A detection method based on LAMP and CRISPR / Cas and its application

Abstract

The present invention relates to the field of nucleic acid detection technology, and discloses a detection method and application based on LAMP and CRISPR / Cas. The detection method comprises the following steps: mutating key amino acids in the PAM domain of the eBrCas12b protein; preparing Cas12b sgRNA by in vitro transcription and purification; evaluating the cis-cleavage ability of eBrCas12b, eBrCas12b-G, and eBrCas12b-GN on the target sequence; constructing a pUC19- without NDM5 The present invention adopts the above-mentioned detection method and application based on LAMP and CRISPR / Cas, optimizes the traditional LAMP-Cas12b one-tube detection system, and significantly improves the sensitivity and speed of one-tube detection.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid detection, and in particular to a detection method and application based on LAMP and CRISPR / Cas. Background Art

[0002] Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification technology that, unlike previous-generation PCR, fluorescent quantitative PCR, and emerging digital PCR technologies, does not require specialized instrumentation for detection. However, false positives are common. Incorrect primer design, contaminated DNA, and non-optimized reaction systems (such as buffer pH, magnesium concentration, and interfering dyes) can reduce detection specificity and sensitivity. CRISPR / Cas technology, by using reporters to detect specific targets, eliminates the limitations of LAMP, reduces the likelihood of false positives, and achieves highly sensitive and specific detection.

[0003] In CRISPR / Cas combined with LAMP technology, the Cas12 endonuclease is particularly preferred due to its collateral cleavage activity. There are three Cas12 enzyme subtypes: Cas12a, Cas12c, Cas12d, Cas12e, Cas12b, Cas12h, Cas12i, and Cas12g. Of these subtypes, Cas12a and Cas12b are most commonly used in LAMP-CRISPR assays. The optimal reaction temperature for the Bst enzyme used in LAMP amplification is 60-65°C, while the optimal reaction temperature for the Cas12a enzyme is around 37-42°C. The entire reaction requires two different temperatures, and using two different reaction processes and reaction systems can increase the risk of contamination. Solutions exist to overcome this limitation. For example, in the HOLMESv2 assay, the thermophilic AapCas12b (isolated from Bacillus acidophilus) is used to achieve a single-tube reaction, addressing the issue of different reaction temperatures.

[0004] A major drawback of the HOLMESv2 (see LI L, LI S, WU N, et al. HOLMESv2: a CRISPR-Cas12b-assisted platform for nucleic acid detection and DNA methylation quantitation[J]. ACS synthetic biology, 2019, 8(10): 2228-2237) detection system is that the trans-cleavage activity of wild-type AacCas12b stops working above 60°C and requires additional additives (such as taurine). Due to suboptimal temperature conditions, longer incubation times are required for stable function, resulting in slower diagnostic speed. In addition, since LAMP reactions are typically optimized at 60°C-65°C, protein performance in this temperature range limits LAMP primer design.

[0005] The wild-type BrCas12b protein used in the present invention still has trans-cleavage activity at 62°C (see NGUYEN LT, MACALUSO NC, PIZZANO BL, et al. A thermostable Cas12b from Brevibacillus leverages one-pot discrimination of SARS-CoV-2 variants of concern[J]. EBioMedicine, 2022, 77), while the engineered eBrCas12b protein still maintains trans-cleavage ability at 68°C (see NGUYEN LT, RANANAWARE SR, YANG LG, et al. Engineering highlythermostable Cas12b via de novo structural analyses for one-pot detection ofnucleic acids[J]. Cell Reports Medicine, 2023, 4(5)). Previous studies have reported the use of the eBrCas12b-LAMP system for virus detection. This paper exploits the characteristics of the LAMP reaction to introduce a PAM site recognized by Cas12b between the outer primers, thereby eliminating the PAM-dependent limitations on target sequence detection. Furthermore, by protein engineering two key residues in the eBrCas12b PAM binding domain, their interference with target amplification is minimized, thereby achieving rapid and highly sensitive Cas12b-LAMP detection. Summary of the Invention

[0006] The purpose of the present invention is to provide a detection method and application based on LAMP and CRISPR / Cas, which optimizes the traditional LAMP-Cas12b one-tube detection system and significantly improves the sensitivity and speed of one-tube detection.

[0007] In the conventional LAMP+Cas12b one-tube rapid detection system, while Bst DNA polymerase amplifies the target sequence, Cas12b binds to the target DNA under the guidance of sgRNA and forms an RNP complex for cleavage. However, this cleavage consumes the target DNA, affecting its continued amplification, thereby reducing the sensitivity and reaction speed of the detection system. To optimize this system, we performed a protein structure comparison of BrCas12b and its two orthologous proteins AacCas12b and BthCas12b, and identified two key residue sites in the BrCas12b PAM binding domain. By protein engineering these sites, the performance of BrCas12b in the one-tube CRISPR-LAMP detection was significantly improved.

[0008] To achieve the above objectives, the present invention provides a modified BrCas12b protein comprising the following mutations:

[0009] a) Asparagine N at position 392 of the sequence shown in SEQ ID NO. 20 is replaced by alanine A;

[0010] b) Glycine G at position 469 of the sequence shown in SEQ ID NO. 20 is replaced by alanine A.

[0011] Furthermore, the mutation of the BrCas12b protein leads to delayed target DNA cleavage activity. When the BrCas12b protein is used in conjunction with loop-mediated isothermal amplification (LAMP), the detection sensitivity of the target DNA is improved.

[0012] The present invention also provides a method for constructing the above-mentioned modified BrCas12b protein, using primers to perform site-directed mutagenesis on eBrCas12b(N392A)-F, eBrCas12b(N392A)-R and eBrCas12b(G469A)-F, eBrCas12b(G469A)-R, the sequence of eBrCas12b(N392A)-F is shown in SEQ ID NO.1, the sequence of eBrCas12b(N392A)-R is shown in SEQ ID NO.2, the sequence of eBrCas12b(G469A)-F is shown in SEQ ID NO.3, and the sequence of eBrCas12b(G469A)-R is shown in SEQ ID NO.4.

[0013] The present invention also provides primers for constructing the above-mentioned BrCas12b protein, including primer pairs eBrCas12b (N392A) -F, eBrCas12b (N392A) -R and primer pairs eBrCas12b (G469A) -F, eBrCas12b (G469A) -R;

[0014] The primer pairs eBrCas12b(N392A)-F and eBrCas12b(N392A)-R replaced asparagine N at position 392 of the BrCas12b protein with alanine A by site-directed mutagenesis;

[0015] The primer pairs eBrCas12b(G469A)-F and eBrCas12b(G469A)-R replaced glycine G at position 469 of the BrCas12b protein with alanine A by site-directed mutagenesis.

[0016] To achieve the above objectives, the present invention also provides a detection method based on LAMP and CRISPR / Cas, comprising the following steps:

[0017] Step 1, key mutation: key amino acid mutations in the PAM domain of BrCas12b protein;

[0018] Step 2, sgRNA preparation: Prepare Cas12b sgRNA by in vitro transcription and purification;

[0019] Step 3, cis-cleavage ability determination: evaluate the cis-cleavage ability of eBrCas12b, eBrCas12b-G, and eBrCas12b-GN on the target sequence;

[0020] Step 4, construct the detection system: construct a pUC19- bla NDM5 coli with the plasmid, and the eBrCas12b-GN one-tube detection system was applied to the detection of NDM-resistant Escherichia coli.

[0021] Furthermore, in step 1, the mutation sites are N392A and G469A.

[0022] Further, in step 1, the mutant primer sequence includes eBrCas12b (N392A) -F, eBrCas12b (N392A) -R, eBrCas12b (G469A) -F and eBrCas12b (G469A) -R; the sequence of eBrCas12b (N392A) -F is shown in SEQ ID NO. 1, the sequence of eBrCas12b (N392A) -R is shown in SEQ ID NO. 2, the sequence of eBrCas12b (G469A) -F is shown in SEQ ID NO. 3, and the sequence of eBrCas12b (G469A) -R is shown in SEQ ID NO. 4.

[0023] Furthermore, in step 3, eBrCas12b-G is a G469A mutant, and eBrCas12b-GN is a N392A and G469A mutant.

[0024] The present invention also provides a kit based on LAMP and CRISPR / Cas detection, comprising the above-mentioned modified BrCas12b protein.

[0025] The present invention also provides the use of the above-mentioned kit in nucleic acid detection, wherein the purpose of the application is not to diagnose and treat diseases.

[0026] Furthermore, the application method is as follows: the sample is added to the broth and shake-cultured at 37°C for 1-2 hours, the bacteria are enriched by centrifugation, and then the LAMP+eBrCas12b-GN reaction mixture is added. The bacteria are lysed and released at a constant temperature of 65°C. As the target DNA is amplified, the eBrCas12b-GN protein binds to the target DNA under the guidance of sgRNA to form an RNP complex. Its trans-cutting activity is activated to cut the ssDNA probe in the cutting system, and the positive sample emits obvious fluorescence under the background of ultraviolet light.

[0027] In the present invention, mutations are introduced into the wild-type BrCas12b protein to make its hydrophobic core more compact, thereby enhancing thermal stability. The wild-type BrCas12b protein sequence is shown in SEQ ID NO.20, as follows:

[0028]

[0029] The advantages and positive effects of the detection method and application based on LAMP and CRISPR / Cas described in the present invention are:

[0030] The present invention utilizes the characteristics of the LAMP reaction in the Cas12b-LAMP one-tube rapid detection system to introduce a PAM site recognized by Cas12b between the outer primers, thereby eliminating the restriction of the target sequence on PAM dependence. In addition, by mutating two key residues in the PAM binding domain of BrCas12b, the damage of Cas12b to the target DNA in the initial stage of isothermal amplification is effectively reduced, thereby significantly improving the sensitivity and speed of the one-tube detection.

[0031] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The gel electrophoresis results of the cis-cleavage ability of eBrCas12b, eBrCas12b-G, and eBrCas12b-GN proteins on target DNA in the embodiments of the present invention are as follows;

[0033] Figure 2 BrCas12b protein one-tube method for detecting pUC19- bla NDM5 Cell analysis results;

[0034] Figure 3 For the eBrCas12b-GN protein one-tube method for detecting pUC19- bla NDM5 Cell analysis results;

[0035] Figure 4 Schematic diagram of the sequence mutation of the BrCas12b protein N392 / G469 site in an embodiment of the present invention. DETAILED DESCRIPTION

[0036] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.

[0037] Unless otherwise defined, all technical terms used in the present invention or all other embodiments obtained by persons of ordinary skill in the art without creative effort based on the embodiments herein are within the scope of protection of the present invention. Experimental methods in the following examples where specific conditions are not specified are generally determined in accordance with national standards. All experimental instruments, equipment, and reagents in the following examples where the sources are not indicated are commercially available.

[0038] Unless otherwise defined or indicated, all professional and scientific terms used in the present invention have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the present invention.

[0039] Example

[0040] 1. Key mutation design: Key amino acid mutations were made in the PAM domain of BrCas12b protein, specifically N392A and G469A. The schematic diagram of the sequence mutation of BrCas12b protein N392 / G469 site is as follows Figure 4 shown.

[0041] The specific operations are:

[0042] 1.1 Construction of plasmid pET28-eBrCas12b:

[0043] According to the DNA sequence of eBrCas12b, it was synthesized by Qingke Biotechnology Co., Ltd. and connected to the pET28 plasmid.

[0044] 1.2eBrCas12b key amino acid N392A and G469A mutations:

[0045] 1.2.1 Design of mutation primers (as shown in Table 1):

[0046] Table 1 Mutation primers

[0047] ;

[0048] 1.2.2 PCR amplification of pET28-eBrCas12b plasmid (Table 2):

[0049] Table 2 Reaction system

[0050] ;

[0051] The reaction program was as follows: 98°C for 3 min; 98°C for 10 s, 61.5°C for 20 s, and 72°C for 4 min 30 s (30 cycles); and stored at 4°C.

[0052] 1.2.3 Digest the amplified product with DpnI to remove the methylated template plasmid (Table 3):

[0053] Table 3 Reaction system

[0054] ;

[0055] The above reaction system was placed in a constant temperature of 37°C for 1 to 2 hours.

[0056] 1.2.4 Recombination reaction (Table 4):

[0057] Table 4 Reaction system

[0058] ;

[0059] The above system was placed at 50°C for 30 minutes. After the reaction was completed, it was transformed into DH5α competent cells.

[0060] 1.3 eBrCas12b-G and eBrCas12b-GN protein expression:

[0061] The mutated plasmids pET28a-eBrCas12b-G and pET28a-eBrCas12b-GN were transformed into BL21 (DE3) competent cells and shaken at 37°C until OD 600 =0.6-0.8, IPTG=0.3mM, 16°C, 150rpm overnight culture followed by ultrasonic disruption and protein purification.

[0062] 2. sgRNA preparation: Prepare Cas12b sgRNA through in vitro transcription and purification. The specific steps are:

[0063] 2.1 Preparation of DNA transcription template (Table 5):

[0064] Table 5 Sequence

[0065] ;

[0066] The underlined part is the target sequence. The above oligo sequence was synthesized by Qingke Biotechnology Co., Ltd.

[0067] 2.2 Annealing system preparation (Table 6):

[0068] Table 6 Annealing system

[0069] ;

[0070] The reaction program was as follows: 95°C for 2 min; 95°C for 15 s, 55°C for 15 s, and 72°C for 10 s (30 cycles); and stored at 4°C.

[0071] 2.3 Cas12b sgRNA in vitro transcription (Table 7):

[0072] Table 7 Reaction system

[0073] ;

[0074] The reagents were prepared in sequence for the in vitro transcription reaction system, mixed thoroughly, and then centrifuged briefly. The transcript was then transcribed overnight at 37°C for 12-16 hours.

[0075] 2.4 Digestion of DNA template with DNaseI (Table 8):

[0076] Table 8 Reaction system

[0077] ;

[0078] Reaction conditions: 37°C, 1-4h.

[0079] 2.5 Magnetic bead purification of Cas12b sgRNA transcripts (Table 9):

[0080] Table 9 Sequence

[0081] ;

[0082] The target sequence is underlined.

[0083] 3. Cis-cleavage ability assay: Evaluate the cis-cleavage ability of eBrCas12b and its mutants (eBrCas12b-G: G469A mutant, eBrCas12b-GN: N392A / G469A mutant) on target sequences.

[0084] The specific operations are:

[0085] 3.1 Determination of the cis-cleavage ability of eBrCas12b / eBrCas12b-G / eBrCas12b-GN proteins on target DNA:

[0086] Prepare Cas12b cis-cleavage buffer (10×Cis-cleavage Buffer): 20mM Tris-HCl, 10mM (NH4)2SO4, 10mM Mg2SO4 and 0.1% Tween 20, the buffer has a pH of 7.5 at room temperature of 25°C.

[0087] Table 10 Reaction system

[0088] ;

[0089] The Cas12b protein reaction system containing different mutants (Table 10) was first incubated at 65°C for 1-6 minutes, then transferred to 85°C for 10 minutes to inactivate the Cas12b protein enzyme activity. 2 μL of 10× Loading Buffer was added and mixed for agarose gel electrophoresis analysis.

[0090] The results are as follows Figure 1As shown, gel electrophoresis showed that eBrCas12b and eBrCas12b-G had a cleavage delay of about 3 minutes and then completely digested the target sequence, while eBrCas12b-GN almost completely digested the target sequence in 6 minutes. This shows that engineering two key residues N392A / G496A in the eBrCas12b PAM binding domain can delay the cleavage of the target sequence, thereby avoiding interference with the initial isothermal amplification.

[0091] 4. Application of detection system: Construction of pUC19- bla NDM5 plasmid, and the eBrCas12b / eBrCas12b-GN one-tube detection system was applied to the carrier bla NDM The E. coli detection experiment was carried out to verify its actual detection effect. The specific operation is:

[0092] 4.1 Construction of pUC19- bla NDM5 Plasmids:

[0093] 4.1.1 Primer design (Table 11):

[0094] Table 11 Primer sequences

[0095] ;

[0096] 4.1.2 PCR amplification (Table 12):

[0097] Table 12 Reaction system

[0098] ;

[0099] The reaction program was as follows: 98°C for 3 m; 98°C for 10 s, 61.5°C for 20 s, and 72°C for 1 m 30 s, for 30 cycles; and storage at 4°C.

[0100] 4.1.2 Homologous recombination and transformation (Table 13):

[0101] Table 13 Reaction system

[0102] ;

[0103] The above system was placed at 50°C for 30 minutes. After the reaction was completed, it was transformed into DH5α competent cells.

[0104] 4.2 pUC19- bla NDM5 The cells were cultured at 37°C with shaking until the OD 600= 0.5, take 1 mL of bacterial solution for centrifugation enrichment, pipette and mix with 200 μL Nuclease-free Water and dilute to 10 -1 -10 -7 50 μL of each gradient was spread onto ampicillin (100 μg / mL) LB agar plates and repeated three times.

[0105] 4.3 New Delhi Metallo-β-lactamase ( bla NDM ) Special primers (Table 14):

[0106] Table 14 Primer sequences

[0107] ;

[0108] Preparation of 10× LAMP primer mix (Table 15):

[0109] Table 15 Configuration system

[0110] ;

[0111] 4.4 LAMP-Cas12b one-tube system preparation (Table 16):

[0112] Table 16 Reaction system

[0113] ;

[0114] Fluorescence quantitative PCR instrument, select FAM as the fluorescence channel, 65℃, 30s / cycle, 30min.

[0115] 4.5 Fluorescence Curve Analysis

[0116] The results are as follows Figure 2 and Figure 3 As shown, Figure 2 One-tube assay for BrCas12b protein detection in pUC19- bla NDM5 cell, Figure 3 One-tube assay for eBrCas12b-GN protein detection in pUC19- bla NDM5 cells. Figure 2 The detection limit is 20 CFU / μL. Figure 3 The detection limit was 10 CFU / μL, and the sensitivity was improved by two times, indicating that protein engineering of two key residues N392A and G469A in the PAM binding domain of BrCas12b can significantly improve the performance of BrCas12b in the one-tube CRISPR-LAMP assay.

[0117] Therefore, the present invention adopts the above-mentioned detection method and application based on LAMP and CRISPR / Cas, optimizes the traditional LAMP-Cas12b one-tube detection system, and significantly improves the sensitivity and speed of one-tube detection.

[0118] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A modified BrCas12b protein, characterized in that: The wild-type BrCas12b protein is obtained by mutation. The protein sequence of the wild-type BrCas12b protein is shown in SEQ ID NO.20, and the mutation sites are asparagine N at position 392 of the sequence shown in SEQ ID NO.20 is replaced by alanine A and glycine G at position 469 of the sequence shown in SEQ ID NO.20 is replaced by alanine A.

2. The modified BrCas12b protein according to claim 1, wherein: The mutation of the wild-type BrCas12b protein results in delayed target DNA cleavage activity, and the modified BrCas12b protein improves the detection sensitivity of the target DNA when used in conjunction with loop-mediated isothermal amplification (LAMP).

3. A detection method based on LAMP and CRISPR / Cas, characterized in that: The following steps are involved: Step 1, key mutation: The PAM domain of the wild-type BrCas12b protein described in claim 1 is subjected to key amino acid mutations, with the mutation sites being N392A and G469A, to obtain eBrCas12b-GN; Step 2, sgRNA preparation: Prepare Cas12b sgRNA by in vitro transcription and purification; Step 3, cis-cleavage ability determination: evaluate the cis-cleavage ability of eBrCas12b-GN on the target sequence; Step 4, construct the detection system: construct a pUC19- bla NDM5 coli with the plasmid, and the eBrCas12b-GN one-tube detection system was applied to the detection of NDM-resistant Escherichia coli.

4. A kit based on LAMP and CRISPR / Cas detection, characterized by: A BrCas12b protein comprising the modification described in claim 1.

5. Use of the kit according to claim 4 in nucleic acid detection, wherein the purpose of the use is not the diagnosis and treatment of a disease, and is characterized in that: The application method is as follows: the sample is added to the broth and shake-cultured at 37°C for 1-2 hours, the bacteria are enriched by centrifugation, and then the LAMP+eBrCas12b-GN reaction mixture is added. The bacteria are lysed and released at a constant temperature of 65°C. As the target DNA is amplified, the eBrCas12b-GN protein binds to the target DNA under the guidance of sgRNA to form an RNP complex, and its trans-cutting activity is activated to cut the ssDNA probe in the cutting system. The positive sample emits fluorescence under the background of an ultraviolet light source. The eBrCas12b-GN protein is the modified BrCas12b protein described in claim 1.

Images