Engineered crRNA and application thereof in preparation of reagent for detecting pathogens or drug-resistant genes related to pathogens

Abstract

The invention relates to the technical field of gene detection, in particular to engineered crRNA and application of the engineered crRNA to preparation of a reagent for detecting pathogens or drug-resistant genes related to the pathogens, the engineered crRNA comprises a crRNA skeleton and a sensitizing sequence, the crRNA skeleton comprises an inherent sequence and a guiding sequence, the inherent sequence comprises a stem-loop structure, the guiding sequence comprises a stem-loop structure, and the stem-loop structure comprises a stem-loop structure. The guiding sequence is used for being combined with a target gene, the sensitizing sequence is connected with the 3'tail end of the guiding sequence, and the sensitizing sequence is a DNA fragment which is 1-15nt and does not contain GC basic groups. By connecting the sensitizing sequence to the 3'terminal of the crRNA skeleton, the binding probability of the guiding sequence and the target gene is improved, so that the sensitivity of the CRISPR / Cas detection system is remarkably improved, and the lower detection limit of the target gene can reach 1 copy / reaction.

Description

Technical Field

[0001] The present invention relates to the field of gene detection technology, and in particular to an engineered crRNA and its application in preparing a reagent for detecting pathogens or drug-resistant genes associated with pathogens. Background Art

[0002] Respiratory diseases seriously endanger human health, among which infection by pathogenic microorganisms represented by bacteria and viruses is the main factor causing respiratory diseases. Respiratory infectious diseases have a high incidence rate, spread rapidly, and are very likely to cause serious public safety incidents. We need faster and more accurate detection methods to achieve the goals of early detection, early isolation, and early treatment. Compared with traditional pathogen identification methods, nucleic acid detection technology is a powerful tool for early diagnosis. Although qPCR has been widely used in front-line detection as the gold standard for nucleic acid detection, the detection technology based on variable temperature amplification requires expensive equipment, and requires standardized laboratories and professional technicians to operate. The reaction time is long, and it is difficult to meet the requirements of real-time detection. Early monitoring cannot be achieved for extremely low pathogen content. How to detect more quickly, simply, accurately, and at a low cost is the future development direction of pathogen nucleic acid detection.

[0003] Some existing rapid nucleic acid detection methods include LAMP (loop-mediated isothermal amplification) and RPA-exo probe method (recombinase polymerase amplification-probe method). Under the mediation of some special constant-temperature nucleic acid amplification enzymes, this type of method gets rid of the dependence on variable temperature equipment, and the target nucleic acid can be amplified and detected at a fixed temperature. Although the nucleic acid constant-temperature amplification detection based on LAMP or RAA gets rid of the limitations of thermal cycling equipment, its high-speed amplification and high sensitivity bring the possibility of false positives.

[0004] The CRISPR / Cas system is an emerging nucleic acid detection tool and shows great potential as a next-generation molecular diagnostic method. The CRISPR sequence is a special DNA repeat sequence that contains highly conserved repeats of approximately 21-48 bp and spacers of 26-72 bp. The repeats are separated by different spacers, and the CRISPR sequence recognizes the target gene through the spacer. Cas is located near the CRISPR site and is a general term for the double-stranded DNA nuclease family. The working principle of the CRISPR / Cas system is that crRNA (CRISPR-derived RNA) binds to tracrRNA (trans-activating RNA) through base pairing to form a tracrRNA / crRNA complex, which guides nucleases such as Cas12 proteins to shear double-stranded DNA at the target sequence site paired with crRNA, guiding the Cas protein to cut the nucleic acid at a specific point.

[0005] At present, many types of Cas proteins have been identified that can cut target sites under the guidance of sgRNA. Cas12a belongs to this family and can specifically cut target genes under the guidance of sgRNA. At the same time, the activated Cas12a can exert non-specific shearing enzyme activity and non-specifically shear the single-stranded DNA (ssDNA) encountered. Taking advantage of the characteristic of Cas12a that can be activated by targeting, it can be specifically designed to be used for specific detection of target genes.

[0006] Constant temperature detection based on the CRISPR / Cas system introduces a highly precise CRISPR system on the basis of the constant temperature amplification method, greatly improving the accuracy and sensitivity of the detection method.

[0007] At present, the constant temperature detection technology based on CRISPR is mainly divided into two categories, namely the nucleic acid constant temperature detection technology called DETECTR developed by Jennifer Doudna, the winner of the 2020 Nobel Prize in Chemistry, which depends on Cas12a, and the nucleic acid constant temperature detection technology called SHERLOCK developed by Zhang Feng, which depends on Cas13a. The principle is to amplify and enrich the target fragments through constant temperature amplification methods such as LAMP and RPA. The amplified target fragments will be targeted and recognized by the Cas protein through a section of sgRNA, activating the Cas protein to become a DNA cutter (Cas12a) or RNA cutter (Cas13a), and cutting all nearby single-stranded DNA (Cas12a) or single-stranded RNA (cas13a). Based on this principle, single-stranded DNA is prepared into a fluorescent quenching probe, and the specific detection of the target sequence is achieved by monitoring the release of fluorescent signals.

[0008] The nucleic acid constant temperature detection technology that relies on Cas12a has more advantages in cost and speed because it does not require reverse transcription in the CRISPR stage, but the detection sensitivity needs to be further improved. Summary of the invention

[0009] In view of the shortcomings of the prior art described above, the object of the present invention is to provide an engineered crRNA and its application in the preparation of a reagent for detecting pathogens or drug-resistant genes associated with pathogens, so as to solve the problems in the prior art.

[0010] To achieve the above-mentioned purpose and other related purposes, the present invention first provides an engineered crRNA, wherein the engineered crRNA comprises a crRNA skeleton and a sensitization sequence, wherein the crRNA skeleton comprises an intrinsic sequence and a guide sequence, wherein the intrinsic sequence comprises a stem-loop structure, wherein the guide sequence is used to bind to a target gene, wherein the sensitization sequence is connected to the 3' end of the guide sequence, and wherein the sensitization sequence is a DNA fragment of 1 to 15 nt and does not contain GC bases.

[0011] The present invention also provides a CRISPR / Cas system, which includes the above-mentioned engineered crRNA.

[0012] The present invention also provides an application of the above-mentioned engineered crRNA or the above-mentioned CRISPR / Cas system in the preparation of a reagent for detecting pathogens or drug-resistant genes associated with pathogens.

[0013] As described above, the engineered crRNA of the present invention and its use in the preparation of reagents for detecting pathogens or drug-resistant genes associated with pathogens have the following beneficial effects: by adding a 7nt single-stranded DNA sensitization sequence to the 3' end of the crRNA, the probability of binding between the guide sequence and the target sequence is increased, thereby significantly improving the sensitivity of the CRISP / Cas detection system, and the detection limit of the target sequence can reach 1 copy / reaction. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is a schematic diagram showing the test results of adding DNA / RNA sensitization sequence to the 5' end / 3' end of crRNA in Example 1 of the present invention.

[0015] Figure 2 Shown is a schematic diagram of the test results of adding different GC content DNA sequences to 3'crRNA in Example 2 of the present invention.

[0016] Figure 3 It is a schematic diagram showing the test results of adding different lengths of AT-only sequences to 3'crRNA in Example 3 of the present invention.

[0017] Figure 4 Shown is a schematic diagram of the test results of different combinations of adding 7 nt AT to 3'crRNA in Example 4 of the present invention.

[0018] Figure 5 to Figure 28Schematic diagram showing the comparison of the differences in detection sensitivity of 24 target sequences in Example 5 of the present invention, which are: influenza A virus (IFUA), influenza B virus (IFUB), Streptococcus pneumoniae (SPN), Acinetobacter baumannii (Aba), Klebsiella pneumoniae (KP), Pseudomonas aeruginosa (PA), Staphylococcus aureus (SA), Mycoplasma pneumoniae (MP), new coronavirus (2019-nCOV), adenovirus (HADV), human metapneumovirus (hMPV), parainfluenza virus (hPIV), Haemophilus influenzae (HI), Stenotrophomonas maltophilia (SM), Escherichia coli (ECO), Moraxella catarrhalis (MC), Legionella pneumophila (MIP), Enterococcus faecalis (EFCA), Enterococcus faecium (EFCI), Chlamydia pneumoniae (CP), Pseudomonas aeruginosa (PJP), carbapenem resistance gene blaKPC and / or methicillin resistance gene mecA / mecC).

[0019] Fig.29 Shown is a schematic diagram of the sensitivity of detecting the crRNA backbone using Mycoplasma pneumoniae as the target.

[0020] Fig.30 Schematic diagram showing the sensitivity of detecting engineered crRNA using Mycoplasma pneumoniae as the target DETAILED DESCRIPTION

[0021] The present invention first provides an engineered crRNA, which includes a crRNA backbone and a sensitization sequence, wherein the crRNA backbone includes an intrinsic sequence and a guide sequence, the intrinsic sequence includes a stem-loop structure, the guide sequence is used to bind to a target gene, the sensitization sequence is connected to the 3' end of the guide sequence, and the sensitization sequence is a DNA fragment of 1 to 15 nt and does not contain GC bases.

[0022] The nucleotide sequence of the scaffold sequence is UAAUUUCUAAGUGUAGAU (SEQ ID NO. 112).

[0023] In the present invention, the portion of crRNA excluding the guide sequence is defined as the intrinsic sequence.

[0024] In certain embodiments of the present invention, the length of the DNA sensitization sequence is 3 to 11 nt.

[0025] In a preferred embodiment of the present invention, the length of the DNA sensitization sequence is 5 to 9 nt.

[0026] In certain embodiments of the present invention, the DNA sensitization sequence includes any combination of A and T bases.

[0027] In a preferred embodiment of the present invention, the DNA sensitization sequence includes A, ATATA, ATATATA, ATATAATATA, TATTATT, TTTTAAT, TTTTTAA, TTTATAT, and TTATTAT.

[0028] Furthermore, the length of the DNA sensitization sequence is 7nt.

[0029] Furthermore, the DNA sensitization sequence is 5'-TTATTAT-3'.

[0030] In certain embodiments of the present invention, the target gene is selected from a pathogenic gene in a pathogen or a drug-resistant gene associated with a pathogen.

[0031] The pathogen resistance gene refers to a nucleotide sequence (DNA fragment) that encodes drug resistance traits. These genes enable pathogens to develop resistance to certain antibiotics, thereby reducing or eliminating the efficacy of these drugs.

[0032] Specifically, the pathogen is selected from one or more of influenza A virus (IFUA), influenza B virus (IFUB), Streptococcus pneumoniae (SPN), Acinetobacter baumannii (Aba), Klebsiella pneumoniae (KP), Pseudomonas aeruginosa (PA), Staphylococcus aureus (SA), Mycoplasma pneumoniae (MP), new coronavirus (2019-nCOV), adenovirus (HADV), human metapneumovirus (hMPV), parainfluenza virus (hPIV), Haemophilus influenzae (HI), Stenotrophomonas maltophilia (SM), Escherichia coli (ECO), Moraxella catarrhalis (MC), Legionella pneumophila (MIP), Enterococcus faecalis (EFCA), Enterococcus faecium (EFCI), Chlamydia pneumoniae (CP) or Pseudomonas japonicus (PJP).

[0033] The drug resistance gene associated with the pathogen is selected from a carbapenem resistance gene or a methicillin resistance gene. The carbapenem resistance gene is blaKPC; the methicillin resistance gene is mecA or mecC.

[0034] In certain embodiments of the present invention, the guide sequence in the crRNA backbone is designed based on the conserved sequence of the pathogenic gene in the pathogen to be detected or the drug-resistant gene associated with the pathogen, so as to complementarily pair with the target gene.

[0035] Specifically, the specific sequence of the pathogen to be detected or the drug-resistant gene associated with the pathogen is selected through the National Center for Biotechnology Information (NCBI) database of the United States, and the corresponding crRNA is designed for each specific sequence to obtain a series of candidate crRNA nucleotide sequences. The candidate crRNA is compared online in the National Center for Biotechnology Information (NCBI) of the United States to check whether the comparison result is the corresponding target sequence, thereby obtaining a crRNA with higher specificity.

[0036] The present invention also provides a CRISPR / Cas system, which includes engineered crRNA.

[0037] In certain embodiments of the present invention, the CRISPR / Cas12a system further includes a buffer, a Cas protease or a reporter probe.

[0038] Furthermore, the Cas protease is Cas12a protease.

[0039] Furthermore, the working concentration of the Cas protease is 20-40 nM. The working concentration of the Cas protease is selected from any of the following ranges: 20-40 nM, 20-40 nM, 20-40 nM, 20-40 nM.

[0040] Furthermore, the working concentration of the Cas protease is 30 nM.

[0041] Furthermore, the working concentration of the crRNA sequence is 20-40 nM. The working concentration of the crRNA sequence is selected from any of the following ranges: 20-40 nM, 20-40 nM, 20-40 nM, 20-40 nM.

[0042] Furthermore, the working concentration of the crRNA sequence is 30 nM.

[0043] Furthermore, the working concentration of the reporter probe is 150-250 nM. The working concentration of the reporter probe is selected from any of the following ranges: 150-170 nM, 170-190 nM, 190-210 nM, 210-230 nM, 230-250 nM.

[0044] Furthermore, the working concentration of the reporter probe is 200 nM.

[0045] The working concentration refers to the concentration of the solution used directly during the experiment.

[0046] Furthermore, the reporter probe is a ssDNA fragment with a fluorescent group connected to one end and a quenching group connected to the other end.

[0047] Furthermore, the reporter probe is 5'FAM-TTATT-3'BHQ1.

[0048] In certain embodiments of the present invention, the CRISPR / Cas12a system also includes a RAA amplification system.

[0049] The present invention also provides the use of the above-mentioned engineered crRNA or the above-mentioned CRISPR / Cas system in the preparation of reagents for detecting pathogens or drug-resistant genes associated with pathogens.

[0050] Furthermore, the pathogen is selected from one or more of influenza A virus (IFUA), influenza B virus (IFUB), Streptococcus pneumoniae (SPN), Acinetobacter baumannii (Aba), Klebsiella pneumoniae (KP), Pseudomonas aeruginosa (PA), Staphylococcus aureus (SA), Mycoplasma pneumoniae (MP), new coronavirus (2019-nCOV), adenovirus (HADV), human metapneumovirus (hMPV), parainfluenza virus (hPIV), Haemophilus influenzae (HI), Stenotrophomonas maltophilia (SM), Escherichia coli (ECO), Moraxella catarrhalis (MC), Legionella pneumophila (MIP), Enterococcus faecalis (EFCA), Enterococcus faecium (EFCI), Chlamydia pneumoniae (CP), and Pseudomonas spp. (PJP).

[0051] Furthermore, the drug-resistant gene associated with the pathogen is selected from a carbapenem resistance gene or a methicillin resistance gene; the carbapenem resistance gene is blaKPC; and the methicillin resistance gene is mecA / mecC.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] Example 1 Screening of base types of sensitization sequences

[0056] Taking Mycoplasma pneumoniae (MP) as the detection target, DNA sensitization sequences were added to the 5' and 3' ends of the crRNA backbone, respectively, and the effects of the DNA sensitization sequence composed of T / A / G / C bases and the RNA modification fragment composed of U / A / G / C bases on improving the detection ability were compared.

[0057] 1. Experimental Materials

[0058] 1. Reagents: The amplification reagent used in Example 1 is the RNA constant temperature rapid amplification kit (article number: WLRE8208KIT) of Weifang Anpu Future Biotechnology Co., Ltd. Primers, reporter probes, and crRNA are synthesized by General Bio (Anhui) Co., Ltd. The primers, reporter probes, and crRNA sequences in Example 1 are shown in SEQ ID NO: 1-7, 113 (Table 3). Cas12 protein is LbCas12a (article number: E00201) of Jiangsu Dongkang Biopharmaceutical Technology Co., Ltd.

[0059] 2. Instruments used: clean bench (SW-CJ-1D, Longyang Scientific Instruments), conventional PCR instrument (A100, Hangzhou Langji Scientific Instruments), LightCycler 480 real-time fluorescence quantitative PCR instrument (Roche).

[0060] 2. Experimental Methods

[0061] 1. crRNA design

[0062] The specific sequence of Mycoplasma pneumoniae was Blast from the National Center for Biotechnology Information (NCBI) database of the United States, and the corresponding crRNA was designed according to its specific sequence, and sensitization sequences were added to the 5' and 3' ends, respectively. The DNA sensitization sequence was TAGCTT and the RNA sensitization sequence was UAGCUU, which were synthesized by General Biotechnology (Anhui) Co., Ltd.

[0063] 2. Sample nucleic acid extraction

[0064] Extract nucleic acid: Collect clinical samples of Mycoplasma pneumoniae targets, use Biori fully automatic nucleic acid extraction and purification instrument to extract and purify nucleic acid from samples for subsequent amplification experiments.

[0065] 3. RAA constant temperature amplification

[0066] The operation was carried out according to the instructions of the kit. The preparation of the RAA system is shown in Table 1. After mixing, amplification was performed at 42°C for 15 min.

[0067] Table 1 RAA system preparation

[0068] Components Volume (μL) Abuffer 29.4 MP-F (10 μM) 0.8 MP-R (10μM) 0.8 template 5 ddH2O 11.5 B buffer 2.5 Total volume 50

[0069] 4. CRISPR detection

[0070] The reagent configuration of the CRISPR / Cas12 system is shown in Table 2. The product is the product after RAA amplification in Example 1. 5 μL is added to 45 μL of CRISPR / Cas12 reagent, mixed and centrifuged before testing. CRISPR detection program: 37°C, 30s, 30 cycles, instrument: Roche 480.

[0071] Table 2 CRISPR / Cas12 system preparation

[0072]

[0073]

[0074] 3. Test results

[0075] The nucleic acid detection results of adding DNA / RNA sensitization sequence to the crRNA 5' end / 3' end in Example 1 are as follows Figure 1 As shown, the best result was obtained by adding a DNA sensitization sequence consisting of T / A / G / C bases to the 3' end of crRNA.

[0076] Table 3 Sequences involved in Example 1

[0077]

[0078] Note: Bold and underlined sequences represent sensitization sequences.

[0079] Example 2 Screening of GC content of DNA sensitization sequence

[0080] From the results of Example 1, it can be seen that adding a DNA sensitization sequence composed of T / A / G / C bases to the 3' end of crRNA can improve the detection sensitivity.

[0081] This example still uses Mycoplasma pneumoniae as the detection target, and studies the effect of different GC contents of DNA sensitization sequences on improving the detection ability.

[0082] 1. Experimental methods:

[0083] 1. crRNA design

[0084] Taking the Mycoplasma pneumoniae target as an example, the GC content of the 3'-end DNA sensitization sequence was adjusted to 0%, 30%, and 50%, respectively. The specific sequences are SEQ ID NOs: 8-10 (Table 4), which were synthesized by General Biotechnology (Anhui) Co., Ltd.

[0085] 2. RAA amplification was performed according to the steps in Example 1

[0086] 3. crRNA testing

[0087] The RAA product from the previous step was subjected to CRISPR detection, and 5 μL of the amplified product was added. After mixing and centrifugation, it can be tested on the machine. The CRISPR detection procedure is the same as in Example 1.

[0088] 2. Results Analysis

[0089] The RAA amplification in Example 1 and the crRNA testing steps in Example 2 were followed to test the results. Figure 2 As shown, the detection result is best when the GC content of the 3'DNA sensitization sequence of crRNA is 0%.

[0090] Table 4 crRNA sequences involved in Example 2

[0091]

[0092] Note: Bold and underlined sequences represent sensitization sequences.

[0093] Example 3 Screening of DNA enhancement sequence length

[0094] 1. crRNA design

[0095] Taking the Mycoplasma pneumoniae target as an example, different lengths of DNA sensitization sequences containing only AT sequences were added to the 3'crRNA, namely A, ATATA, ATATATA, and ATATATATA. The specific sequences are SEQ ID NOs: 11-14 (Table 5), which were synthesized by General Biotechnology (Anhui) Co., Ltd.

[0096] 2. RAA amplification was performed according to the steps in Example 1

[0097] 3. crRNA testing

[0098] The RAA product of the previous step was subjected to CRISPR detection, and 5 μL of the amplified product was added. After mixing and centrifugation, it can be tested on the machine. The CRISPR detection procedure is the same as in Example 1.

[0099] 2. Results Analysis

[0100] The RAA amplification in Example 1 and the crRNA testing steps in Example 3 were followed to test. The results are as follows: Figure 3 As shown, the results show that the detection result is best when the length of the 3'DNA sensitization sequence (AT composition) of crRNA is 7nt.

[0101] Table 5 crRNA sequences involved in Example 3

[0102]

[0103]

[0104] Note: Bold and underlined sequences represent sensitization sequences.

[0105] Example 4 Screening of DNA sensitization sequence base combination order

[0106] From the results of Example 3, it can be seen that adding a DNA sensitization sequence with a GC content of 0% and a length of 7 nt to the 3' end of crRNA can improve the detection sensitivity.

[0107] This example still takes the Mycoplasma pneumoniae target as an example to compare the effects of different base combination orders of a DNA sensitization sequence with a length of 7 nt and composed of only AT bases on the detection ability.

[0108] 1. Experimental Methods

[0109] 1. crRNA design

[0110] Taking the Mycoplasma pneumoniae target as an example, the combinations are ATATATA, TATTATT, TTATTA, TTTATAT, TTTTAAT, TTTTTAA, and the specific base sequences are SEQ ID NOs: 13, 15-19 (Table 6), which are synthesized by General Biotechnology (Anhui) Co., Ltd.

[0111] 2. RAA amplification was performed according to the steps in Example 1

[0112] 3. crRNA testing

[0113] The RAA product of the previous step was subjected to CRISPR detection, and 5 μL of the amplified product was added. After mixing and centrifugation, it can be tested on the machine. The CRISPR detection procedure is the same as in Example 1.

[0114] 2. Results Analysis

[0115] The RAA amplification in Example 1 and the crRNA testing steps in Example 3 were followed to test. The results are as follows: Figure 4As shown, the DNA sensitization sequence composed of the TTATTAT combination showed the best detection result.

[0116] Table 6 crRNA sequences involved in Example 4

[0117]

[0118]

[0119] Note: Bold and underlined sequences represent sensitization sequences.

[0120] Example 5 Comparison of target detection sensitivity based on CRISPR / Cas12 system

[0121] The DNA sensitization sequence composed of the TTATTAT combination was added to the 3' end of the cRNA of other pathogens to examine its applicability.

[0122] Twenty-four common pathogen targets were selected: influenza A virus (IFUA), influenza B virus (IFUB), Streptococcus pneumoniae (SPN), Acinetobacter baumannii (Aba), Klebsiella pneumoniae (KP), Pseudomonas aeruginosa (PA), Staphylococcus aureus (SA), Mycoplasma pneumoniae (MP), new coronavirus (2019-nCOV), adenovirus (HADV), human metapneumovirus (hMPV), parainfluenza virus (hPIV), Haemophilus influenzae (HI), Stenotrophomonas maltophilia (SM), Escherichia coli (ECO), Moraxella catarrhalis (MC), Legionella pneumophila (MIP), Enterococcus faecalis (EFCA), Enterococcus faecium (EFCI), Chlamydia pneumoniae (CP), Pseudomonas japonicus (PJP), carbapenem resistance genes blaKPC, methicillin resistance genes mecA and mecC, and the detection sensitivity of the engineered crRNA with and without DNA sensitization sequences was compared.

[0123] 1. Design and screening of crRNA

[0124] The specific sequence of the detected microorganism was selected through the National Center for Biotechnology Information (NCBI) database, and the corresponding crRNA was designed for each specific sequence to obtain a series of candidate crRNA nucleotide sequences. The candidate crRNA was compared online in the National Center for Biotechnology Information (NCBI) to check whether the comparison result was the corresponding target sequence, thereby obtaining a crRNA with higher specificity. In this embodiment, 24 crRN skeletons A and corresponding 24 engineered crRNAs (3'crRNA+TTATTAT) were obtained, and the specific nucleotide information was: SEQ ID NO: 1-3, 16, 20-111 (Table 7).

[0125] 2. Extract nucleic acid from samples

[0126] 24 clinical samples with different targets were collected, and the samples were extracted and purified using the Biori fully automatic nucleic acid extraction and purification instrument for subsequent amplification experiments.

[0127] 3. RAA constant temperature amplification of the extracted nucleic acid

[0128] According to the principle of isothermal amplification, the gene was amplified isothermally, the upstream primer F and the downstream primer R were designed according to the gene sequence, and the target sequence fragment was amplified using a isothermal amplification reaction system at room temperature 37°C for 15 minutes.

[0129] 4. Comparison of crRNA detection results before and after optimization

[0130] According to the isothermal shearing target in Example 1: the amplified target sequence obtained after isothermal amplification, different functional complexes: engineered crRNA-Cas12a and crRNA-Cas12a are mixed separately, and the difference in nucleic acid detection ability is determined according to the measured fluorescence value.

[0131] like Figure 5 to Figure 28 As shown, the real-time fluorescence signal monitoring results of 24 targets in this embodiment. Among them, the gray line represents the no target nucleic acid control (NTC), the blue line represents the traditional crRNA-Cas12a detection result, and the red line represents the engineered crRNA-Cas12a detection result.

[0132] As can be seen from the figure, the engineered crRNA-Cas12a has higher detection sensitivity than the traditional crRNA-Cas12a.

[0133] Table 7 crRNA sequences involved in Example 5

[0134]

[0135]

[0136]

[0137] Example 6 Sensitivity comparison test after crRNA addition-TTATTAT-DNA sensitization sequence

[0138] From the results of Example 4, it can be seen that adding a DNA sensitization sequence of TTATTAT to the 3' end of crRNA can improve the detection sensitivity.

[0139] This example continues to take the Mycoplasma pneumoniae target as an example to compare the detection sensitivity with and without the addition of a DNA sensitization sequence.

[0140] 1. Test methods

[0141] 1. crRNA design

[0142] Taking the Mycoplasma pneumoniae target as an example, the base sequence is specifically SEQ ID NO: 13, 16, which is synthesized by General Biotechnology (Anhui) Co., Ltd.

[0143] 2. RAA amplification was performed according to the steps in Example 1.

[0144] 3. Sensitivity comparison test of crRNA (SEQ ID NO: 13) / SEQ ID NO: 16

[0145] The RAA product of the previous step was subjected to CRISPR detection, and 5ul of the amplified product was added. After mixing and centrifugation, it can be tested on the machine. The CRISPR detection procedure is the same as in Example 1.

[0146] 2. Result analysis:

[0147] The RAA amplification in Example 1 and the crRNA testing steps in Example 4 were followed and the results were as follows: Figure 4 As shown, the results showed that the sensitivity after adding DNA sensitization sequence was better than the control group, and the detection limit could reach 1 copy / reaction.

[0148] 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. An engineered crRNA, characterized in that The engineered crRNA includes a crRNA skeleton and a sensitization sequence, wherein the crRNA skeleton includes an intrinsic sequence and a guide sequence, wherein the intrinsic sequence includes a stem-loop structure, wherein the guide sequence is used to bind to a target gene, wherein the sensitization sequence is connected to the 3' end of the guide sequence, and wherein the sensitization sequence is a DNA fragment of 1 to 15 nt and does not contain GC bases.

2. The engineered crRNA according to claim 1, characterized in that The length of the DNA sensitization sequence is 3 to 11 nt.

3. The engineered crRNA according to claim 1, characterized in that The intrinsic sequence is shown as SEQ ID NO.

112.

4. The engineered crRNA according to claim 1, characterized in that The target gene is selected from pathogenic genes in pathogens or drug-resistant genes related to pathogens.

5. The engineered crRNA according to claim 4, wherein the pathogen is selected from one or more of influenza A virus, influenza B virus, Streptococcus pneumoniae, Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Mycoplasma pneumoniae, novel coronavirus, adenovirus, human metapneumovirus, parainfluenza virus, Haemophilus influenzae, Stenotrophomonas maltophilia, Escherichia coli, Moraxella catarrhalis, Legionella pneumophila, Enterococcus faecalis, Enterococcus faecium, Chlamydia pneumoniae, and Yeersporocystis.

6. According to the engineered crRNA according to claim 4, the drug resistance gene associated with the pathogen is selected from carbapenem resistance genes and / or methicillin resistance genes; preferably, the carbapenem resistance gene is blaKPC; preferably, the methicillin resistance gene is mecA or mecC.

7. A CRISPR / Cas system, characterized in that: The CRISPR / Cas system comprises the engineered crRNA described in any one of claims 1 to 6.

8. The CRISPR / Cas system according to claim 7, characterized in that The CRISPR / Cas system includes one or more of a buffer, a Cas protease, or a reporter probe.

9. The CRISPR / Cas system according to claim 8, characterized in that The Cas protease is Cas12a protease.

10. Use of the engineered crRNA according to any one of claims 1 to 6, or the CRISPR / Cas system according to any one of claims 7 to 9 in the preparation of reagents for detecting pathogens or drug-resistant genes associated with pathogens.

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