括靶标F、靶标R、Cas蛋白反应缓冲液。所述退火预混反应液中各组分的终浓度分别为：靶标F终浓度为10μM、靶标R终浓度为10μM、Cas蛋白反应缓冲液的终浓度为1×。将上述预混反应液置于核酸扩增仪上。在70℃条件下温育5min，并逐步降温直至温度降至室温，得到靶标DNA。
(3)配制Cas蛋白预混反应液
Cas蛋白预混反应液包括Cas蛋白反应缓冲液，Cas12蛋白(购自广州博徕斯生物科技股份有限公司，下同)，沉默引导RNA，FQ探针，靶标DNA。反应液中各组分的终浓度分别为：Cas蛋白反应缓冲液终浓度为1×，Cas12蛋白的终浓度为100nM，沉默引导RNA的终浓度为10nM，FQ探针的终浓度为400nM，靶标的终浓度为1nM。本实施例中所使用FQ探针为FQC6(购自湖州河马生物科技有限公司)，序列为FAM-CCCCCC-BHQ1。
所述Cas蛋白预混反应液在37℃下孵育30分钟。测定所述Cas蛋白预混反应液每分钟的荧光信号强度。
图1中，阳性组(P)中加入了靶标DNA，阴性组(N)用无RNase水代替靶标DNA，CG组不含有沉默DNA。
实验结果如图1所示，与引导RNA互补配对不同位置的沉默DNA对其活性进行封闭时，并不能起到很好的活性封闭效果。
然而，图1中，当使用S15、S16、S17三条沉默DNA进行引导RNA的活性封闭时，即使是不加入靶标DNA的阴性对照组也有荧光信号产生，可能是这三条沉默DNA已足以作为靶标，进而激活Cas蛋白的切割活性。
实施例2
沉默RNA(未修饰PC-linker，与引导RNA的间隔区序列完全互补配对)的封闭效果验证
(1)制备沉默引导RNA
采用退火杂交的方式将引导RNA失活。退火预混反应液包括引导RNA、沉默RNA、Cas蛋白反应缓冲液(NEBuffer 2.1，购买于纽英伦生物技术(北京)有限公司，下同)。所述退火预混反应液中各组分的终浓度分别为：引导RNA终浓度为10μM、沉默RNA终浓度为20μM、Cas蛋白反应缓冲液的终浓度为1×。将上述预混反应液置于核酸扩增仪上。在70℃条件下温育5min，并逐步降温直至温度降至室温，得到沉默引导RNA；
沉默RNA的序列为：uguaauggaccucaaacccc(SEQ.ID.NO.33)，引导RNA(即表2中的“配对crRNA”)为SEQ.ID.NO.1。
(2)制备靶标DNA，同实施例1。
(3)配制Cas蛋白预混反应液，同实施例1。
(4)所述Cas蛋白预混反应液在37℃下孵育60分钟。测定所述Cas蛋白预混反应液每分钟的荧光信号强度。
图2中各实验组的主要加样配方如表2所示。表2中的“错配crRNA”是与沉默RNA(SEQ.ID.NO.33)不完全互补配对的引导RNA，序列为 uaauuucuacuaaguguagauaaaaauuacagaagagguug(SEQ.ID.NO.34)。不含靶标的实验组则以无RNase水代替靶标。
表2
  配对crRNA 错配crRNA 沉默RNA Cas12a蛋白 靶标
一 +     + +
二 +     +  
三 +   + + +
四 +   + +  
五   +   + +
六   +   +  
七   + + + +
八   + + +  
结果如图2所示，与引导RNA的间隔区序列完全互补配对的沉默RNA取得了极好的沉默效果，几乎不产生荧光信号(实验三)；而与沉默RNA不完全互补配对的引导RNA在封闭后，仍旧具有很强的荧光信号(实验七)。
说明使用与引导RNA的间隔序列完全互补配对的沉默RNA，能够实现引导RNA活性的完全封闭，且沉默RNA加入到引导RNA不配对的Cas12a切割体系中，不会对该体系产生影响。
实施例3
探究沉默RNA(不同数量的PC-linker，不同的封闭位置，不同用量比)对于引导RNA的活性封闭效果，及其光照之后的活性恢复效果
(1)采用杂交的方式将引导RNA失活，按照沉默RNA(SEQ.ID.NO.33)与引导RNA(SEQ.ID.NO.1)按摩尔浓度比分别为1：1，1.5：1，2：1的比例加入。将10uM引导RNA、沉默RNA、1×Cas蛋白反应缓冲液混合配制预混反应液。将上述预混反应液置于核酸扩增仪上。在70℃条件下反应5min，并逐步降温直至温度降至室温，得到沉默引导RNA；
本发明所使用的修饰PC-linker的序列购自湖州河马生物科技有限公司。
(2)制备靶标DNA，同实施例1。
(3)配制Cas蛋白预混反应液，同实施例1。
(4)在37℃下所述Cas蛋白预混反应液孵育60min。测定所述Cas蛋白预混反应液每分钟的荧光信号强度。
本实施例所使用的沉默RNA 6PC、3PC、2PC的序列同SEQ.ID.NO.33，在其碱基中连接不同数量的PC-linker。R5-3PC除了与引导RNA间隔区序列完全互配之外，还往重复区多配对了5个碱基。
具体地，各沉默RNA的代号和序列如表3所示：
表3
Figure PCTCN2022091068-appb-000001
在图3中，+代表在步骤3之前，使用UV光对该预混反应液照射30s，-代表在步骤3之前，未进行光照处理。
结果如图3所示，使用R5-3PC沉默引导RNA时，能够达到最佳的效果。
从图中可以看出，不论所使用的沉默RNA与引导RNA的比例是1：1；1.5：1；2：1，在光照后，其活性都能与阳性对照组持平，但是从不光照组就能看出封闭效果，即信号值越低，代表封闭效果越好，从图中可以看出，当沉默RNA与引导RNA的加入比例为2：1，并且所使用的封闭RNA为R5-3PC时，封闭效果最佳。
实施例4
验证光控的CRISPR-Cas12检测体系的可行性(靶标DNA为含有非洲猪瘟病毒(ASFV)的B646L基因部分序列的质粒DNA(SEQ.ID.NO.35))
(1)针对非洲猪瘟病毒(ASFV)的B646L基因，设计引导RNA(SEQ.ID.NO.1)。使用的沉默RNA序列为R5-3PC。
(2)制备沉默引导RNA，同实施例2。
(3)配制Cas蛋白预混反应液，所述反应液包括Cas蛋白反应缓冲液，Cas12蛋白，沉默引导RNA，FQ探针。所述反应液的终浓度为：Cas蛋白反应缓冲液终浓度为1×，Cas蛋白的终浓度为100nM，沉默引导RNA的终浓度为100nM，FQ探针的终浓度为400nM。本实例中所使用FQ探针为FQC6，序列为FAM-CCCCCC-BHQ1。
(4)配制扩增反应液。本实施例采用重组酶聚合酶等温扩增方式进行DNA靶标的扩增，使用TwistAmp Basic Kit(货号111781)。所述重组酶聚合酶等温扩增反应液包括引物，扩增缓冲液，扩增酶，含有非洲猪瘟病毒(ASFV)B646L基因部分序列的质粒DNA(由南 京擎科生物科技有限公司合成)、醋酸镁。所述反应液的终浓度为：引物的终浓度为480nM，扩增缓冲液终浓度为1×，靶标终浓度为100ag/μL，醋酸镁终浓度为14mM。
前引物序列：gccgaagggaatggatactgagggaatagcaa(SEQ.ID.NO.36)；
后引物序列：tcccgagaactctcacaatatccaaacagcag(SEQ.ID.NO.37)；
(5)将上述Cas蛋白反应液和扩增反应液混合配制预混反应液，在37℃条件下孵育所述预混反应液。30min后，将其取出，使用UV光对该预混反应液照射30s。采用实时定量PCR仪测定预混反应液的荧光信号强度。
本实施例中，阳性组(P)加入靶标，阴性组(N)加入的是无RNase水。
实验结果如图4所示，阳性组和阴性组的信号差距很大，说明阳性组具有良好的信噪比和反应速率。
实施例5
验证光控的CRISPR-Cas12检测体系的可行性(靶标RNA为新型冠状病毒(SARS-CoV-2)的O基因(基因组坐标：13201-15600，GenBank No.NC_045512)
(1)针对新型冠状病毒(SARS-CoV-2)的O基因，设计引导RNA的序列为uaauuucuacuaaguguagauggugauuucauacaaaccac(SEQ.ID.NO.38)，斜体的碱基为重复区，其余为间隔区。所使用的沉默RNA序列为gugguu(PC-linker)uguaug(PC-linker)aaauca(PC-linker)ccaucua。
(2)制备沉默引导RNA，同实施例4；
(3)配制Cas蛋白预混反应液，同实施例4；
(4)配制反转录扩增反应液，采用反转录重组酶聚合酶等温扩增方式(RT-RPA)进行RNA靶标的扩增，使用TwistAmp Basic Kit(货号111781)。所述反转录重组酶聚合酶等温扩增反应液包括引物，扩增缓冲液，扩增酶，反转录酶，靶标，醋酸镁。所述反应液的终浓度为：引物的终浓度为480nM，扩增缓冲液终浓度为1×，反转录酶终浓度为5U/μL，靶标终浓度为100aM，醋酸镁终浓度为14mM。
(5)同实施例4；
前引物序列为：tgatgccatgcgaaatgctggtattgttgg(SEQ.ID.NO.39)；
后引物序列为：ctgcagttaaagccctggtcaaggttaata(SEQ.ID.NO.40)；
本实施例所使用的靶标为购自中国计量科学研究院的、含有部分新型冠状病毒(SARS-CoV-2)O基因序列(基因组坐标：13201-15600，GenBank No.NC_045512)的RNA标准品(下同)。
本实施例中，阳性组加入的是靶标，阴性组加入的是无RNase水。
如图5所示，在运用于SARS-CoV-2的检测时，阳性组和阴性组的信号差距很大，说明 阳性组具有良好的信噪比和反应速率。
实施例6
基于新型冠状病毒(SARS-CoV-2)N基因的特异性检测
(1)针对新型冠状病毒(SARS-CoV-2)的N基因(基因组坐标：28274-29533，GenBank:MN908947.3)，设计引导RNA的序列为uaauuucuacuaaguguagaucccccagcgcuucagcguuc(SEQ.ID.NO.41)，斜体的碱基为重复区，其余为间隔区。所使用的沉默RNA序列为gaacgc(PC-linker)ugaagc(PC-linker)gcuggg(PC-linker)ggaucua。
余下步骤同实施例5。
前引物序列为：agacgtggtccagaacaaacccaaggaaatt(SEQ.ID.NO.42)；
后引物序列为：tgtgtaggtcaaccacgttcccgaaggtgt(SEQ.ID.NO.43)；
加入的靶标分别为：新型冠状病毒(SARS-CoV-2)RNA标准品(购自中国计量科学研究院)，冠状病毒(SARS-CoV)RNA(SEQ.ID.NO.66)，蝙蝠SARS样冠状病毒(bat-SL-CoVZC45)RNA(SEQ.ID.NO.67)。
冠状病毒(SARS-CoV)RNA和蝙蝠SARS样冠状病毒(bat-SL-CoVZC45)RNA是由生物公司合成N-gene序列的质粒，并通过转录得到了相应的RNA。
NTC组加入无RNase水。
如图6所示，在运用于SARS-CoV-2的特异性检测时，本方法具有良好的检测特异性。另外，值得指出的是本方法将有望运用于单碱基突变的检测。
实施例7
基于新型冠状病毒(SARS-CoV-2)O基因的灵敏度检测
实验材料和步骤同实施例5。
加入的靶标量分别为：10 4copies、10 3copies、10 2copies、10 1copies、10 0copies、0copies(NTC)。
如图7所示，在运用于SARS-CoV-2的灵敏度检测时，本方法具有良好的检测灵敏度，其最低检测限可达10copies。
对比例1
本对比例为常规的单管的基于CRISPR-Cas12的DNA检测方法，选用实施例1的靶标DNA、Cas12a蛋白、引导RNA、荧光报告探针和缓冲液。
(1)针对非洲猪瘟病毒(ASFV)的B646L基因，设计引导RNA的序列(SEQ.ID.NO.1)。
(2)配制Cas蛋白预混反应液，所述反应液包括Cas蛋白反应缓冲液，Cas蛋白，引导 RNA，FQ探针。所述反应液的终浓度为：Cas蛋白反应缓冲液终浓度为1×，Cas蛋白的终浓度为100nM，引导RNA的终浓度为100nM，FQ探针的终浓度为400nM。本实施例中所使用FQ探针为FQC6，序列为FAM-CCCCCC-BHQ1。
图8中“本发明方法”则在Cas蛋白预混反应液中加入了沉默引导RNA，其制法同实施例1，沉默RNA序列为R5-3PC。
除不进行光照处理外，余下步骤同实施例4。
本实施例中，阳性组加入的是靶标，阴性组加入的是无RNase水。
如图8所示，在使用常规的单管的基于CRISPR-Cas12的DNA检测方法进行与本方法相同浓度(100ag/μL)的靶标的检测时，本发明方法具有很强的荧光信号强度(图8)，而常规的CRISPR-Cas12检测方法几乎没有荧光信号产生(图8)。该结果进一步阐明本发明所提出方法能够显著的提高单管检测法的灵敏度。
对比例2
本对比例为使用琼脂糖凝胶电泳验证RT-RPA扩增新型冠状病毒(SARS-CoV-2)RNA标准品，冠状病毒(SARS-CoV)RNA，蝙蝠SARS样冠状病毒(bat-SL-CoVZC45)RNA的特异性。使用实施例6的靶标RNA，和RT-RPA扩增体系。
RT-RPA扩增反应液同实施例6，将上述RT-RPA扩增反应液在37℃条件下孵育30min。随后使用琼脂糖凝胶电泳，验证扩增产物。
如图9所示，当使用琼脂糖凝胶电泳进行新型冠状病毒(SARS-CoV-2)RNA标准品，冠状病毒(SARS-CoV)RNA，蝙蝠SARS样冠状病毒(bat-SL-CoVZC45)RNA三种病毒RNA的RT-RPA扩增产物的验证时，可观察到除NTC(以无RNase代替靶标)外，均有扩增条带出现，说明仅仅依赖于RT-RPA扩增的特异性，是不足以区分这三种病毒的。
而在实施例6中，使用本专利所提出方法进行这三种病毒的特异性分析时，除扩增靶标为新型冠状病毒(SARS-CoV-2)RNA标准品外，其它均没有荧光信号产生(图6)，说明本专利所提出方法具有更好的特异性。
实施例8
针对Cas13系统的沉默DNA(不带PC-linker)的筛选
(1)制备沉默引导RNA。采用退火杂交的方式将引导RNA失活。退火预混反应液包括引导RNA、沉默DNA、Cas蛋白反应缓冲液(PCR缓冲液，购买于宝日医生物技术(北京)有限公司，下同)。所述退火预混反应液中各组分的终浓度分别为：引导RNA终浓度为10μM、沉默DNA终浓度为20μM、Cas蛋白反应缓冲液的终浓度为1×。将上述预混反应液置于核酸扩增仪上。在70℃条件下温育5min，并逐步降温直至温度降至室温，得到沉默引导RNA；
引导RNA：gaccaccccaaaaaugaaggggacuaaaaccaacaucagucugauaagcua(SEQ.ID.NO.44)，斜体的碱基为重复区，其余为间隔区；
沉默DNA的代号、序列、序号如表4所示：
表4
代号 序列 序号
S11 tagcttatcag SEQ.ID.NO.45
S16 tagcttatcagactga SEQ.ID.NO.46
S21 tagcttatcagactgatgttg SEQ.ID.NO.47
R12 gttttagtcccc SEQ.ID.NO.48
R17 ccccttcatttttgggg SEQ.ID.NO.49
R18 ttcatttttggggtggtc SEQ.ID.NO.50
R21 gttttagtccccttcattttt SEQ.ID.NO.51
R30 gttttagtccccttcatttttggggtggtc SEQ.ID.NO.52
S21R1 tagcttatcagactgatgttgg SEQ.ID.NO.53
S21R2 tagcttatcagactgatgttggt SEQ.ID.NO.54
S21R3 tagcttatcagactgatgttggtt SEQ.ID.NO.55
S21R4 tagcttatcagactgatgttggttt SEQ.ID.NO.56
S21R5 tagcttatcagactgatgttggtttt SEQ.ID.NO.57
S20R5 agcttatcagactgatgttggtttt SEQ.ID.NO.58
S19R5 gcttatcagactgatgttggtttt SEQ.ID.NO.59
S18R5 cttatcagactgatgttggtttt SEQ.ID.NO.60
S17R5 ttatcagactgatgttggtttt SEQ.ID.NO.61
S15R5 atcagactgatgttggtttt SEQ.ID.NO.62
S15R10 atcagactgatgttggttttagtcc SEQ.ID.NO.63
S8R12 tgatgttggttttagtcccc SEQ.ID.NO.64
关于沉默DNA代号的含义，引导RNA的序列分为两个部分，重复区(repetitive sequence)和间隔区(spacer sequence)。因此，沉默DNA与引导RNA之间的互补配对，对应重复区的简称为R，对应间隔区的简称为S。不同的数字代表互补配对的碱基长度。
(2)配制Cas蛋白预混反应液，所述反应液包括Cas蛋白反应缓冲液，Cas13蛋白(购自广州博徕斯生物科技股份有限公司，下同)，沉默引导RNA，FQ探针，靶标RNA(同实施例1)。所述反应液中各组分的终浓度分别为：Cas蛋白反应缓冲液终浓度为1×，Cas13蛋白的终浓度为100nM，沉默引导RNA的终浓度为10nM，FQ探针的终浓度为400 nM，靶标的终浓度为1nM。本实施例中所使用FQ探针为FQU5(购自湖州河马生物科技有限公司)，序列为FAM-UUUUU-BHQ1。
所用靶标为单链RNA，靶标序列为：uagcuuaucagacugauguuga(SEQ.ID.NO.65)。
(3)在37℃下所述Cas蛋白预混反应液孵育30分钟。测定所述Cas蛋白预混反应液每分钟的荧光信号强度。
图10中，阳性组(P)中加入了靶标RNA，阴性组(N)用无RNase水代替靶标DNA，CG组不含有沉默DNA。
结果如图10所示，使用R30序列(与引导RNA的重复区序列完全配对)进行引导RNA的活性沉默时，荧光信号强度最低，说明该序列的沉默效果最佳。
实施例9
针对Cas13系统探究沉默DNA(带有PC-linker，不同用量比)对于引导RNA的活性封闭效果，及其光照之后的活性恢复效果。
(1)制备沉默引导RNA。采用杂交的方式将引导RNA失活，按照沉默RNA与引导RNA摩尔浓度比分别为1：1，1.5：1，2：1的比例加入。将10uM引导RNA(SEQ.ID.NO.43)、沉默RNA(gttttagt(PC-linker)ccccttc(PC-linker)atttttg(PC-linker)gggtggtc)、1×Cas蛋白反应缓冲液混合配制预混反应液。将上述预混反应液置于核酸扩增仪上。在70℃条件下反应5min，并逐步降温直至温度降至室温；
步骤(2)-(3)同实施例7。
图11中，阳性组(P)中加入了靶标RNA，阴性组(N)用无RNase水代替靶标DNA，CG组不含有沉默DNA。图中所示荧光强度为Cas蛋白预混反应液反应60min时的荧光信号强度。在图11中，+代表在步骤3之前，使用UV光对该预混反应液照射30s，-代表在步骤3之前，未进行光照处理。
从图中可以看出，不论所使用的沉默RNA与引导RNA的比例是1：1；1.5：1；2：1，在光照后，其活性都能与阳性对照组持平，但是从不光照组就能看出封闭效果，即信号值越低，代表封闭效果越好，从图中可以看出，当沉默RNA与引导RNA的加入比例为2：1时，封闭效果最佳。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (10)

  1. 一种基于光控的CRISPR-Cas核酸检测试剂盒，其特征在于包括沉默引导RNA和Cas蛋白；
    所述的沉默引导RNA，是由沉默核苷酸和引导RNA通过退火杂交形成；
    所述引导RNA是根据靶标核酸序列设计的，包含重复区和间隔区两个区域；
    所述沉默核苷酸与引导RNA的间隔区序列完全互补配对，或者与引导RNA的重复区序列完全配对；
    所述沉默核苷酸的碱基之间采用PC linker连接；
    所述的Cas蛋白为Cas12蛋白或Cas13蛋白。
  2. 根据权利要求1所述的试剂盒，其特征在于：所述沉默核苷酸除了与引导RNA间隔区序列完全互配之外，还往重复区多配对若干个碱基。
  3. 根据权利要求1所述的试剂盒，其特征在于：所述沉默核苷酸中含有1-6个PC linker，每隔5-10个碱基嵌入一个PC linker。
  4. 根据权利要求1所述的试剂盒，其特征在于：所述沉默核苷酸与引导RNA的摩尔浓度比为(1～2):1。
  5. 根据权利要求1所述的试剂盒，其特征在于：所述沉默核苷酸中含有3个PC linker。
  6. 根据权利要求1所述的试剂盒，其特征在于：所述沉默核苷酸中，每隔6个碱基嵌入一个PC linker。
  7. 根据权利要求1所述的试剂盒，其特征在于：所述沉默核苷酸与引导RNA的质量比为2:1。
  8. 根据权利要求1所述的试剂盒，其特征在于：所述的试剂盒还包括荧光报告探针、扩增引物、酶或缓冲液中的一种以上。
  9. 一种基于上述试剂盒的光控CRISPR-Cas核酸检测方法，其特征在于使用权利要求1-8任一项所述的试剂盒。
  10. 根据权利要求9所述的检测方法，其特征在于包括以下步骤：
    (1)将沉默引导RNA、Cas蛋白、荧光报告探针、扩增引物、酶与缓冲液混合配制成预混反应液，加入包含靶标核酸的待测物形成单管内的混合体系；
    (2)将上述混合体系进行等温扩增，待等温扩增完成，启动UV光照，连接子断裂，沉默核苷酸从引导RNA上脱离，CRISPR-Cas核酸切割反应启动，最后检测所述混合体系中的荧光信号。
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