CRISPR / Cas system construction and application based on DNA triple-helix molecular probe

Abstract

The invention relates to construction and application of a CRISPR (clustered regularly interspaced short palindromic repeats) / Cas system based on a DNA (deoxyribonucleic acid) triple helix molecular probe. According to the CRISPR / Cas system construction based on the DNA triple-helix molecular probe, a triple-helix molecular beacon reporter probe and Cas protein are coupled; the triple-helix molecular beacon reporter probe is formed by hybridization of stem single-stranded ssDNA and loop single-stranded ssDNA, the loop single-stranded ssDNA is as shown in SEQ ID NO: 1-4, and the stem single-stranded ssDNA is as shown in SEQ ID NO: 5-6. According to the biosensor disclosed by the invention, the detection limit is as low as 31fM when double genes of the ORF1ab gene and the N gene are detected without amplification, so that the detection sensitivity is greatly improved.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to the construction and application of a CRISPR / Cas system based on a DNA triple helix molecular probe. Background Art

[0002] Clustered regularly interspaced short palindromic repeats (CRISPR) is an acquired immune system found in bacteria and archaea. Due to the trans-cutting nuclease activity of the CRISPRV type (Cas12) and type VI (Cas13) systems, and the advantages of simple operation, low cost, and strong specificity, it has been widely used in the field of medical molecular testing in recent years. CRISPR-Cas 12a performs target-specific DNA cleavage (called cis-cleavage) and non-specific cleavage of surrounding single-stranded DNA (called trans-cleavage) under the guidance of CRISPR RNA (crRNA). Its trans-cutting nuclease activity can only be activated when the target is complementary to the crRNA base pairing. Due to the characteristic that the activated Cas12a cuts single-stranded DNA (ssDNA) indiscriminately, researchers have widely used it in auxiliary bioassays and obtained relatively good analytical performance. Based on this principle, many diagnostic tools such as DETECTER, HOLMES, etc. have been developed for the detection of DNA samples. Most fluorescent reporters are still short ssDNA (Repoter) (5-7nt) based on the principle of fluorescence resonance energy transfer (FRET). When the Cas12a / crRNA complex is activated with the corresponding target DNA, the ssDNA reporter molecule will be cut, resulting in the recovery of fluorescence. Without pre-amplification, the sensitivity of the CRISPR-Cas12a system is so low that it can only detect the target DNA in the picomolar range.

[0003] In order to improve the detection sensitivity of the CRISPR / Cas system, researchers have conducted extensive research from various aspects, such as integrating various bio-amplification technologies (such as PCR, LAMP, RPA, NASBA, etc.), optimizing the parameters of the CRISPR / Cas system and digital detection technology. Among them, optimizing the parameters of the CRISPR / Cas system is a hot topic of research, and developing new reporter probes is the simplest, most economical and effective way to improve sensitivity and shorten detection time. In order to overcome the shortcomings of current short-chain probes, researchers have tried to develop various types of signal probes. Systematically investigating the fluorescent signal readout of CRISPR-Cas12a for highly sensitive SARS-CoV-2 detection (Systematically investigating the fluorescent signal readout of CRISPR-Cas12a for highly sensitive new coronavirus detection) disclosed that the trans-cleavage of Cas12a is not limited to ssDNA and dsDNA reporters, but can be extended to molecular beacons (MBs), and disclosed a highly sensitive new coronavirus detector that successfully achieved a sensitivity as low as 100fM without an amplification strategy, but the detection limit of the highly sensitive new coronavirus detector is still high. Summary of the invention

[0004] The first object of the present invention is to provide a CRISPR / Cas system construction based on a DNA triple helix molecular probe to solve the technical problem that the detection limit of the CRISPR / Cas system is high in the absence of pre-amplification.

[0005] The second object of the present invention is to provide an application of a CRISPR / Cas system based on a DNA triple helix molecular probe.

[0006] In order to achieve the above purpose, the technical solution adopted by the present invention is:

[0007] In the CRISPR / Cas system based on DNA triple-helix molecular probes, a triple-helix molecular beacon reporter probe and a Cas protein are coupled; the triple-helix molecular beacon reporter probe is formed by hybridization of a stem single-stranded ssDNA and a loop single-stranded ssDNA, the loop single-stranded ssDNA is shown in SEQ ID NO:1-4, and the stem single-stranded ssDNA is shown in SEQ ID NO:5-6.

[0008] Furthermore, the ends of the circular single-stranded ssDNA are labeled with fluorescein and a quencher, respectively.

[0009] Furthermore, the preparation method of the triple-helix molecular beacon reporter probe is: thermally cycle the stem single-stranded ssDNA and the loop single-stranded ssDNA at 95°C for 5 minutes, annealing to obtain the stem single-stranded ssDNA and the loop single-stranded ssDNA, cooling to 25°C, and incubating in a Tris-HCl buffer solution for 1.5-3 hours to obtain a triple-helix molecular beacon reporter probe; the pH of the Tris-HCl buffer solution is 7.9.

[0010] Furthermore, the final concentration ratio of the stem single-stranded ssDNA and the loop single-stranded ssDNA is 1:1.

[0011] Furthermore, the number of bases in the loop single-stranded ssDNA is 10-40 nt, and the number of bases in the stem single-stranded ssDNA is 8-10 nt.

[0012] Application of the CRISPR / Cas system based on DNA triple helix molecular probes in detecting ORF1ab gene, N gene, ORF1ab gene and N gene double gene in the SARS-CoV-2 genome.

[0013] Furthermore, the method comprises the following steps: incubating the Cas protein with crRNA to form a complex, and then adding the target gene and a triple-helix molecular beacon reporter probe for cutting; the target gene is the ORF1ab gene, the N gene, or a double gene of the ORF1ab gene and the N gene.

[0014] Furthermore, the final concentration of the Cas protein is 1 μM, the final concentration of the crRNA is 1.2 μM, the final concentration of the target gene is 10 nM, and the final concentration of the triple-helix molecular beacon reporter probe is 0.5 μM.

[0015] Furthermore, the Cas protein is LbCas12a.

[0016] Beneficial effects of the present invention:

[0017] The detection limit of the biosensor of the present invention is as low as 31 fM when detecting ORF1ab gene and N gene without amplification, which greatly improves the detection sensitivity.

[0018] The present invention uses intramolecular triple helix molecular beacon as a reporter probe for Cas 12a, and compared with linear single-stranded ssDNA reporter probe (Repoter), triple helix molecular beacon reporter probe can enhance the trans-cleavage activity of Cas 12a. In the same time, the triple helix molecular beacon reporter probe of the present invention produces a faster and stronger fluorescent signal than Repoter, and can achieve detection as low as fM without amplification, proving that the sensitivity and specificity of Cas12a detection can be enhanced in triple helix molecular beacon reporter probe. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a design and characterization diagram of the triple-helix molecular beacon reporter probe in Example 1, wherein A is a design diagram of a triple-helix molecular beacon reporter probe, B is a design diagram of a triple-helix molecular beacon reporter probe with a stem single-stranded ssDNA of 10 nt, C is a design diagram of a triple-helix molecular beacon reporter probe with a stem single-stranded ssDNA of 8 nt, D is a diagram of the interaction between crRNA and activated Cas 12a, E is a schematic diagram of the Cas12a trans-cutting activity of THMB, and F is a schematic diagram of the Cas 12a trans-cutting activity of Repoter;

[0020] Figure 2 A diagram showing the selection of a triple-helix molecular beacon reporter probe for a buffer solution;

[0021] Figure 3 A diagram showing the selection of incubation time for triple helical molecular beacon reporter probes;

[0022] Figure 4 is the quenching rate of the triple-helix molecular beacon reporter probe;

[0023] Figure 5 It is the melting curve of the triple-helix molecular beacon reporter probe;

[0024] Figure 6 Kinetic curves of Cas12a trans-cleavage of linear ssDNA and triple-helix molecular beacon reporter probes;

[0025] Figure 7 It is a cutting efficiency diagram of the DNA probe trans-cut by the LbCas12a / crRNA / dsDNA complex;

[0026] Figure 8 The Michaelis-Menten plot is a graph of the Cas12a trans-cutting activity of Repoter and triple-helix molecular beacon reporter probes using ssDNA activators;

[0027] Fig. 9 It is a polyacrylamide gel electrophoresis diagram of the time dependence of the trans-cleavage activity of activated LbCas12a on DNA reporter molecules;

[0028] Fig.10 is a schematic diagram of a biosensor;

[0029] Fig.11 The feasibility diagram of the biosensor for detecting ORF1ab gene and N gene separately and ORF1ab gene and N gene dual gene detection;

[0030] Fig.12 This is the CD spectrum identification diagram of THMB (Loop 20nt: Stem 10nt);

[0031] Fig.13 This is the long-term stability graph of THMB (Loop20nt: Stem10nt) at 4°C;

[0032] Fig.14 Optimization diagram of triple helix molecular beacon reporter probe concentration conditions for detecting ORF1ab gene;

[0033] Fig.15 Optimization diagram of triple helix molecular beacon reporter probe concentration conditions for detecting N gene;

[0034] Fig.16 This is the standard curve diagram for detecting ORF1ab gene;

[0035] Fig.17 This is the detection limit diagram for detecting ORF1ab gene;

[0036] Fig.18 This is the standard curve diagram for detecting N gene;

[0037] Fig.19 This is the detection limit diagram for detecting N gene;

[0038] Fig. 20 This is the standard curve diagram for detecting the ORF1ab gene and N gene double gene;

[0039] Fig.21 This is the detection limit diagram for detecting ORF1ab gene and N gene;

[0040] Fig. 22 To analyze the specificity of biosensors in detecting the novel coronavirus;

[0041] Fig.23 Fluorescence signals of triple-helix molecular beacon reporter probes and classic single-stranded probes. DETAILED DESCRIPTION

[0042] The present invention will be further described below in conjunction with embodiments and drawings.

[0043] Example 1

[0044] The construction of the CRISPR / Cas system based on DNA triple helix molecular probe in Example 1 includes the following steps:

[0045] 1. Preparation of triple-helix molecular beacon reporter probe

[0046] 1.1 Design and sequence of triple helix molecular beacon reporter probe (THMB)

[0047] The triple-helix molecular beacon reporter probe includes stem single-stranded ssDNA (sfO) and circular single-stranded ssDNA (pre-THMB). The ends of pre-THMB are labeled with fluorescein and a quencher, respectively. In the absence of sfO, a triple-helix molecular beacon reporter probe cannot be formed. The fluorescein is far away from the quencher and displays a strong fluorescence signal in the solution. When sfO is present in the solution, one end of pre-THMB forms a double strand with sfO under the action of the Watson-Crick bond, while the other end folds into the major groove of the double helix due to the Hoogsteen bond, forming a stem triple-helix structure. At the same time, the distance between the fluorescein and the quencher is shortened, resulting in fluorescence quenching, thereby reducing the fluorescence signal in the solution. The fluorescein is FAM, and the quencher is BHQ1. The nucleotide sequences of the stem single-stranded ssDNA and the circular single-stranded ssDNA are shown in Table 1. The formation of THMB is as shown in Figure 1 shown.

[0048] Table 1 Nucleotide sequences of stem single-stranded ssDNA (sfO) and loop single-stranded (Loop) ssDNA

[0049]

[0050]

[0051] 1.2 Preparation and identification of THMB

[0052] Preparation: The stem single-stranded ssDNA and the loop single-stranded ssDNA are thermally cycled at 95°C for 5 minutes to anneal to obtain a mixture of the stem single-stranded ssDNA and the loop single-stranded ssDNA, which is then cooled to room temperature (~25°C) and incubated in a Tris-HCl buffer solution for 2 hours to obtain THMB. The Tris-HCl buffer solution includes a final concentration of 10 mM trishydroxymethylaminomethane hydrochloride (Tris-HCl), 50 mM sodium chloride (NaCl), and 10 mM magnesium chloride hexahydrate (MgCl2·6H2O), and the pH of the Tris-HCl buffer solution is 7.9. The final concentrations of the stem single-stranded ssDNA and the loop single-stranded ssDNA in the mixture of the stem single-stranded ssDNA and the loop single-stranded ssDNA are both 2.5 μM.

[0053] Identification: The Cas12a-mediated cleavage test was carried out in a Tris-HCl buffer solution. First, LbCas12a (2 μL, 10 μM) was pre-incubated with crRNA (4.8 μL, 5 μM) at 37 ° C for 30 min to form a LbCas12a / crRNA complex, and the target DNA or non-target DNA or cleavage buffer (2 μL) and THMB (2 μL, 2.5 μM) were added to form a 20 μL reaction solution. The cleavage reaction was performed at 37 ° C for 1 h. The LbCas12a / crRNA complex was formed by pre-incubation with crRNA (4.8 μL, 5 μM) at 37 ° C for 30 min, and the target DNA added was a self-designed single-stranded target DNA or SARS-CoV-2ORF1ab gene or SARS-CoV-2N gene, and the target DNA was added in an amount of 2 μL, with a final concentration of 10 nM. The sequence of the single-stranded target DNA is shown in SEQ ID NO: 8.

[0054] 1.3 Identification of THMB

[0055] Fluorescence spectrum measurement

[0056] For FRET determination, the circular ssDNA and stem ssDNA labeled with 5'-FAM and 3'-BHQ1 were diluted to 50 mL with a final concentration of 250 nM. Fluorescence measurements were performed using SpectraMax i3X (Shanghai, China) at room temperature (~25 °C). The excitation wavelength was set to 480 nm, and the emission wavelength scan range was from 506 nm to 650 nm. In experiments to test the effect of target DNA concentration on cleavage efficiency, the final target concentration ranged from 0 to 1 nM.

[0057] Measurement of circular dichroism spectra (CD measurement)

[0058] Circular dichroism spectroscopy experiments were performed on Chriascan (Applied Photophysics, UK). Data collection was performed by scanning from 220 nm to 300 nm at a scanning rate of 100 nm per minute at room temperature (~25°C) through a cuvette with a 0.1 cm optical path, taking the average of three scans. LbCas12a (2 μL, 10 μM) was pre-incubated with crRNA (4.8 μL, 5 μM) at 37°C for 30 minutes. THMB (2 μL, 25 μM) and target DNA (2 μL, 100 nM) were then added to Tris-HCl buffer solution (pH 7.9) and the cleavage reaction was performed at 37°C for 1 hour. FRET detection was performed before CD measurement to ensure that THMB was completely cleaved, and the mixture was set to 200 μL with a final concentration of 2.5 μM for CD detection. All spectra were blank-corrected with matching buffer solutions.

[0059] 1.4 Optimization of THMB formation conditions

[0060] The procedure is consistent with the measurement of fluorescence spectroscopy.

[0061] The fluorescence melting curve was obtained by heating a solution containing 250 nM THMB from 20°C to 60°C at a rate of 1°C per minute and monitoring the fluorescence every 0.5°C. In order to estimate the transition equilibrium constant (Ks) of THMB, a two-state model was assumed in which THMB exists in two states, folded and unfolded, in the solution and is independent of temperature. The changes in the folded and unfolded fractions of THMB with temperature were calculated according to the formula:

[0062] α=(B U -F T ) / (B U -B T )

[0063] Where α represents the folding rate of THMB at temperature (T), B T and B U are the baseline fluorescence intensities when the probe is fully folded and fully unfolded, respectively, and F T is the fluorescence value of THMB at a given temperature. Then, the equilibrium constant Ks of the reaction is calculated based on α between 0.15 and 0.85 as Ks = (1–α) / α. According to the van't Hoff equation:

[0064] ln(K S )=-ΔH° / R×1 / T+ΔS° / R

[0065] The obtained linear equation about (ln(Ks)) and (1 / T). The equilibrium constant (Ks) and free energy (ΔG°) at 37°C were obtained by the van't Hoff equation, where K S =exp(-G° / RT), ΔG°=ΔH°-TΔS°.

[0066] 1.5 Nonspecific cleavage of the CRISPR / Cas12a system

[0067] Measurement of kinetic curves

[0068] The self-designed single-stranded target DNA was selected as the target. The Cas12a / crRNA complex was first incubated at 37°C for 30 minutes, with a final concentration of 1μM and 1.2μM. In a series of Eppendorf tubes, 1μL of Cas12a / crRNA complex, 2μL of single-stranded target DNA (10nM) was transferred to a final volume of 20μL, and a solution containing 250nM of DNA reporter gene (Repoter, THMBs) was placed in a real-time fluorescence quantitative PCR instrument, and the cutting reaction was carried out at 37°C for 1 hour, and the fluorescence signal was collected.

[0069] Michaelis-Menten enzyme kinetic measurements and data analysis

[0070] The cleavage reaction was performed by adding 1 μL of Cas12a / crRNA complex and 2 μL of single-stranded target DNA (final concentration of 10 nM) to THMB solutions of different final concentrations (final volume 20 μL) (THMB concentration range 23 nM to 750 nM). Each reaction was repeated three times, and fluorescence measurements were collected every 20 seconds. The average reaction rate for the first 180 seconds was calculated as the initial reaction rate (V0) from the fluorescence data collected from probes of different concentrations during the Michaelis enzyme kinetic experiment. The fluorescence intensity F(t) measured in the cleavage reaction experiment is the sum of the fluorescence intensities of the cleaved probe and the uncut probe. The calculated reaction rate is converted from au / min to nM / min according to the formula:

[0071] F(t)=F cleaved (t)+F uncleaved (t)

[0072] where F cleaved (t) and F uncleaved (t) are the fluorescence intensities of the cut and uncut probes, respectively. It indicates that under the same experimental conditions, the Cas12a / crRNA complex containing single-stranded target DNA and the Cas12a / crRNA complex without single-stranded target DNA were completely cut after incubation of 20 μL solution for 24 h (F cleaved ) and uncut (F uncleaved ) The endpoint fluorescence intensity of the probe at different concentrations. The F cleaved and F uncleaved Standard curve with increasing THMB concentration (concentration range 23nM to 750nM). The initial reaction rate (V0) can be converted from au / min to nM / min using the formula:

[0073] F(t)=m1×[C cleaved ]+m2×(1-[C cleaved ])

[0074] Where m1 and m2 are Figure 7 and Figure 8 The slope of the standard curve equation in was obtained by measuring the concentration at different time points, and the reaction rate (nM / min) was obtained by taking the derivative of the above formula with respect to time. The calculated reaction rate (nM / min) was fitted with the FRET-based THMB reporter gene (substrate) concentration, and the maximum reaction rate (V max ) and the Michaelis constant (K m ), according to the formula: Y = (V max ×X) / (K m +X). where X is the concentration of THMB and Y is the enzyme reaction rate. Finally, the turnover constant (k cat ) is calculated as k cat =V max / E, where E is the concentration of active Cas12a / crRNA complex (1 nM).

[0075] Native-PAGE analysis

[0076] LbCas12a (2 μL, 10 μM) was pre-incubated with crRNA (4.8 μL, 5 μM) at 37 ° C for 10 minutes. THMB (2 μL, 25 μM) and single-stranded target DNA (2 μL, 100 nM) were then added to the reaction solution and incubated at 37 ° C for an appropriate time. The reaction system was incubated at 65 ° C for 15 minutes to inactivate LbCas12a. The cleavage products were separated by electrophoresis at 110 V / 200 mA for 80 minutes using a native PAGE gel (15%, w / v) prestained with Gelred (Bioti μM). The DNA Native-PAGE gel was exposed to ChemiDoc MP to obtain a gel map.

[0077] 2. Results and Discussion

[0078] 2.1 Design of THMB hairpin DNA reporter gene based on fluorescence resonance energy transfer (FRET)

[0079] THMB is constructed by mixing pre-THMB and sfO in a concentration ratio of 1:1 and annealing, wherein pre-THMB is labeled with fluorescein FAM and quencher BHQ1 at both ends. After annealing, sfO and pre-THMB are mixed and hybridized to form a double chain, and then a triple helix structure is formed at the stem under the Hoogsteen parallel interaction. Based on the principle of fluorescence resonance energy transfer dependence on distance, when THMB is formed, the distance between the two ends of pre-THMB is close, and the fluorescence of FAM (λEm=520nm) is quenched by BHQ1, such as Figure 1A. Based on this principle, a group of 8 triple helix molecular beacon reporter probes were designed, with the stem fixed at 10nt or 8nt, and the loops were designed as permutations of 40nt, 30nt, 20nt, and 10nt, respectively. Figure 1 B and 1C.

[0080] Taking THMB (Loop40nt:Stem10nt) as an example, PBS (1×), PBS (1×) + 1mM Mg 2+ , PBS (1×) + 2.5 mM Mg 2+ , Tris-HCl (pH7.4), Tris-HCl (pH7.9) and PBST buffer solutions were incubated under the same conditions. Figure 2 It can be seen that THMB (Loop40nt: Stem10nt) has the best formation effect in Tris-HCl (pH7.4) and Tris-HCl (pH7.9), with a fluorescence quenching factor of up to 29.68. However, studies have shown that the trans-cleavage activity of Cas12a is highest in the Tris-HCl (pH7.9) environment.

[0081] Taking THMB (Loop 40nt: Stem 10nt) as an example, Tris-HCl (pH 7.9) was selected as the buffer solution, and the incubation time was selected as 0.5, 1, 1.5, 2, 3, and 4 h, respectively. Figure 3 It can be seen that the formation effect of THMB (Loop40nt: Stem10nt) is best when the incubation time is 2h. When the buffer solution is Tris-HCl (pH7.9) and the incubation time is 2h, the remaining 7 THMBs are verified. When the number of stem bases is fixed at 10nt or 8nt, the 8 THMBs can form triple helical hairpin structures in the buffer solution Tris-HCl (pH7.9), and the fluorescence quenching multiples are approximately between 10-40 times (such as Figure 4 ). Figure 4 It can be seen that when the stem of THMB is fixed at 6 nt, the fluorescence quenching multiple does not exceed 10 times. The reason is that when the stem is too short, the force of the Hoogsteen bond in the stem is difficult to counteract the tension between the molecules in the ring part, which makes the formation of the triple helix more difficult, so the fluorescence quenching multiple is lower.

[0082] From Table 2 and Figure 5It can be seen that the thermal stability of THMB is closely related to the number of bases in the loop and stem, and THMB with 10 nt in the stem all show higher Tm values. The melting temperatures of THMB (Loop20 nt: Stem8 nt), THMB (Loop30 nt: Stem8 nt), and THMB (Loop40 nt: Stem8 nt) are low, and the THMB folding state ratio α calculated based on the equilibrium constant Ks at the optimal temperature (37°C) of Cas12a trans-cutting activity is lower than 40%. It can be seen that THMB (Loop20nt: Stem8 nt), THMB (Loop30 nt: Stem8 nt), and THMB (Loop40 nt: Stem8 nt) have high fluorescence background values ​​at 37°C, and the triple helix DNA hairpin structure is in an unfolded state, which does not meet the requirements of probe physical stability and is not suitable for probe research in the CRISPR / Cas12a system.

[0083] Table 2 Melting temperatures (Tm), equilibrium constants (K) at 37°C of 8 THMBs s ) Free energy and folded state ratio

[0084]

[0085] 2.2 Study on the cleavage dynamics of THMB by CRISPR-Cas12a

[0086] from Figure 6It can be seen that compared with other THMBs, THMB (Loop20nt: Stem10nt) produces the highest fluorescence signal change in the shortest time and reaches the plateau phase the fastest (about 20min), while the rate of trans-cutting of THMB (Loop10nt: Stem10nt) is closely followed, which may be related to the excessively high thermal stability of the probe itself. The triple helix structure of the stem is relatively stable. Even if the loop is trans-cut, the double-stranded stem is not easily cut, and it is difficult to unwind. The reason why the rates of THMB (Loop30nt: Stem10nt) and THMB (Loop40nt: Stem10nt) are relatively low is that the larger loop leads to a larger molecular volume, which makes it difficult to enter the Cas12a cleavage domain, and the activity of the molecule is hindered, resulting in a slower fluorescence recovery and a lower cutting rate. As the shearing time increases, the fluorescence recovery rate of all selected THMBs is higher than that of Repoter, which may be attributed to the fact that the molecular weight of THMBs is much larger than that of ssDNA, the probability of contact and collision between molecules is greater, and the single-stranded region of the loop is more likely to be digested by trans-cleavage, thereby showing a better signal output efficiency. However, the excessive stability of THMB does not mean that it will show higher performance when used as a Cas12a probe. The reason is that the higher the stability of THMB, the smaller the proportion of hairpin structures in an unfolded state, resulting in a lower fluorescence background value. At this point, after Cas12a in the activated state trans-cuts its single-stranded loop, it is difficult for the overly stable stem structure of THMB to convert to an unfolded state, the fluorescence conduction effect is blocked, and the fluorescence recovery efficiency of the probe is reduced.

[0087] 3.2THMB faster and stronger signal change and sensitivity analysis

[0088] Prepare 10× pre-incubated LbCas12a protein / crRNA / single-stranded target DNA complexes (final concentrations of 1 μM, 1.2 μM, and 10 nM, respectively), add THMB or Repoter without amplification, and perform fluorescence reading. Figure 7 It can be seen that compared with Repoter, the THMB of the present invention shows a better shear rate. cat / K m The ratio was about 4-13 times higher than that of Repoter (Table 3). The reason for this difference is that the K m These results show that THMB (Loop20nt: Stem10nt) has the highest affinity with CRISPR / Cas12a.

[0089] As can be seen from Table 3, the K of THMB (Loop 20nt: Stem 10nt) cat / Km The value is the highest, 13 times that of Repoter, because its highest affinity and high catalytic efficiency make it show the highest cleavage efficiency. Polyacrylamide gel electrophoresis was used to characterize the degree of cleavage of THMB (Loop 20nt: Stem 10nt) at different reaction times, and it was basically all trans-cleaved within 20 minutes.

[0090] In summary, THMB (Loop 20nt: Stem 10nt) has good stability, higher trans-cleavage efficiency, faster fluorescence signal change rate and highest affinity.

[0091] Table 3 Kcat, Km values ​​and Kcat / Km ratios of THMBs

[0092]

[0093]

[0094] 3.3 Establishment of the biosensing platform “TH-CRISPR”

[0095] THMB (Loop20nt:Stem10nt) was coupled with CRISPR / Cas 12a to construct a biosensor named TH-CRISPR. SARS-CoV-2 was used as a target to evaluate the performance of TH-CRISPR, with the ORF1ab gene and N gene fragment in the SARS-CoV-2 genome map as a model as the target. The schematic diagram is shown in the figure. Fig.10 As shown. Without the need for complex instruments, a crRNA of Cas 12a that specifically matches the specific base sequences of the ORF1ab gene and the N gene fragment is designed. In order to improve the efficiency of targeted binding, a dual crRNA is designed to target and bind to different sites of the SARS-CoV-2 gene. After the extracted viral RNA is reverse transcribed into cDNA or the DNA model target is mixed with the CRISPR / Cas 12a complex, an activated Cas12a with trans-cutting activity is obtained, and a THMB reporter probe is added to the detection environment for fluorescence reading to achieve qualitative and quantitative detection of the target. For the synthetic DNA model target, the two selected genes are detected separately and in double detection. The crRNA sequence of the ORF1ab gene is shown in SEQ ID NO:9, and the crRNA sequence of the N gene is shown in SEQ ID NO:10.

[0096] from Fig.11It can be seen that when only THMB is present in the solution, the fluorescence reading at 520 nm is low. After the addition of Cas12a and the synthetic DNA model target, THMB is trans-cut by the activated Cas12a based on FRET. The fluorescent groups FAM and the quenching group BHQ1 at both ends of THMB are far apart. Under the irradiation of excitation light with a wavelength of 480 nm, the fluorescence value of the fluorescent group FAM at a wavelength of 520 nm is detected. Fig.11 The target concentration added was low (only 1 nM). It can be seen that when ORF1ab gene or N gene exists alone, the fluorescence value is significantly lower than the fluorescence detected when both genes exist. Therefore, it can be preliminarily deduced that dual gene detection can achieve the effect of 1+1>2. Fig.12 It can be seen that when only pre-THMB single strand exists in the solution, the CD spectrum is irregular. After THMB is formed, a strong negative peak at 212nm and a positive peak at 279nm (red line) with triple helix characteristic peaks appear. After adding activated Cas12a, the strong negative peak at 212nm shifts to the right and decreases, a positive peak appears at 220nm, and two negative peaks at 245nm and 210nm are characteristic peaks of DNA double strands, indicating that the triple helix structure is cleaved. THMB (Loop20nt: Stem10nt) itself shows excellent stability, from Fig.13 It can be seen that the selected THMB can maintain high stability within 50 days, and the background fluorescence remains at a low level, showing great potential for long-term storage. The feasibility and condition optimization of the detection of the ORF1ab gene and the N gene (final concentration of 10nM target gene) were verified respectively. When the two target genes exist alone, the output of fluorescence signals can be achieved. The concentration of THMB probe added during the detection of the two targets was optimized. Fig.14 and 15 It can be seen that when the THMB probe concentration is 0.5 μM, the maximum output of the fluorescence signal can be reached.

[0097] 3.4THMB has strong sensitivity and high specificity in detecting SARS-CoV-2

[0098] The ORF1ab gene and the N gene were detected separately without amplification. Fig.16 It can be seen that as the concentration of the ORF1ab target gene changes from 0 to 5 nM, the fluorescence intensity at 520 nm increases, showing a linear relationship in the pM range, with a correlation coefficient R 2 is 0.996, and the minimum detection limit (LOD) is 8.15pM ( Fig.17 ). When the target is changed to N gene, Fig.18It can be clearly seen that in the concentration gradient from 0 to 1 nM, the fluorescence intensity at 520 nm increases and is linear in the pM range, with a correlation coefficient R 2 is 0.9997 and the minimum detection limit is 1.55pM ( Fig.19 ). Using the same detection method, the target is a double gene (i.e., the Cas12a complex activated by the ORF1ab gene and the N gene and their corresponding RNA is added at the same time), from Fig. 20 and Fig.21 As can be seen in the figure, in the concentration range of 0 to 1 nM, the fluorescence intensity gradient at 520 nM increases and is linear in the pM range, with a correlation coefficient R 2 The detection limit was 0.9949 and the lowest detection limit was 31fM. Since the THMB probe has a high sensitivity, linear single-stranded DNA and THMB were compared at low concentrations (0-12pM). Fig.23 It can be seen that the fluorescence signal of THMB is higher than that of linear single-stranded DNA, the slope is about 2 times higher, and the difference in fluorescence signal change is higher.

[0099] Coronaviruses similar to SARS-CoV-2, such as severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS-CoV), human coronavirus (HCoV-HKU1), and human coronavirus OC43 (HCoV-OC43), were selected for comparison with the same method. Fig. 22 It can be seen that the THMB probe has a high specificity for the SARS-CoV-2 virus.

Claims

1. Construction of CRISPR / Cas system based on DNA triple helix molecular probe, characterized in that: The triple-helix molecular beacon reporter probe and the Cas protein are coupled; the triple-helix molecular beacon reporter probe is formed by hybridization of a stem single-stranded ssDNA and a loop single-stranded ssDNA, the loop single-stranded ssDNA is shown in SEQ ID NO: 1-4, and the stem single-stranded ssDNA is shown in SEQ ID NO: 5-6.

2. The construction of the CRISPR / Cas system based on DNA triple helix molecular probe according to claim 1, characterized in that: The ends of the circular single-stranded ssDNA are labeled with fluorescein and a quencher, respectively.

3. The construction of the CRISPR / Cas system based on DNA triple helix molecular probe according to claim 1, characterized in that: The preparation method of the triple-helix molecular beacon reporter probe is as follows: thermally cycle the stem single-stranded ssDNA and the loop single-stranded ssDNA at 95°C for 5 minutes to anneal the stem single-stranded ssDNA and the loop single-stranded ssDNA, cool them to 25°C, and incubate them in a Tris-HCl buffer solution for 1.5-3 hours to obtain the triple-helix molecular beacon reporter probe; the pH of the Tris-HCl buffer solution is 7.

9.

4. The construction of the CRISPR / Cas system based on DNA triple helix molecular probe according to claim 3, characterized in that: The final concentration ratio of the stem single-stranded ssDNA and the loop single-stranded ssDNA is 1:

1.

5. The construction of the CRISPR / Cas system based on DNA triple helix molecular probe according to claim 1, characterized in that: The number of bases in the loop single-stranded ssDNA is 10-40 nt, and the number of bases in the stem single-stranded ssDNA is 8-10 nt.

6. Application of the CRISPR / Cas system based on DNA triple helix molecular probes in detecting ORF1ab gene, N gene, ORF1ab gene and N gene double gene in the SARS-CoV-2 genome.

7. The use of the CRISPR / Cas system based on DNA triple helix molecular probe according to claim 6, characterized in that: The following steps are involved: The Cas protein is incubated with crRNA to form a complex, and then the target gene and triple-helix molecular beacon reporter probe are added for cleavage; The target gene is ORF1ab gene, N gene or ORF1ab gene and N gene.

8. The use of the CRISPR / Cas system based on DNA triple helix molecular probe according to claim 7, characterized in that: The final concentration of the Cas protein is 1 μM, the final concentration of the crRNA is 1.2 μM, the final concentration of the target gene is 10 nM, and the final concentration of the triple-helix molecular beacon reporter probe is 0.5 μM.

9. The use of the CRISPR / Cas system based on DNA triple helix molecular probe according to claim 7, characterized in that: The Cas protein is LbCas12a.

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