LbCas12a variant and application thereof in nucleic acid detection

Abstract

The invention relates to the technical field of biology, in particular to an LbCas12a variant and application thereof in nucleic acid detection. According to the invention, a series of LbCas12a mutants with differentiated enzyme digestion activities are obtained by modifying key amino acid sites related to substrate recognition and conformation activation in LbCas12a protein. Compared with an existing method, a one-tube nucleic acid detection system based on the mutant has better system stability, sensitivity, specificity and universality, and high-sensitivity one-tube nucleic acid detection can be achieved under the condition that exogenous addition is not needed, and special consumables or complex equipment are not needed. The mutant set provides a more advantageous tool for the on-site application of a CRISPR (clustered regularly interspaced short palindromic repeats) detection system.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to the LbCas12a variant and its application in nucleic acid detection. Background Technology

[0002] Molecular detection technology based on the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system has shown broad application potential in the field of molecular diagnostics due to its high specificity, programmability, and significant signal amplification effect. In recent years, CRISPR has been used to construct various sensitive, rapid, and visualized detection platforms, demonstrating unique advantages in early screening of infectious diseases, pathogen environmental monitoring, and genetic variation analysis. With the optimization of related reaction systems and the development of portable reading devices, CRISPR detection technology is gradually transitioning from laboratory research to practical applications and is considered a next-generation molecular diagnostic technology.

[0003] However, traditional CRISPR assays typically employ a two-step process: first, the target nucleic acid is pre-amplified in a separate reaction tube, and then the amplified product is opened and transferred to another reaction system for CRISPR detection. This opening operation easily leads to the amplified product escaping in the form of an aerosol, resulting in serious false positive problems and limiting its widespread use in clinical diagnosis, environmental pathogen monitoring, and other scenarios.

[0004] To address this issue, the "one-pot" detection strategy proposed in recent years aims to simultaneously achieve nucleic acid amplification and CRISPR detection within a single sealed reaction tube, thus avoiding the contamination risks associated with opening the tube. However, the one-pot strategy still faces significant challenges in practical applications. The most critical contradiction lies in the strong Cas protein cleavage activity, which can easily inhibit the amplification reaction or prematurely consume the template, leading to decreased detection sensitivity and prolonged reaction time. Existing technologies have attempted to address these contradictions from two directions: physical isolation or molecular regulation. For example, spatial separation between the amplification system and the CRISPR detection system can be achieved in the early stages of the reaction through special container structures, viscosity regulation, or solid-phase localization, or the activity of the Cas protein can be adjusted through suboptimal PAM sequences and photocontrol elements. Although these methods alleviate the interference between amplification and detection to some extent, they generally suffer from problems such as reliance on special consumables or equipment, high system complexity, and insufficient stability and sensitivity, making it difficult to meet the requirements of versatility, sensitivity, and ease of operation for rapid on-site detection. Therefore, it is still necessary to optimize and realize one-pot detection at a more fundamental level. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide the LbCas12a variant and its application in nucleic acid detection.

[0006] The LbCas12a variants provided by this invention include mutations that affect conformational activation and mutations that affect substrate recognition.

[0007] The mutation affecting conformational activation is located at position 930 of the wild-type LbCas12a protein or its equivalent site. The mutation includes changing the G residue at position 930 of the wild-type LbCas12a protein to a C residue, D residue, T residue, or W residue.

[0008] The mutation affecting substrate recognition is located at position 380, 386, or 390 of the wild-type LbCas12a protein, or its equivalent site. The mutation includes changing an amino acid residue at position 380, 386, or 390 of the wild-type LbCas12a protein, or its equivalent site, to an A residue.

[0009] Specifically, the LbCas12a variant provided by this invention has a mutation at least at position 930 of the wild-type LbCas12a protein and at the following positions: position 380, position 386, or position 390.

[0010] In some embodiments, the mutation site is G930D or G930D and K380A.

[0011] In some embodiments, the amino acid sequence of the wild-type LbCas12a protein is shown in SEQ ID NO:1.

[0012] Specifically, the variant includes the G930D mutation in the amino acid sequence of the wild-type LbCas12a protein. Alternatively, the mutant includes both the G930D and K380A mutations in the amino acid sequence of the wild-type LbCas12a protein.

[0013] This invention achieves controllable regulation of the cleavage activity of the LbCas12a protein by mutating key conserved sites, thereby significantly improving the overall performance of the single-tube detection system. Compared to other single-point mutations, LbCas12a-G930C, LbCas12a-G930D, LbCas12a-G930T, and LbCas12a-G930W exhibit stronger fluorescence signals and thus higher sensitivity. Among the variants containing double mutation sites, LbCas12a-G930D / K380A, LbCas12a-G930D / R386A, and LbCas12a-G930D / K390A retain stable detection performance. The LbCas12a-G930D / K380A double mutant, in particular, still exhibits a significant fluorescence signal at a target concentration of 1 aM, with detection sensitivity several orders of magnitude higher than the wild type.

[0014] The present invention also provides a nucleic acid that encodes the LbCas12a variant as described above.

[0015] The nucleic acid described in this invention is the coding sequence of the LbCas12a variant as described above. It can be a sequence that has undergone codon optimization and encodes the variant as described above. This invention does not limit the specific sequence.

[0016] The present invention also provides an expression box that includes a promoter and a nucleic acid as described above.

[0017] The expression frame described in this invention includes a promoter, which includes, but is not limited to, the T7 promoter, CMV promoter, EF1α promoter, lac promoter, and tet-on promoter. Optionally, the expression frame may also include a terminator, which includes, but is not limited to, the T7 terminator, SV40 terminator, and bGH terminator. Furthermore, the expression frame may also include regulatory elements such as enhancers and Kozak sequences.

[0018] The present invention also provides plasmid vectors comprising nucleic acids as described above, or comprising expression cassettes as described above.

[0019] The plasmid vectors described in this invention are used for the storage, expression, or amplification of the variants and / or nucleic acids as described above. They can be pET series vectors, or commonly used cloning or expression vectors such as pUC series, pGEX series, and pEGFP series.

[0020] The present invention also provides a host cell containing the plasmid vector as described above, or whose genome integrates the nucleic acid as described above or the expression cassette as described above.

[0021] The host cell described in this invention is used for the storage, expression, or amplification of the variants and / or nucleic acids as described above, and can be a prokaryotic cell or a eukaryotic cell. Prokaryotic cells can be *Escherichia coli* (e.g., *Rosetta*, BL21, DH5α, etc.), and eukaryotic cells can be yeast cells, insect cells, or mammalian cells (e.g., HEK293, CHO, etc.). By introducing a plasmid vector containing the nucleic acid or expression cassette of this invention into the host cell, efficient expression and purification of the LbCas12a variant can be achieved.

[0022] This invention also provides the application of the LbCas12a variant, the nucleic acid, the expression cassette, and the plasmid vector as described above in the preparation of nucleic acid detection reagents.

[0023] In this invention, the nucleic acid detection is based on RPA reaction and CRISPR system. RPA reaction can rapidly amplify target nucleic acid sequence under isothermal conditions. As mentioned above, after the LbCas12a variant recognizes and binds to the specific target nucleic acid amplified by RPA, its non-specific cleavage activity is activated, thereby cleaving the oligonucleotide chain with fluorescent and quenching groups labeled at both ends, resulting in the separation of fluorescent and quenching groups and the generation of a detectable fluorescent signal, thereby realizing the qualitative or quantitative detection of target nucleic acid.

[0024] The present invention also provides a nucleic acid detection reagent, comprising: RPA reagent, amplification primers, crRNA, the LbCas12a variant as described above, and ssDNA reporter molecule.

[0025] The RPA reagent involved in this invention is commercially available. The core components of the RPA reagent are recombinase, polymerase, ssDNA binding protein, deoxynucleoside triphosphate (dNTP), buffer, and DEPC water.

[0026] As a feasible example, each 30 μL reaction system includes: 24 μL RPA reagent, 20 nM LbCas12a or its mutant, 30 nM crRNA, 400 nM FQ ssDNA reporter molecule (FAM-CCCCCCCC-BHQ), 3 μL buffer B from the RPA amplification kit, and 3 μL of the sample to be tested.

[0027] In this invention, buffer A comprises 6.5% polyethylene glycol, 100 mM Tris-HCl, and 1.5% mannitol. Buffer B comprises 21 mM magnesium acetate.

[0028] The RPA mixture was prepared by resuspending one tube of RPA reagent lyophilized powder in 30 μL of buffer A, adding 2 μL each of 10 μM RPA amplification primers, and then adding 28 μL of DEPC-treated water.

[0029] In this invention, the amplification primers include an upstream primer and a downstream primer, both of which are 30-35 bp in length. In a specific embodiment, the CG% in the amplification primer sequence is 30%-70%.

[0030] The crRNA described in this invention is a classic crRNA, wherein the PAM site is TTTV. In specific embodiments, the sequence of the crRNA is shown in SEQ ID NO:15 or 16.

[0031] The present invention also provides a nucleic acid detection method, which includes: mixing the sample to be tested with the detection reagent as described above, incubating and then detecting the fluorescence intensity.

[0032] In this invention, the incubation temperature is 35~40℃ and the incubation time is 30~65 min. In a specific embodiment, the incubation temperature is 37℃ and the incubation time is 30 min or 62.5 min.

[0033] This invention modifies key amino acid sites in the LbCas12a protein related to substrate recognition and conformational activation, resulting in a series of LbCas12a mutants with differentiated enzymatic activity. The one-tube nucleic acid detection system based on these mutants exhibits better system stability, sensitivity, specificity, and universality compared to existing methods, enabling highly sensitive one-tube nucleic acid detection without the need for exogenous additives, special consumables, or complex equipment. This mutant set provides a more advantageous tool for the field application of CRISPR detection systems. Attached Figure Description

[0034] Figure 1 Agarose gel electrophoresis analysis of plasmid cleavage products from LbCas12a-G930 mutant site;

[0035] Figure 2 Fluorescence curve of cis-cleavage activity of LbCas12a-G930 site mutant against labeled double-stranded DNA;

[0036] Figure 3 Fluorescence curves of one-tube nucleic acid detection of the f3l gene of 1 fM Mpox for each mutant at the LbCas12a-G930 site;

[0037] Figure 4 Fluorescence curve of a single-tube nucleic acid detection of the f3l gene of Mpox by a partial mutant of the LbCas12a-G930 site;

[0038] Figure 5Fluorescence curve of f3l gene nucleic acid detection in one tube of Mpox for LbCas12a-G930D mutant;

[0039] Figure 6 The detection limit of the P1 gene nucleic acid detection of LbCas12a partial mutants for Mycoplasma pneumoniae in a single tube;

[0040] Figure 7 The specificity of the LbCas12a-G930D / K380A single-tube nucleic acid detection system; where A represents the specificity of the single-tube nucleic acid detection system for the monkeypox virus f3l gene; and B represents the specificity of the single-tube nucleic acid detection system for the Mycoplasma pneumoniae P1 gene.

[0041] Figure 8 qPCR and LbCas12a-G930D / K380A were used to detect different concentrations of monkeypox pseudovirus in saliva samples. The threshold was set as three times the mean of the fluorescence signal of the negative control plus its standard deviation. Fluorescence signals above the threshold were considered positive, and those below the threshold were considered negative. Detailed Implementation

[0042] This invention provides the LbCas12a variant and its application in nucleic acid detection. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0043] Unless otherwise defined in this invention, the scientific and technical terms associated with this invention shall have the meanings understood by one of ordinary skill in the art.

[0044] The terms “comprising,” “including,” and “having” are used interchangeably to indicate the inclusiveness of a scheme, meaning that the scheme may contain elements other than those listed. It should also be understood that the use of “comprising,” “including,” and “having” herein also provides for schemes “consisting of…”.

[0045] The term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can mean: A exists alone, A and B exist simultaneously, or B exists alone. A and B can be singular or plural.

[0046] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items.

[0047] The numerical ranges and parameters involved in this invention have been presented as precisely as possible in the specific embodiments. However, any numerical value inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all numerical ranges or specific data used in this disclosure may have a reasonable deviation within a certain range, such as ±10%, ±5%, ±1%, or ±0.5%.

[0048] All test materials used in this invention are common commercially available products. The RPA reagent is from Anpu Future (Changzhou) Biotechnology Co., Ltd., and its product name is DNA Isothermal Rapid Amplification Kit.

[0049] The amino acid and nucleic acid sequences involved in the embodiments include:

[0050] The amino acid sequence of wild-type LbCas12a:

[0051]

[0052] Table 1 Primers and crRNA sequences

[0053]

[0054] It should be understood that in the various embodiments of this application, the sequence numbers of the above processes do not imply the order of execution. Some or all steps can be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application. The present invention is further illustrated below with reference to embodiments:

[0055] Example 1: Screening of LbCas12a mutants

[0056] First, based on protein sequence conservation analysis and structural feature comparison, a highly conserved key amino acid site, G930, on LbCas12a was identified (sequence number as SEQ ID NO: 1). Then, saturation mutagenesis was performed on this site to construct and express mutants containing 19 amino acid substitutions (G930A, G930C, G930D, G930E, G930F, G930H, G930I, G930K, G930L, G930M, G930N, G930P, G930Q, G930R, G930S, G930T, G930V, G930W, and G930Y). The catalytic activity of these mutants was systematically characterized using in vitro fluorescence cleavage assays and plasmid substrate cleavage assays. The experimental results showed that different amino acid substitutions led to significant differences in the specific cleavage activity of Cas12a, indicating that this site plays an important regulatory role in the Cas12a catalytic process.

[0057] Example 2: LbCas12a mutant used for one-tube monkeypox virus nucleic acid detection

[0058] The nucleic acid detection system in this embodiment includes polymerase, deoxynucleoside triphosphates (dNTPs), primers for amplifying target nucleic acids, LbCas12a mutant, crRNA, and single-stranded oligonucleotide probes labeled with fluorescent groups and quenchers at both ends. This system was used to detect the Mpox gene of monkeypox virus.

[0059] LbCas12a and its various mutants were expressed and purified as follows: Gene fragments encoding LbCas12a and its various mutants were cloned into a pET-based expression vector containing a C-terminal 6His tag. E. coli strain Rosetta, transformed with the recombinant plasmid, was cultured to OD600 = 0.8, then incubated with 0.2 mM isopropyl β-D-1-thiogalactoside (IPTG) at 16 °C for 16 h. Proteins were isolated from cell lysates using Ni-NTA resin and eluted with buffer 1 (40 mM Tris-HCl, 500 mM NaCl, 5% glycerol and 500 mM imidazole, pH = 7.5), followed by buffer 2 (20 mM Tris-HCl, 200 mM NaCl, 5% glycerol, pH = 7.5). Glycerol was then added to a final concentration of 20%, and the mixture was stored at -80 °C.

[0060] The nucleic acid amplification method used in this embodiment is RPA, and the relevant reagents are from Amp-Future Biotech (Changzhou) Co., Ltd. (China). However, the one-tube nucleic acid detection method based on the LbCas12a mutant can be combined with other nucleic acid amplification methods, and is not limited to RPA. The RPA amplification primer sequences used are shown in SEQ ID NO: 4-5.

[0061] In this embodiment, the single-stranded oligonucleotide probe labeled with fluorescent groups and quenchers at both ends is a single-stranded DNA with the sequence 5'-CCCCCCCC-3', wherein the 5' end is labeled with the fluorescent group FAM and the 3' end is labeled with the fluorescent group BHQ1.

[0062] Nucleic acid testing was performed according to the following steps: Following the manufacturer's instructions, one tube of lyophilized RPA reagent powder was resuspended in 30 μL of buffer A, and 2 μL each of 10 μM RPA amplification primers and 28 L of DEPC-treated water were added to form an RPA mixture. Each detection reaction system contained 24 μL of the RPA mixture, 20 nM LbCas12a or its mutant, 30 nM crRNA, 400 nM FQ ssDNA reporter molecule (FAM-CCCCCCCC-BHQ), and 3 μL of buffer B from the RPA amplification kit. 3 μL of the sample to be tested was added to the obtained reaction system, and the reaction system was then placed in a QuantStudio™ 1 real-time quantitative PCR instrument (Applied Biosystems, Thermo Fisher Scientific, USA) and read at 37 °C. It is worth noting that although a real-time quantitative PCR instrument was used to read the test results in this example, the method of this invention is also compatible with other signal output methods, such as flow chromatography strips, small fluorescence reading devices, etc.

[0063] The above-described detection system was used to detect serially diluted monkeypox virus nucleic acid. LbCas12a variants included: LbCas12a-G930C, LbCas12a-G930D, LbCas12a-G930T, and LbCas12a-G930W. Experimental results showed that rapid one-tube nucleic acid detection could be achieved using LbCas12a mutants, with results output in approximately 20-30 minutes. The detection limits for the mutants LbCas12a-G930C, LbCas12a-G930T, LbCas12a-G930W, and LbCas12a-G930D / R386A were 100 aM (60 cp / µL), while the detection limit for the mutant LbCas12a-G930D was 10 aM (6 cp / µL).

[0064] Further optimization was performed on the best-performing LbCas12a-G930D mutant. Mutations were performed again at sites affecting DNA recognition, such as K380, R386, and K390, to construct a series of double mutants (including LbCas12a-K380A, LbCas12a-R386A, LbCas12a-K390A, LbCas12a-G930D / K380A, LbCas12a-G930D / R386A, and LbCas12a-G930D / K390A). The above detection system was used to detect serially diluted monkeypox virus nucleic acid. The results showed that the detection limit for the LbCas12a-G930D / K380A mutant was 1 aM (0.6 cp / µL), comparable to qPCR, sufficient to provide accurate detection results. Figure 3 , Figure 4 , Figure 5 The above detection system was used to detect nucleic acids from the novel coronavirus, Pseudomonas aeruginosa, Shewanella putrefactiveis, Mycoplasma pneumoniae, Helicobacter pylori, influenza A virus, Aeromonas hydrophila, and adenovirus. All results were negative, indicating that the detection method has high specificity. Figure 7 ).

[0065] Example 3: LbCas12a mutant used for one-tube mycoplasma pneumoniae nucleic acid detection

[0066] The nucleic acid detection system in this embodiment includes polymerase, deoxynucleoside triphosphates (dNTPs), primers for amplifying target nucleic acids, LbCas12a mutant, crRNA, and single-stranded oligonucleotide probes labeled with fluorescent groups and quenchers at both ends. This system was used to detect the P1 gene of Mycoplasma pneumoniae.

[0067] LbCas12a and its various mutants were expressed and purified as follows: Gene fragments encoding LbCas12a and its various mutants were cloned into a pET-based expression vector containing a C-terminal 6His tag. E. coli strain Rosetta, transformed with the recombinant plasmid, was cultured to OD600 = 0.8, then incubated with 0.2 mM isopropyl β-D-1-thiogalactoside (IPTG) at 16 °C for 16 h. Proteins were isolated from cell lysates using Ni-NTA resin and eluted with buffer 1 (40 mM Tris-HCl, 500 mM NaCl, 5% glycerol and 500 mM imidazole, pH = 7.5), followed by buffer 2 (20 mM Tris-HCl, 200 mM NaCl, 5% glycerol, pH = 7.5). Glycerol was then added to a final concentration of 20%, and the mixture was stored at -80 °C.

[0068] The nucleic acid amplification method used in this embodiment is RPA, and the relevant reagents are from Weifang Amp-Future Biotech (Changzhou) Co., Ltd. (China). However, the one-tube nucleic acid detection method based on the LbCas12a mutant can be combined with other nucleic acid amplification methods, and is not limited to RPA. The RPA amplification primer sequences used are shown in SEQ ID NO: 10-11.

[0069] In this embodiment, the single-stranded oligonucleotide probe labeled with fluorescent groups and quenchers at both ends is a single-stranded DNA with the sequence 5'-CCCCCCCC-3', wherein the 5' end is labeled with the fluorescent group FAM and the 3' end is labeled with the fluorescent group BHQ1.

[0070] Nucleic acid detection was performed according to the following steps: Following the manufacturer's instructions, one tube of lyophilized RPA reagent powder was resuspended in 29.4 μL of buffer A, and 2 μL each of 10 μM RPA amplification primers and 14.1 L of DEPC-treated water were added to form an RPA mixture. Each detection reaction system contained 18 μL of the RPA mixture, 20 nM LbCas12a or its mutant, 30 nM crRNA, 400 nM FQ ssDNA reporter molecule (FAM-CCCCCCCC-BHQ), and 2 μL of buffer B from the RPA amplification kit. 3 μL of the sample to be tested was added to the obtained reaction system, and the reaction system was then placed in a QuantStudio™ 1 real-time quantitative PCR instrument (Applied Biosystems, Thermo Fisher Scientific, USA) and read at 37 °C. It is worth noting that although a real-time quantitative PCR instrument was used to read the test results in this example, the method of this invention is also compatible with other signal output methods, such as flow chromatography strips, small fluorescence reading devices, etc.

[0071] The above detection system was used to detect serially diluted Mycoplasma pneumoniae nucleic acid. Experimental results showed that the LbCas12a mutant can achieve rapid one-tube nucleic acid detection, with results output in approximately 20-30 minutes. The detection limits of the LbCas12a-G930D and LbCas12a-G930D / K380A mutant detection systems were 1 aM (0.6 cp / µL), comparable to qPCR, sufficient to provide accurate detection results. Figure 6 The above detection system was used to detect nucleic acids from the novel coronavirus, Pseudomonas aeruginosa, Shewanella putrefactiveis, Helicobacter pylori, influenza A virus, Aeromonas hydrophila, and adenovirus. All results were negative, indicating that the detection method has high specificity. Figure 7 ).

[0072] To verify the detection capability of the method of the present invention on monkeypox virus pharyngeal swab samples, a simulated clinical sample was constructed for detection. Pharyngeal swab samples were placed in nucleic acid lysis buffer and thoroughly mixed to release the nucleic acid from the sample. Then, monkeypox virus pseudovirus was added to the lysed sample and mixed thoroughly to obtain a simulated monkeypox virus pharyngeal swab sample for testing the detection method. qPCR and LbCas12a-G930D / K380A were used to detect different concentrations of monkeypox virus in saliva samples. For the detection system based on LbCas12a-G930D / K380A, the threshold was set to three times the mean plus standard deviation of the negative control fluorescence signal; fluorescence signals above this threshold were considered positive, and signals below this threshold were considered negative. Results are as follows: Figure 8 The results showed that the bCas12a-G930D / K380A-mediated CRISPR-Dx detection method was highly consistent with the qPCR method. In the detection of simulated monkeypox virus throat swab samples, the CRISPR-Dx method had a sensitivity of 97.6% and a specificity of 100%, with only one false negative and no false positives. Furthermore, in samples corresponding to different qPCR Ct values, the fluorescence intensity (FL Intensity) detected by CRISPR-Dx showed a good correlation with the qPCR Ct value; the lower the Ct value (the higher the viral nucleic acid concentration), the stronger the detected fluorescence signal, indicating that this method can reflect the differences between samples with different viral loads within a certain range.

[0073] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A variant of LbCas12a with a mutation at least at position 930 of the wild-type LbCas12a protein and at position 380, 386 or 390.

2. The LbCas12a variant according to claim 1, characterized in that, The mutation site is G930D, or a combination of G930D and K380A.

3. The LbCas12a variant according to claim 1 or 2, characterized in that, The amino acid sequence of the wild-type LbCas12a protein is shown in SEQ ID NO:

1.

4. A nucleic acid encoding the LbCas12a variant as described in any one of claims 1 to 3.

5. An expression box comprising a promoter and the nucleic acid of claim 4.

6. A plasmid vector comprising the nucleic acid of claim 4, or comprising the expression cassette of claim 5.

7. A host cell containing the plasmid vector of claim 6, or having the nucleic acid of claim 4 or the expression cassette of claim 5 integrated into its genome.

8. The use of the LbCas12a variant according to any one of claims 1 to 3, the nucleic acid according to claim 4, the expression cassette according to claim 5, and the plasmid vector according to claim 6 in the preparation of nucleic acid detection reagents.

9. Nucleic acid testing reagents, including: RPA reagent, amplification primers, crRNA, the LbCas12a variant and ssDNA reporter molecule as described in any one of claims 1 to 3.

10. Nucleic acid detection methods, including: The sample to be tested is mixed with the nucleic acid detection reagent according to claim 9, and the fluorescence intensity is detected after incubation.

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