Application of Cas12Max in preparation of gene detection product

Abstract

The invention relates to the field of nucleic acid detection, in particular to application of Cas12Max in preparation of a gene detection product, the gene detection product is a gene detection kit, the gene detection kit comprises an RPA reaction composition and a CRISPR / Cas12Max reaction composition, the RPA reaction composition comprises an RPA reaction primer group, and the CRISPR / Cas12Max reaction composition comprises an RPA reaction primer group. The CRISPR / Cas12Max reaction composition comprises Cas12Max, gRNA (guide Ribonucleic Acid) and a reporter gene colloidal gold probe or a fluorescent probe. The kit is a nucleic acid home detection system, realizes high-sensitivity, low-cost and instrumentation-free detection of nucleic acid samples under the assistance of simple, convenient and universal heat insulation equipment, droppers and the like, has wider PAM limitation, and can be suitable for nucleic acid detection of PAM lacking TTV motifs.

Description

Technical Field

[0001] The present invention relates to the field of nucleic acid detection, and in particular to the use of Cas12Max in the preparation of gene detection products. Background Art

[0002] With the continuous development of the biomedical field, nucleic acid detection technology plays a key role in disease diagnosis. The SARS-CoV-2 virus spreads quickly and covers a wide range, which puts higher requirements on nucleic acid detection methods in terms of detection speed, convenience and sensitivity. At the same time, with the demand for precision treatment of tumors, new detection methods are often needed to quickly, accurately and reasonably priced molecular typing of patients. Therefore, there is an urgent need for a fast, convenient, low-cost, highly specific and sensitive detection method to guide the rapid diagnosis, prevention and control or rational use of drugs for infectious diseases and tumors.

[0003] The CRISPR / Cas system composed of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated proteins (Cas proteins) is an acquired immune system inherent in bacteria or archaea. The modified CRISPR / Cas system is widely used in gene editing in eukaryotic and prokaryotic cells. It is worth noting that the Type V (such as Cas12a~k) and Type VI (such as Cas13a~d) family Cas effector proteins not only have cis-cleavage activity, but also have trans-cleavage activity. Therefore, the CRISPR / Cas editing system constructed based on such effector proteins can be applied to gene detection. Specifically, under the guidance of guide RNA (gRNA), Cas proteins specifically bind to the target sequence and activate trans-cleavage activity, thereby indiscriminately cutting nonspecific nucleic acids. An artificially designed reporter gene consists of short, single-stranded nucleotides with different modification groups at both ends. This reporter gene is cleaved under the action of the trans-cleavage activity of the Cas protein, so that the modification groups at both ends are separated. This separated modification group can be detected by fluorescence means or colloidal gold technology, thereby indirectly indicating the presence or absence of the target sequence and achieving the purpose of gene detection.

[0004] CRISPR / LbaCas12a is the most studied mainstream CRISPR detection system and is widely used in nucleic acid detection research and product development such as HPV and SARS-CoV-2. However, the system has strict TTV (V represents C, G or A base) motif PAM restrictions on the target sequence, which limits its scope of application. On the other hand, the existing CRISPR methods have shortcomings in home nucleic acid testing, such as the need for constant temperature heating equipment and precise sample guns. Therefore, it is necessary to establish a new detection system that relies on RPA and CRISPR / Cas12Max, and combines common insulation equipment with convenient pipetting devices. It can detect a wider range of target nucleic acids (lower PAM restrictions) and is closer to the requirements of home testing, and use it for nucleic acid detection. Summary of the invention

[0005] In view of the shortcomings of the prior art described above, an object of the present invention is to provide the use of Cas12Max in the preparation of genetic testing products to solve the problems in the prior art.

[0006] To achieve the above-mentioned purpose and other related purposes, the present invention provides the use of Cas12Max in the preparation of gene detection products.

[0007] The present invention also provides a gene detection kit, which includes an RPA reaction composition and a CRISPR / Cas12Max reaction composition, wherein the RPA reaction composition includes an RPA reaction primer set, and the CRISPR / Cas12Max reaction composition includes Cas12Max, gRNA, and a reporter gene colloidal gold probe or a fluorescent probe.

[0008] As described above, the use of Cas12Max of the present invention in the preparation of gene detection products has the following beneficial effects: by improving the detection method and optimizing the use of equipment, a simple insulation device is used as a container, and by pouring a suitable temperature liquid, the insulation device can maintain the liquid temperature as much as possible, providing the temperature environment required for detection, which is different from the traditional electric-driven heating device. The container is simpler and more cost-effective. The present invention constructs a CRISPR / Cas12Max nucleic acid home detection system with the assistance of simple and universal insulation equipment and droppers, and realizes high sensitivity, low cost and de-instrumentation detection of nucleic acid samples, and has a wider PAM restriction, which can be applied to EGFR-T790M (lack of TTV motif PAM) nucleic acid detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 Design of the CRISPR / Cas12Max targeting SARS-CoV-2 system and optimization of the detection system of the present invention;

[0010] Figure 2 The results of SARS-CoV-2 clinical sample detection by CRISPR / Cas12Max of the present invention;

[0011] Figure 3 Design of the CRISPR / hfCas12Max targeting SARS-CoV-2 system and optimization of the detection system of the present invention;

[0012] Figure 4 The results of SARS-CoV-2 clinical sample detection by CRISPR / hfCas12Max of the present invention;

[0013] Figure 5 The CRISPR / hfCas12Max home nucleic acid detection system, process and performance evaluation results of the present invention;

[0014] Figure 6 The results of detecting the EGFR-T790M target by CRISPR / hfCas12Max and CRISPR / LbaCas12a of the present invention. DETAILED DESCRIPTION

[0015] The present invention first provides the use of Cas12Max in preparing gene detection products.

[0016] In certain embodiments of the present invention, the Cas12Max is xCas12i-N243R or hfCas12Max (i.e., high-fidelity Cas12Max).

[0017] The xCas12i-N243R is a mutant xCas12i-N243R of the xCas12i in application number 202380012151.6 (SEQ ID NO: 1 in the patent is the amino acid sequence of xCas12i).

[0018] The hfCas12Max is a mutant xCas12i-N243R+E336R+D892R of xCas12i in application number 202380012151.6 (SEQ ID NO: 1 in the patent is the amino acid sequence of xCas12i).

[0019] The hfCas12Max can also be purchased directly from Kaika Biotechnology (Shanghai) Co., Ltd. with the product number CAS-EE128.

[0020] Compared with traditional Cas9 and Cas12, hfCas12Max has a smaller molecular weight, a wider range of PAM recognition, and a more efficient editing efficiency, with equivalent or lower off-target effects. hfCas12Max recognizes 5'-TN or 5'-TNN PAM sequences.

[0021] The genetic testing products are reagents used to analyze and identify specific gene sequences, mutations, expression levels or their functional characteristics in the genetic material (DNA or RNA) in a sample. The genetic testing products can be used for a variety of purposes, including but not limited to disease risk assessment, diagnosis, prediction of treatment response, screening for genetic disease carriers, forensic identification, agricultural genotype selection, etc.

[0022] The detection sample source of the genetic detection product is selected from any one or more of the following: virus sampling tube, nasopharyngeal swab, oropharyngeal swab, saliva, sputum, feces or blood.

[0023] The genetic testing product is user-friendly and easy to use. It does not require specific laboratory equipment or trained technicians and can be used by ordinary patients at home.

[0024] The gene detection product is not restricted by the TTV (V represents C, G or A base) motif PAM.

[0025] The present invention also provides a gene detection kit, which includes an RPA reaction composition and a CRISPR / Cas12Max reaction composition, wherein the RPA reaction composition includes an RPA reaction primer set, and the CRISPR / Cas12Max reaction composition includes Cas12Max, gRNA, and a reporter gene colloidal gold probe or a fluorescent probe.

[0026] The gene detection kit is a portable kit and patients can perform the detection by themselves.

[0027] The gene detection kit can be used to detect any target gene, and only the corresponding RPA reaction primer set and gRNA need to be designed according to the target gene. In certain embodiments of the present invention, the gene detection kit is used to detect coronavirus genes, influenza virus genes, etc. The coronavirus is, for example, SARS-CoV virus, SARS-CoV-2 virus or MERS-CoV virus.

[0028] The RPA reaction primer set includes an upstream primer and a downstream primer. The RPA reaction primer set can be designed according to the gene to be detected.

[0029] In certain embodiments of the present invention, the gene to be detected is the SARS-CoV-2 NSP12 gene, the nucleotide sequence of the RPA upstream primer is shown in SEQ ID NO: 4, and the nucleotide sequence of the RPA downstream primer is shown in SEQ ID NO: 5.

[0030] In certain embodiments of the present invention, the gene to be detected is the SARS-CoV-2En gene, the nucleotide sequence of the RPA upstream primer is shown in SEQ ID NO: 7, and the nucleotide sequence of the RPA downstream primer is shown in SEQ ID NO: 8.

[0031] In certain embodiments of the present invention, the gene to be detected is the EGFR-T790M gene, the nucleotide sequence of the RPA upstream primer is shown in SEQ ID NO: 17, and the nucleotide sequence of the RPA downstream primer is shown in SEQ ID NO: 20.

[0032] In certain embodiments of the present invention, the working concentration of the RPA upstream primer and the RPA downstream primer is 0.4-0.6 μM. "Working concentration" refers to the concentration of the upstream primer or the downstream primer based on the total volume of the reaction system during the reaction.

[0033] In certain embodiments of the present invention, the RPA reaction composition further comprises magnesium acetate, a dNTP mixture, a quality control substance, a buffer and / or an aqueous medium.

[0034] In certain embodiments of the present invention, the working concentration of magnesium acetate is 10-16 mM.

[0035] The dNTP mixture is usually used as a raw material in DNA synthesis, and specifically may include dATP, dGTP, dTTP, dCTP, and the like.

[0036] The quality control products include positive quality control products and negative quality control products.

[0037] The positive control product is the genome sequence of the target gene. In certain embodiments, the positive control product is a plasmid containing the target gene, which also contains a partial backbone sequence; in certain embodiments, the positive control product is a viral vector containing the plasmid.

[0038] The negative control product is any substance that does not contain the target gene. In some embodiments, the negative control product may be a solvent, such as water or PBS; in some embodiments, the negative control product is a plasmid vector, and the vector skeleton is consistent with the corresponding positive control product vector, but does not contain the target gene sequence; in some embodiments, the negative control product is a viral vector containing the plasmid.

[0039] The buffer can generally provide the most suitable reaction conditions for the RPA reaction system. The buffer only needs to play the above role. For example, the buffer can be the Rehydration Buffer in TwistDx, TABAS03KIT.

[0040] The aqueous medium can usually be used to adjust the concentration of each component in the RPA system, and can usually be used as a dilution solvent. The aqueous medium is, for example, RNAse-free H2O.

[0041] In certain embodiments of the present invention, the quality control product, buffer, and magnesium acetate in the RPA reaction composition can be a commercial kit, such as TwisDx Basic kit (TABAS03KIT), or you can make your own.

[0042] In certain embodiments of the present invention, when the target gene is RNA, the RPA reaction composition further includes reverse transcriptase.

[0043] In certain embodiments of the present invention, the RPA reaction composition further includes a reverse transcription primer, which is an RPA forward and reverse primer for amplifying the target gene, or a random short fragment primer known in the art, which is usually used according to the instructions of the commercial instructions. If the target gene is RNA, it needs to be reverse transcribed into cDNA in the presence of reverse transcriptase, and then amplified by RPA. If the target gene is DNA, reverse transcriptase is not required, and RPA amplification can be performed directly.

[0044] In certain embodiments of the present invention, the working concentration of Cas12Max in the CRISPR / Cas12Max reaction composition is 30 to 200 nM. For example, the working concentration of Cas12Max is 30 to 40 nM, 40 to 50 nM, 50 to 60 nM, 60 to 70 nM, 70 to 80 nM, 80 to 90 nM, 90 to 100 nM, 100 to 120 nM, 120 to 140 nM, 140 to 160 nM, 160 to 180 nM, 180 to 200 nM. In a preferred embodiment of the present invention, the probe in the CRISPR / Cas12Max reaction composition is a reporter gene colloidal gold probe, and the working concentration of hfCas12Max in the CRISPR / Cas12Max reaction composition is 30 to 40 nM.

[0045] In certain embodiments of the present invention, the gRNA in the CRISPR / Cas12Max reaction composition can be designed according to the gene to be detected.

[0046] In certain embodiments of the present invention, when the gene to be detected is the SARS-CoV-2 NSP12 gene, the nucleotide sequence of the gRNA is shown in SEQ ID NO: 2. When the gene to be detected is the En gene, the nucleotide sequence of the gRNA is shown in SEQ ID NO: 6. When the gene to be detected is EGFR-T790M, the nucleotide sequence of the gRNA is shown in any one of SEQ ID NOs: 14 to 16.

[0047] In certain embodiments of the present invention, the working concentration of gRNA in the CRISPR / Cas12Max reaction composition is 30 to 200 nM. For example, the working concentration of gRNA is 30 to 40 nM, 40 to 50 nM, 50 to 60 nM, 60 to 70 nM, 70 to 80 nM, 80 to 90 nM, 90 to 100 nM, 100 to 120 nM, 120 to 140 nM, 140 to 160 nM, 160 to 180 nM, 180 to 200 nM. In certain embodiments of the present invention, the probe in the CRISPR / Cas12Max reaction composition is a reporter gene colloidal gold probe, and the working concentration of gRNA in the CRISPR / Cas12Max reaction composition is 30 to 40 nM.

[0048] In certain embodiments of the present invention, one end of the reporter gene fluorescent probe is labeled with a fluorescent reporter group, and the other end is labeled with a fluorescent quencher group. Specifically, the fluorescent reporter group is selected from one of the following: FAM, HEX, VIC, CY3, ROX, 610, TEXAS RED, CY5. Specifically, the fluorescent quencher group corresponds to the fluorescent reporter group and can quench the corresponding fluorescent reporter group. For example, when the fluorescent reporter group is FAM, the fluorescent quencher group is selected from one of the following: BHQ1, Dabcyl, TAMRA, MGB.

[0049] In certain embodiments of the present invention, the nucleotide sequence of the reporter gene fluorescent probe is shown in SEQ ID NO:9.

[0050] In certain embodiments of the present invention, one end of the reporter gene colloidal gold probe is labeled with a fluorescent reporter group, and the other end is labeled with biotin.

[0051] In certain embodiments of the present invention, the sequence of the reporter gene colloidal gold probe is shown in SEQ ID NO:10.

[0052] In certain embodiments of the present invention, the CRISPR / Cas12Max reaction composition further comprises any one or more of the following: Mg 2+ , RNase inhibitors, aqueous media, buffer.

[0053] The Mg 2+ It can be provided by MgCl2. 2+ The working concentration is 10-16 mM.

[0054] The working concentration of the RNase inhibitor is 2000 U / ml.

[0055] The aqueous medium is, for example, RNAse-free H2O.

[0056] The buffer can usually provide the most suitable reaction conditions for the reaction. The buffer only needs to play the above role. For example, the buffer can be NEBuffer TM 2.1 or NEBuffer TM r2.1 (NEB, B6002V).

[0057] In certain embodiments of the present invention, the RPA reaction composition and the CRISPR / Cas12Max reaction composition may include various independently packaged reagents described above, or may be prepared as premixed solutions, that is, the RPA reaction composition is prepared as premixed solution A, and the CRISPR / Cas12Max reaction composition is prepared as premixed solution B. It is more convenient to use after being prepared as a premixed solution.

[0058] The gene detection kit also includes any one or more of a sample genome DNA extraction reagent, a colloidal gold test strip, and a disposable Pasteur pipette.

[0059] The gene detection kit also includes a sample collection device and / or a sample preservation solution. The sample collection device includes a sample collection tube and a swab. The sample collection tube, swab and sample preservation solution can be integrated commercial consumables or homemade. The commercial reagent is, for example, a disposable virus sampling tube, which includes a collection tube, a swab and a sample preservation solution, for example, purchased from Origin Cell Technology (Chuzhou) Co., Ltd., model VTM, UTM, ATM, MTM, PTM, KTM, NTM or TTM-B1).

[0060] The colloidal gold test strip includes a sample pad, a gold pad, a nitrocellulose membrane (NC membrane), and a blotting paper pasted on a bottom plate. The gold pad is fixed with a colloid gold-labeled FITC (FAM) antibody, and the nitrocellulose membrane is pre-coated with a detection line and a quality control line for immunoreaction with the colloidal gold marker. The blotting paper allows the liquid sample to flow upward by absorbing water, and the gold-labeled FITC (FAM) antibody on the gold pad recognizes and binds to the FAM group on the reporter gene and moves upward together, and adsorbs the Biotin group at the other end of the reporter gene with the pre-fixed Streptavidin avidin on the quality control line, thereby fixing the gold label on the quality control line. If the reporter group is cut by the Cas protein, the gold-labeled particles continue to move upward and are adsorbed by the antibody of the anti-FITC (FAM) antibody on the detection line. The colloidal gold test strip is a commercial product, such as Milenia Hybri Detect1 (Twist Dx. Cat. No. MILLENIA01). The disposable Pasteur pipette includes 5μl and 6μl specifications. Disposable Pasteur pipette is a disposable stretched plastic dropper that can be used to transfer trace amounts of liquid. It is easy to operate, has a volume scale, and is inexpensive.

[0061] The gene detection kit also includes a heat preservation device, which can provide a detection environment of 37°C to 42°C and can be maintained for more than 40 minutes.

[0062] The heat preservation device can be a simple heat-insulating container that can display the temperature and contains a non-toxic, non-volatile liquid such as water, and has the characteristics of being simple and cheap. The heat preservation device can be a commercial digital display heat preservation water cup.

[0063] The detection sample source of the gene detection kit is selected from any one or more of the following: nasopharyngeal swab, oropharyngeal swab, saliva, sputum, feces or blood.

[0064] The present invention also provides a simple and multifunctional nucleic acid detection method based on CRISPR / Cas12Max, comprising the following steps:

[0065] S1, performing RPA isothermal amplification reaction on the target gene using the RPA reaction composition;

[0066] S2, reacting the RPA amplification product obtained in step S1 with the CRISPR / Cas12Max reaction composition;

[0067] S3. Perform fluorescence or colloidal gold detection on the reaction product of step S2.

[0068] In certain embodiments of the present invention, in the RPA isothermal amplification reaction system of S1, the amount of viral template used is generally at the aM level. In certain embodiments, in order to clarify the effect of sample concentration and enzyme activity, the starting concentration can be 0.77Copy / μL, 7.72Copy / μL, 77.2Copy / μL and 772Copy / μL, such as Figure 1 g.

[0069] In certain embodiments of the present invention, the reaction system in S2 is as described in Table 4. In certain embodiments, in order to clarify the effect of the amount of RPA product and the enzyme activity, the amount of RPA product can be 4 μl to 10 μl ( Figure 1 e); In certain embodiments, to clarify the effect of enzyme concentration and enzyme activity, the starting concentration of Cas12Max enzyme may be 0.50 μM, 1.00 μM and 3.60 μM ( Figure 1 f).

[0070] Preferably, the RPA isothermal amplification reaction and CRISPR / Cas12Max detection temperature are 37°C to 42°C.

[0071] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0072] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0073] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0074] Example 1: Verification of CRISPR / xCas12i-N243R detection system

[0075] The relatively conservative region of NSP12 or En gene was used as the target fragment ( Figure 1 a). First, according to the SARS-CoV-2 viral genome sequence (NC_045512), a partial nucleic acid sequence (SEQ ID NO: 1) including NSP12, NSP14, ORF3a, En and N genes was taken for biosynthesis. Then the SEQ ID NO: 1 sequence was cloned into the same lentiviral vector containing the long terminal repeat (LTR) element to form a plasmid vector containing the target gene, and the vector was packaged into a lentivirus to form a pseudovirus that simulates SARS-CoV-2 ( Figure 1 b). Set up blank plasmid and virus as negative control, the virus does not contain the sequence component of SEQ ID NO: 1 ( Figure 1 b). Lentivirus is cleaved by protease to release nucleic acid components. Take an appropriate amount of virus into LysisBuffer (add 1x PK protease), lyse at 55℃ for 10min, and then heat to 90℃ for 5min to inactivate it;

[0076] Secondly, the xCas12i-N243R protein (hereinafter referred to as Cas12Max) used in this embodiment is obtained by biological purification from Nanjing GeneScript Biotechnology Co., Ltd. (GeneScript), and its purity is about 90% ( Figure 2 a).

[0077] Thirdly, RPA primers targeting NSP12 gene were designed, the sequences of which are shown in SEQ ID NO: 4 and SEQ ID NO: 5, and their specific gRNA, the sequence of which is shown in SEQ ID NO: 2. RPA primers targeting En gene were designed, the sequences of which have the characteristics described in SEQ ID NO: 7 and SEQ ID NO: 8, and their specific gRNA, the sequence of which has the characteristics described in SEQ ID NO: 6. RPA primers and gRNA were obtained by chemical synthesis from Nanjing GenScript Biotechnology Co., Ltd.;

[0078] Finally, the enzyme activity was verified in two systems, one with plasmid as template and the other with pseudovirus as template:

[0079] 1.1 Validation in a plasmid-based template system

[0080] Step 1): RPA isothermal amplification was performed using an RPA kit (TwistDx, TABAS03KIT) according to the instructions. The reagents were prepared as shown in Table 1, and then the prepared reagents were mixed with TwistAmp Basic reaction to obtain a reaction system. Among them, the RPA Forward Primer sequence is SEQ ID NO: 4, and the RPA Reverse Primer sequence is SEQ ID NO: 5. The reaction system was placed in a PCR instrument or a constant temperature water bath device and reacted at 42°C for 20 minutes.

[0081] Table 1

[0082] Rehydration Buffer 29.5μL RPA Forward Primer (10μM) 2.5μL RPA Reverse Primer (10μM) 2.5μL MgOAc (280 mM) 2.5μL Plasmid template (aM level) 5μL <![CDATA[RNAse-free H2O]]> Add to final 50μL

[0083] Step 2): gRNA contains SEQ ID NO: 2, Blank is RNAse-free H2O, and Neg is a blank plasmid without a target sequence. The reaction system is placed in a 384-well plate, and the fluorescent reporter group is the reporter gene fluorescent probe described in SEQ ID NO: 9. Each reaction contains 1 to 3 biological replicates.

[0084] The system is shown in Table 2 and includes the following components:

[0085] Table 2

[0086] <![CDATA[MgCl2(100mM)]]> 2.0μL NEBuffer 2.1(10x) 2.0μL RNase inhibitor (40000U / mL) 2.0μL gRNA (1 μM) 2.0μL Cas12Max(1μM) 2.0μL RPA amplification products 4μL Reporter gene fluorescent probe (1μM) 2μL <![CDATA[RNAse-free H2O]]> 4μL

[0087] 1.2 Parameter verification in a system using viruses as templates:

[0088] Step i): The RPA isothermal amplification system introduces a reverse transcription system based on Table 1, which can reverse transcribe the viral RNA genome into cDNA and then perform RPA amplification. The specific reaction system is shown in Table 3 below, and includes the following components:

[0089] Table 3

[0090]

[0091] The reaction system was placed in a PCR instrument or a constant temperature water bath and reacted at 42°C for 20 min.

[0092] Step ii): The sequence of gRNA is as shown in SEQ ID NO: 2, Blank is RNAse-free H2O, and Neg is a blank viral vector without a target sequence. The reaction system is placed in a 384-well plate, and the fluorescent reporter group is a reporter gene fluorescent probe described in SEQ ID NO: 9. Each reaction contains 1 biological replicate.

[0093] The reaction system is shown in Table 4 below, and comprises the following components:

[0094] Table 4

[0095] <![CDATA[MgCl2(100mM)]]> 2.0μL NEBuffer 2.1(10x) 2.0μL RNase inhibitor (40000U / mL) 2.0μL gRNA (1 μM) 2.0μL Cas12Max(1μM) 2.0μL RPA amplification products 6μL Reporter gene fluorescent probe (1μM) 2μL <![CDATA[RNAse-free H2O]]> 2μL

[0096] This example also uses pseudovirus as a template to study a series of factors affecting the reaction activity according to the above steps. The results and conclusions are as follows:

[0097] a) RPA reaction at 42°C can achieve better amplification effect ( Figure 1 c), the amplified product can activate the enzymatic activity of xCas12i-N243R ( Figure 1 d); and 6 to 8 μl of the product of step 1) can achieve a good shearing effect in step 2) ( Figure 1 e);

[0098] b) The final concentration of Cas12Max protein is 100nM, the detection system is cost-effective and can achieve good shearing effect ( Figure 1 f);

[0099] e) CRISPR / Cas12Max, i.e. CRISPR / xCas12i-N243R, can detect viral vectors at aM concentration level ( Figure 1 g).

[0100] Example 2: Performance evaluation of CRISPR / xCas12i-N243R in detecting SARS-CoV-2 clinical samples

[0101] In order to evaluate the performance of CRISPR / xCas12i-N243R in detecting SARS-CoV-2 clinical samples, the test results were interpreted by means of fluorescent signals.

[0102] 10 extracted SARS-CoV-2 clinical nucleic acid samples were selected, and the SARS-CoV-2 pseudovirus described in the present invention was used as a positive reference (Pos), and the blank pseudovirus was used as a negative reference (Neg). Considering that the evolution of the SARS-CoV-2 virus may cause the NSP12 target sequence to mutate, the conservative fragment of the En gene selected in Example 1 was used as the target fragment for clinical detection in this embodiment. The reference virus was lysed and the nucleic acid was released by the method described in Example 1 of the present invention, and the detection was performed by the following steps:

[0103] a) The RPA amplification system is consistent with the method described in step i) of Example 1 of the present invention, and the selected RPA primers targeting the En gene are shown in SEQ ID NO: 7 and SEQ ID NO: 8; the selected RPA primers targeting the NSP12 gene are shown in SEQ ID NO: 4 and SEQ ID NO: 5.

[0104] b) The CRISPR fluorescence recognition system is consistent with the method described in step ii) of Example 1 of the present invention. The selected gRNA targeting the En gene is shown in SEQ ID NO: 6, and the selected gRNA targeting the NSP12 gene is shown in SEQ ID NO: 2.

[0105] The results suggest that CRISPR / xCas12i-N243R has good performance in fluorescent detection of En gene in extracted SARS-CoV-2 clinical nucleic acid samples ( Figure 2 b).

[0106] Example 3: Verification of CRISPR / hfCas12Max detection system

[0107] hfCas12Max is a double mutation of E336R and D892R introduced on the basis of Cas12Max protein, which has been shown to have higher fidelity activity and editing efficiency (PMID: 37378659). This example constructs and verifies the CRISPR / hfCas12Max detection system, in which hfCas12Max is a commercial product provided by Kaixia Biotechnology (Shanghai) Co., Ltd. (CAS-EE128) ( Figure 2 a).

[0108] First, in this example, a target gene-containing plasmid (0.52 pMol concentration) was used as a template, and the Cas12Max enzyme activity and hfCas12Max enzyme activity were compared and verified by the step 1) and step 2) systems in Example 1.

[0109] Secondly, the correlation between parameters and enzyme activity was verified by the following system:

[0110] Step 1): RPA isothermal amplification was performed using an RPA kit (TwistDx, TABAS03KIT) according to the instructions. The reagents were prepared as shown in Table 5, and then the prepared reagents were mixed with TwistAmp Basic reaction to obtain a reaction system. Among them, the RPA forward primer is SEQ NO ID: 7, and the RPA reverse primer is SEQ ID NO: 8. The reaction system was placed in a PCR instrument or a constant temperature water bath device and reacted at 42°C for 20 minutes.

[0111] Table 5

[0112]

[0113]

[0114] Step 2): The detection system is shown in Table 6, where the gRNA sequence is SEQ ID NO: 6, Blank is RNAse-free H2O, and Neg is a blank plasmid without a target sequence. The system described in Table 6 is a standard system. To clarify the effect of gRNA on enzyme activity, the starting concentration of gRNA can be 0.75 μM, 1.5 μM, 3.0 μM, and 6.0 μM ( Figure 3 e,f); To clarify the effect of hfCas12Max on enzyme activity, the starting concentration of hfCas12Max protein can be 1.33μM, 1.0μM, 0.67μM and 0.33μM ( Figure 3 d);

[0115] The reaction system was placed in a 384-well plate, and the fluorescent reporter group was a reporter gene fluorescent probe described in SEQ ID NO: 9. Each reaction contained 1 to 3 biological replicates.

[0116] Table 6

[0117] <![CDATA[MgCl2(100mM)]]> 2.0μL NEBuffer 2.1(10x) 2.0μL RNase inhibitor (40000U / mL) 2.0μL gRNA (3 μM) 1.0μL hfCas12Max(1μM) 3.0μL RPA amplification products 6μL Reporter gene fluorescent probe (1μM) 2μL <![CDATA[RNAse-free H2O]]> 2μL

[0118] Place the reaction system in an ELISA reader and set the temperature to 42°C (or a temperature gradient, Figure 3 b) for detection. The excitation and emission wavelengths were 480 nm and 520 nm, respectively. The test results suggest that CRISPR / hfCas12Max has specific cleavage activity and nonspecific cleavage activity in vitro, and this activity is affected by the target sequence and gRNA ( Figure 3 d~h).

[0119] Furthermore, this example further studies a series of factors affecting the reactivity, and the results suggest that:

[0120] a) hfCas12Max has better protease activity ( Figure 3 a); and 4 to 8 μl of the product of step 1) can achieve a good shearing effect in step 2) ( Figure 3 c);

[0121] b) CRISPR / hfCas12Max has a high reaction activity at 37-48°C ( Figure 3 b);

[0122] c) When the final concentration of hfCas12Max protein is 150nM and the final concentration of gRNA is 150nM, the detection system is cost-effective and can achieve better shearing effect ( Figure 3 d~f);

[0123] Finally, the hfCas12Max enzyme activity and LbaCas12a enzyme activity comparison verification were performed by the step 1) and step 2) systems in this embodiment. In particular, the LbaCas12a protein gRNA targeting the NSP12 gene has a sequence shown in SEQ ID NO: 11; the LbaCas12a protein gRNA targeting the En gene has a sequence described in SEQ ID NO: 12.

[0124] The results suggest that CRISPR / LbaCas12a has good reaction activity when detecting PAM target sequences with TTN (N represents any base) motifs ( Figure 3 g, h).

[0125] Example 4: CRISPR / hfCas12Max sensitivity detection and SARS-CoV-2 clinical sample detection performance evaluation

[0126] First, the present invention verifies the sensitivity of CRISPR / hfCas12Max detection. To clarify the correlation between plasmid template concentration and enzyme activity, the template starting concentrations are 7.72Copy / μL, 77.2Copy / μL and 772Copy / μL. The reaction is carried out by the system of step 1) and step 2) in Example 3.

[0127] The results showed that CRISPR / hfCas12Max can detect plasmid vectors and viral vectors at aM concentration level ( Figure 4 a)

[0128] Secondly, 8 unextracted SARS-CoV-2 clinical nucleic acid samples (all positive samples) were selected, and the test results were interpreted by colloidal gold signal. The SARS-CoV-2 pseudovirus described in the present invention was used as a positive reference (Pos), and the blank pseudovirus was used as a negative reference (Neg). The reference virus was lysed and nucleic acid released by the method of Example 1 of the present invention.

[0129] Finally, the correlation between parameters and enzyme activity was verified by the following system:

[0130] a) The RPA amplification system described in step 1) is consistent with the method described in step i) of Example 1 of the present invention, and the selected RPA primers targeting the En gene are shown in SEQ ID NO: 7 and SEQ ID NO: 8.

[0131] b) In step 2), the CRISPR colloidal gold recognition system, the selected gRNA sequence targeting the En gene is shown in SEQ ID NO: 6. The structure and sequence of the reporter gene colloidal gold probe are shown in SEQ ID NO: 10.

[0132] c) The CRISPR colloidal gold recognition is completed by the following reaction, and the reaction system is shown in Table 7:

[0133] Table 7

[0134] <![CDATA[MgCl2(100mM)]]> 2.0μL NEBuffer 2.1(10x) 10.0μL RNase inhibitor (40000U / mL) 1.0μL gRNA (3 μM) 1.0μL hfCas12Max(1μM) 4.0μL RPA amplification products 6μL Reporter gene colloidal gold probe (4μM) 2μL <![CDATA[RNAse-free H2O]]> 74μL

[0135] The product after the reaction was mixed with colloidal gold test strips for color development. The results showed that the colloidal gold color development method can well indicate the presence of SARS-CoV-2 viral nucleic acid in unextracted samples ( Figure 4 b), which is consistent with the clinical test results.

[0136] Example 5: CRISPR / hfCas12Max nucleic acid home detection system and sensitivity and specificity experiments

[0137] This example constructs a CRISPR / hfCas12Max nucleic acid home detection system ( Figure 5 a) and achieved highly sensitive, low-cost and instrument-free detection of SARS-CoV-2 nucleic acid samples. Specifically:

[0138] First, a disposable virus sampling tube (virus sampling tube, origin cell, TTM-B1) was used for sampling. After the sample (sample number: case19 and case20) was dipped into the slow-release solution in the sampling tube with a cotton swab, it was placed in a 60°C environment for 15 minutes to inactivate the virus. In this embodiment, the sensitivity test was completed based on the SARS-CoV-2 pseudovirus and its control group virus, and two SARS-CoV-2 virus clinical samples were taken for testing.

[0139] Secondly, a common digital thermostatic water cup was used as an insulated container, and an appropriate amount of hot water was added and naturally cooled to 42°C to start the test. The results showed that the CRISPR / hfCas12Max detection system can maintain a high activity at 37°C to 42°C, and the digital thermostatic water cup can meet the requirements of the present invention.

[0140] Thirdly, a simple and inexpensive pipetting device was used to absorb a certain amount of liquid and mix the mixture by blowing. The pipetting device is a disposable brushed plastic Pasteur pipette with two specifications, which respectively transfers 5 μl of sample to liquid A (all components except the template in Table 5 in Example 3) for mixing reaction (i.e. RPA isothermal amplification reaction) and transfers 6 μl of reaction product to liquid B (all components except the RPA amplification product in Table 7 in Example 4) for mixing reaction.

[0141] Finally, through Figure 5 b The correlation between the reaction parameters and enzyme activity was verified.

[0142] The RPA amplification system is consistent with step i) in Example 1 of the present invention, and the reaction is carried out in an insulated container.

[0143] The CRISPR recognition system is consistent with the content described in part c) of Example 4 of the present invention, and the reaction is carried out in an insulated container.

[0144] Furthermore, the present invention provides a CRISPR / hfCas12Max home nucleic acid detection device ( Figure 5 a) Capable of detecting nucleic acids at aM concentrations ( Figure 5 c) and is specific. Case 19 and case 20 samples were positive, proving that the system can be used for home testing of SARS-CoV-2 clinical samples ( Figure 5 d).

[0145] Example 6: CRISPR / hfCas12Max detection of non-TTN motif PAM target genes

[0146] When CRISPR / hfCas12Max edits non-TTN motif PAM target genes in vivo, it has better cleavage activity than CRISPR / LbaCas12a, and CRISPR / hfCas12Max has a wider range of applications. However, detecting CRISPR / hfCas12Max editing non-TTN motif PAM target genes in vivo is a difficulty in CRISPR detection. The present invention constructs an EGFR-T790M mutation site, whose adjacent region lacks TTN motif PAM, and then uses CRISPR / hfCas12Max to detect the EGFR-T790M site system, and compares its performance with that of the CRISPR / LbaCas12a system.

[0147] Specifically:

[0148] First, based on the EGFR-T790M genome sequence, a portion of the nucleic acid sequence was biosynthesized to obtain the target gene (SEQ ID NO: 13).

[0149] Secondly, RPA primers F1, F2, R1 and R2 targeting the EGFR-T790M gene of CRISPR / hfCas12Max were designed, and the sequences thereof had the characteristics described in SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, respectively, and specific gRNA1 to gRNA3 thereof, and the sequences thereof had the characteristics described in SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively ( Figure 6a). In particular, the present invention also describes the performance of CRISPR / LbaCas12a in detecting the EGFR-T790M target, wherein the specific guide sequences gRNA4 to gRNA6 have SEQ ID NO: 21, SEQ ID NO: 23, and SEQ ID NO: 24 of the present invention, which have the same Spacer sequence as gRNA1 to gRNA3, respectively.

[0150] Finally, the correlation between parameters and enzyme activity was verified by the following system:

[0151] The RPA amplification system is consistent with the method described in step 1) of Example 1 of the present invention.

[0152] The CRISPR recognition system shown is consistent with the method described in step 2) of Example 1 of the present invention.

[0153] From the results, we can see that

[0154] a) The primer combination F1+R2 can achieve better amplification effect ( Figure 6 b~c);

[0155] b) CRISPR / hfCas12Max can achieve better detection results under the guidance of gRNA3 ( Figure 6 d~f);

[0156] c) hfCas12Max has an advantage over LbaCas12a in recognizing non-TTN motif PAM targets ( Figure 6 d~f).

[0157] As can be seen from the above, CRISPR / hfCas12Max can be applied to genetic testing, and has high sensitivity in detecting SARS-CoV-2 virus. After system optimization, it can meet the needs of home nucleic acid testing. It also has a wider PAM restriction and can be applied to EGFR-T790M (lacking TTV motif PAM) nucleic acid detection.

[0158] The nucleotide and amino acid sequences used in the present invention are as follows:

[0159] SARS-CoV-2 template base sequence (SEQ ID NO: 1):

[0160]

[0161]

[0162]

[0163]

[0164]

[0165] The above examples are for the purpose of illustrating the embodiments disclosed by the present invention and are not to be construed as limiting the present invention. In addition, the various modifications listed herein and the variations of the methods in the invention are obvious to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of the present invention.

Claims

1. The use of Cas12Max in the preparation of genetic testing products.

2. The use according to claim 1, characterized in that The Cas12Max is xCas12i-N243R or hfCas12Max.

3. A gene detection kit, characterized in that: The gene detection kit includes an RPA reaction composition and a CRISPR / Cas12Max reaction composition, wherein the RPA reaction composition includes an RPA reaction primer set, magnesium acetate and water, and the CRISPR / Cas12Max reaction composition includes Cas12Max, gRNA, a reporter gene colloidal gold probe or a fluorescent probe.

4. The gene detection kit according to claim 3, characterized in that: The RPA reaction primer set includes an upstream primer and a downstream primer, and the working concentrations of the upstream primer and the downstream primer are 0.4-0.6 μM.

5. The gene detection kit according to claim 3, characterized in that: The RPA reaction composition also includes magnesium acetate, a dNTP mixture, a quality control product, a buffer and / or an aqueous medium. Preferably, the working concentration of the magnesium acetate is 10 to 16 mM.

6. The gene detection kit according to claim 3, characterized in that: The RPA reaction composition also includes reverse transcriptase and reverse transcription primers.

7. The gene detection kit according to claim 3, characterized in that: The working concentration of Cas12Max in the CRISPR / Cas12Max reaction composition is 30 to 200 nM, and / or the working concentration of gRNA is 30 to 200 nM.

8. The gene detection kit according to claim 3, characterized in that: One end of the reporter gene fluorescent probe is labeled with a fluorescent reporter group, and the other end is labeled with a fluorescent quencher group, and / or the nucleotide sequence of the reporter gene fluorescent probe is as shown in SEQ ID NO:

9.

9. The gene detection kit according to claim 3, characterized in that: One end of the reporter gene colloidal gold probe is labeled with a fluorescent reporter group, and the other end is labeled with biotin, and / or the sequence of the reporter gene colloidal gold probe is shown in SEQ ID NO:

10.

10. The gene detection kit according to claim 3, characterized in that: The CRISPR / Cas12Max reaction composition also includes any one or more of the following: Mg 2+ , RNase inhibitors, aqueous media, buffer.

11. The gene detection kit according to claim 3, characterized in that: The gene detection kit also includes any one or more of a sample collection device, a sample preservation solution, a sample genomic DNA extraction reagent, a colloidal gold test strip, and a disposable Pasteur pipette. Preferably, the disposable Pasteur pipette has specifications of 5μl and 6μl.

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