Two gene editing tool enzymes with large PAM recognition range and application of two gene editing tool enzymes

Abstract

The invention discloses two novel endonuclease Gs12-15 and Gs12-17 of a CRISPR (clustered regularly interspaced short palindromic repeats) / Cas system and an application of the two novel endonuclease Gs12-15 and Specifically, the invention provides two novel guide RNA dependent type endonucleases excavated based on a metagenomics method, and the novel guide RNA dependent type endonucleases have the advantages that the novel guide RNA dependent type endonucleases have the genome editing capability with a wider target site coverage range, the reaction temperature range is wider, and target DNA is cut in a genome in a high-activity and high-specificity manner. Researches find that PAM of Gs12-15 nuclease is preferentially NHHA, and the PAM recognition range of Gs12-17 is NNHV (N is a basic group T / C / G / A, H is a basic group T / C / A, and V is a basic group G / A / C). The invention establishes a nucleic acid visual detection technology based on CRISPR / Gs12-15 and CRISPR / Gs12-17 system mediation, and has a wide application prospect in the fields of genome site-directed modification and nucleic acid detection.

Description

Technical Field

[0001] This invention belongs to the field of genome editing technology, specifically relating to two newly identified endonucleases Gs12-15 and Gs12-17 of the CRISPR / Cas system and their applications. Background Technology

[0002] The CRISPR / Cas system boasts advantages such as high efficiency, low cost, and ease of operation. Its emergence has led to a breakthrough in genome editing technology, with broad application prospects in fields such as medicine and synthetic biology. In 2015, researchers discovered eight novel class 2 type V CRISPR proteins with cleavage activity against double-stranded DNA, termed Cpf1 (later named Cas12a), leading to the development of a new generation of CRISPR genome editing tools.

[0003] The discovery of the "trans-cleavage activity" of the Cas12a family has broadened the application scope of CRISPR technology. Introducing fluorescently modified ssDNA probes into detection systems allows trans-activated Cas12a to non-specifically cleave them, thus the CRISPR / Cas12a system is widely used in nucleic acid detection. Compared with traditional nucleic acid diagnostic techniques, CRISPR / Cas12a-mediated nucleic acid detection has many advantages. For example, combining the trans-cleavage activity of Cas12a with techniques such as isothermal amplification enables real-time detection of viruses. Researchers extended crRNA with phosphate-thiolated ssDNA (PS-DNA), ssDNA, and ssRNA of different lengths, combined with different concentrations of divalent cations and different reporter genes to construct the ENHANCE system. Combining this system with isothermal amplification significantly improved the detection performance of SARS-CoV-2 RNA and increased the specificity of Cas12a.

[0004] Although numerous studies have found that modifying and altering the existing CRISPR / Cas12a system can change its function and achieve certain effects, artificially modified nucleases still have certain limitations. It is still necessary to continuously develop novel Cas12a proteins using bioinformatics techniques, expand the PAM range of Cas12a, and improve the Cas12a family system. Summary of the Invention

[0005] This invention is the first to develop novel endonucleases Gs12-15 and Gs12-17 of the CRISPR / Cas system and the nucleic acid visualization detection technology they mediate.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The endonucleases in the CRISPR / Cas system include the following proteins:

[0008] Ⅰ. Gs12-15 nuclease with the amino acid sequence shown in SEQ ID NO.1; Gs12-17 nuclease with the amino acid sequence shown in SEQ ID NO.2;

[0009] II. Proteins that have more than 80% sequence identity with the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2, and that essentially retain the biological functions derived from the sequence;

[0010] III. Proteins with one or more amino acid substitutions, deletions, or additions compared to the amino acid sequences shown in SEQ ID NO.1 and SEQ ID NO.2, and which substantially retain the biological functions derived from their sequences.

[0011] A fusion protein comprising the aforementioned endonuclease and a polypeptide linked to the N-terminus or C-terminus of the protein.

[0012] The polynucleotide is either a polynucleotide encoding the aforementioned endonuclease or a polynucleotide encoding the aforementioned fusion protein.

[0013] A carrier containing the polynucleotide.

[0014] A visual nucleic acid detection kit includes the aforementioned endonuclease, a single-stranded DNA fluorescence-quenching reporter gene, and a guide RNA that pairs with the target nucleic acid.

[0015] A CRISPR / Cas gene editing system includes the aforementioned endonuclease, or the aforementioned fusion protein, or the aforementioned polynucleotide, or the aforementioned vector, and further includes a homologous repeat sequence capable of binding the aforementioned endonuclease and a guide sequence capable of targeting a target sequence.

[0016] The technical solution of the present invention has the following main beneficial effects:

[0017] 1. This invention provides for the first time two novel new members of the CRISPR / Cas12a system family, Gs12-15 and Gs12-17, discovered by combining metagenomics and experimental methods.

[0018] 2. This invention has found that the endonucleases Gs12-15 and Gs12-17 have the advantage of having a wider range of genome editing capabilities covering target sites compared to known Cas12a nucleases (PAM is TTTV). Attached Figure Description

[0019] Figure 1Amino acid conservation and phylogenetic analysis of guide RNA-dependent endonucleases Gs12-15 and Gs12-17 predicted using metagenomics methods. A. Amino acid sequence similarity analysis of the two new proteins and three known Cas12a proteins; B. CRISPR / Cas12a phylogenetic tree.

[0020] Figure 2 Loci and domains of nucleases Gs12-15 and Gs12-17. A. Locus analysis of novel nucleases; B. Domain analysis of nucleases; C. Comparison of amino acid conservation between novel nucleases and LbCas12a.

[0021] Figure 3 Secondary structure folding and multiple sequence alignment of guide RNA DR sequence. A. Secondary structure of nuclease DR; B. DR sequence alignment of nuclease.

[0022] Figure 4 Identify and compare the PAM recognition characteristics of Gs12-15 and Gs12-17. A. Flowchart of bacterial attenuation experiment; B. Characteristics of Gs12-15 and Gs12-17 in recognizing PAM.

[0023] Figure 5 Verify the in vitro trans-cleavage activity of Gs12-15 and Gs12-17 nucleases for classical PAM. A. Trans-cleavage pattern of Gs12-15 recognizing classical PAM; B. Trans-cleavage pattern of Gs12-17 recognizing classical PAM.

[0024] Figure 6 Cis-cleavage activities of Gs12-15 and Gs12-17 nucleases on non-classical PAM targets. The target is the African swine fever P72 gene amplification fragment. A. Cis-cleavage diagram of Gs12-17 on non-classical PAM; B. Cis-cleavage diagram of LbCas12a on non-classical PAM; C. Cis-cleavage diagram of Gs12-15 on non-classical PAM.

[0025] Figure 7 Trans-cleavage activity of Gs12-15 and Gs12-17 nucleases on non-classical PAM targets. The target is the African swine fever P72 gene amplification fragment. A. Trans-cleavage diagram of Gs12-17 on non-classical PAM; B. Trans-cleavage diagram of Gs12-15 on non-classical PAM.

[0026] Figure 8 Temperature ranges for cis-cleavage activity of two novel nucleases. A. Gel electrophoresis images of cis-cleavage at different temperatures (Gs12-15); B. Gel electrophoresis images of cis-cleavage at different temperatures (Gs12-17).

[0027] Figure 9. Temperature ranges of the trans-cleavage activities of two novel nucleases. A. Diagrams of the trans-cleavage of Gs12-15 under blue light and UV light and the fluorescence histogram; B. Diagrams of the trans-cleavage of Gs12-17 under blue light and UV light and the fluorescence histogram. Detailed implementation manners

[0028] Term explanations

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0030] The Genie scissor endonuclease family, where Genie means elf, representing a bacterial origin, and scissor represents gene scissors, indicating its possible gene editing function. The Chinese name corresponding to the Genie scissor endonuclease is the "Genie Scissor" endonuclease, and the Genie scissor gene editing system represents the gene editing system mediated by the "Genie Scissor" endonuclease, abbreviated as "Genie gene editing".

[0031] The protospacer adjacent motif (PAM) is a short DNA sequence (usually 2-6 base pairs in length). Traditionally, it is considered that PAM is necessary for Cas nuclease cleavage, usually 3-4 nucleotides downstream of the cleavage site. There are many different Cas endonucleases that can be purified from different bacteria, and each enzyme may recognize a different PAM sequence.

[0032] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer.

[0033] Example 1. Mining of two novel Cas12a homologous endonucleases based on bioinformatics strategies

[0034] Based on the bioinformatics identification workflow of a novel guide RNA-dependent endonuclease developed by the inventors, deep mining of bacterial-encoded proteins was performed on massive metagenomic sequencing data from public databases such as the NCBI nr (Non-Redundant Protein Sequence Database) and the Global Microbial Gene Catalogue Database (GMGC). The general analysis workflow was as follows: for all contig sequences in the target database, the minced software was used to search for and locate CRISPR arrays; then, the prodigal software was used to predict proteins expressed near the CRISPR array; redundancy was removed from all predicted proteins using CD-hit software; protein clustering analysis was performed using mega software; and CRISPR-Cas similarity protein identification and classification were performed using hmmer software. Ultimately, two new unknown bacterial proteins were obtained.

[0035] This invention names these newly discovered proteins from different bacteria as Genie scissor (Gs) endonucleases. For ease of subsequent research, and based on bacterial species origin, the inventors have named two new, unknown bacterial proteins Gs12-15 and Gs12-17, following the naming convention of "endonuclease + numerical code". The amino acid sequence of Gs12-15 is shown in SEQ ID NO: 1, and its nucleotide sequence is shown in SEQ ID NO: 3; the amino acid sequence of Gs12-17 is shown in SEQ ID NO: 2, and its nucleotide sequence is shown in SEQ ID NO: 4. First, sequence similarity analysis was performed between the predicted nuclease and three known nucleases. The amino acid homology of Gs12-15 with LbCas12a, FnCas12a, and AsCas12a were 31.59%, 36.04%, and 34.24%, respectively. The amino acid homology of Gs12-157 with LbCas12a, FnCas12a, and AsCas12a were 34.72%, 42.27%, and 37.75%, respectively. Figure 1 A). Phylogenetic analysis revealed that these two new bacterial proteins are located on different CRISPR-Cas12a phylogenetic branches. Figure 1 B) It is speculated that they may be novel RNA-guided endonucleases.

[0036] Furthermore, the inventors analyzed the gene locus of this protein using CRISPRCasFinder software. The results showed that both Gs12-15 and Gs12-17 nucleases possess CRISPR array sequences and Cas4, Cas1, and Cas2 proteins. Figure 2A). Next, the domains of the novel nuclease were compared with those of LbCas12a. HMMER software and the Pfam database were used for comparative analysis to predict the functional domains of the nuclease. The results showed that the novel nuclease and LbCas12a have similar domain characteristics, such as REC, WED, RuvC, and NUC. Figure 2 B). Finally, the predicted Nuc and RuvC domains of the Gs12-15 and Gs12-17 nucleases were compared with the amino acid sequences of LbCas12a. The results showed that the Nuc and RuvC domain sequences of the Gs12-15 and Gs12-17 nucleases differed significantly from those of LbCas12a. Figure 2 C). Therefore, further experiments are urgently needed to determine whether they possess targeted nucleic acid cleavage activity.

[0037] Finally, the inventors predicted and performed multiple sequence alignment of the DR sequence secondary structures of the Gs12-15 and Gs12-17 nucleases using the RNAfold webserver (http: / / rna.tbi.univie.ac.at / cgi-bin / RNAWebSuite / RNAfold.cgi). The results showed that the DR secondary structures of the novel nucleases were similar to those of the known LbCas12a, FnCas12a, and AsCas12a. Figure 3 A), Gs12-15 and Gs12-17 differ from the DR sequences of the three known Cas12a species only at the eleventh base. Figure 3 B), and the eleventh base of both Gs12-15 and Gs12-17 nucleases is adenine (A).

[0038] Example 2. Identification of PAM sequence characteristics of a novel CRISPR / Cas12a nuclease

[0039] First, DNA sequences encoding Gs12-15 and Gs12-17 were synthesized after codon optimization for *E. coli*, and NLS nuclear localization signals were added to their C-termini, respectively. The DNA sequences are shown in SEQ ID NO. 5 and SEQ ID NO. 6. These sequences were then ligated into the pET-28a prokaryotic expression vector and transformed into *E. coli* strain BL21. After identifying positive clones, the cells were induced with IPTG (final concentration 0.5 mM) at 16°C for 16 h, and the target proteins were obtained by affinity chromatography.

[0040] Next, PAM sequences recognized by Gs12-15 and Gs12-17 endonucleases, which exhibit low homology and in vitro target nucleic acid cleavage activity, were identified through bacterial PAM library subtraction experiments. The construction procedure for the randomized mixed PAM vector library was as follows: Synthesizing the DNA oligo sequence 5'-GGCCAGTGAATTCGAGCTCGGTACCCGGGNNNNNNN GAGAAGTCATTT A ATAAGGCCACT GTTAAAAAGCTTGGCGTAATCATGGTCATAGCTGTTT-3', where N is a random deoxyribonucleotide. Using Oligo-F: 5'-GGCCAGTGAATTCGAGCTCGG-3' and Oligo-R: 5'-AAACAGCTATGACCATGATTACGCCAA-3' as upstream and downstream primers, PCR amplification was performed, followed by homologous recombination into the pUC19 vector. After transformation into E. coli, plasmid extraction yielded a random mixed PAM vector library. The guide RNA sequence used was: 5'-AAUUUCUACUAUUGUAGAUU GAGAA GUCAUUUAAUAAGGCCACU -3' (The underlined area is the target recognition sequence).

[0041] Bacterial PAM library reduction experiment: The constructed vector pACYC-Duet-1-Gs12-X-crRNA, co-expressing the predicted two endonucleases (Gs12-15, Gs12-17) and crRNA, was transformed into DE3(BL21) competent cells to prepare stably expressing bacterial strains. Stable-transforming bacterial strains constructed using the expression vector pACYC-Duet-1-Gs12-X without crRNA served as negative controls. 100 ng of PAM library plasmid was electroporated into the stably expressing bacterial strains, and selection was performed using plates with both ampicillin and chloramphenicol antibiotics. After 16 h, the colonies on the plates were scraped off for plasmid extraction. Using 100 ng of extracted plasmid as templates, PCR amplification was performed using library sequencing primers Seq-F: 5'-GGCCAGTGAATTCGAGCTCGG-3' and PAM-Seq-R: 5'-CAATTTCACACAGGAAACAGCTATGACC-3'. After product recovery, the experimental and control groups were subjected to next-generation high-throughput sequencing. The sequencing results were analyzed and displayed using Weblogo 3.0. Figure 4 A).

[0042] The characteristics of PAM sequences recognized by two nucleases were identified: For the 16384 different types of PAM sequences contained in the starting vector library, their frequency of occurrence in the experimental and control groups during high-throughput sequencing was statistically analyzed, and normalized using the total number of PAM sequences in each group. The change in PAM consumption for each sequence was calculated as log2(normalized value of control group / normalized value of experimental group). A value greater than 3.5 was considered a significantly consumed PAM. Weblogo 3.0 was then used to visualize the base frequencies at each position in the significantly consumed PAM sequences. The results are as follows: Figure 4 As shown in B, the Gs12-15 and Gs12-17 nucleases exhibit cleavage activity for some non-classical PAMs, not only for classical PAMs. Gs12-15 recognizes PAMs in the range of NHHA, while Gs12-17 recognizes PAMs in the range of NNHV (N represents T / C / G / A, V represents G / A / C, and H represents T / C / A), which differs from the previously reported PAMs specifically recognized by Cas12a nucleases with a "TTTV" base sequence.

[0043] Example 3. Evaluation of PAM preference characteristics of novel Gs12s nuclease

[0044] This embodiment tests the cleavage activity of two nucleases on double-stranded DNA through in vitro experiments. Guide RNAs paired with the target nucleic acid guide the two nucleases to recognize and bind to the target nucleic acid, thereby activating their cleavage activity and cutting the double-stranded target nucleic acid in the system. The cleavage activity is then identified by observing changes in the size of the target band during agarose gel electrophoresis. In this embodiment, the target double-stranded DNA (dsDNA) is the African swine fever P72 gene. Multiple classic guide RNAs recognizing different PAM sites were designed (Table 1). The sequence of the African swine fever P72 gene is: 5'-CTGTAACGCAGCACAGCTGAACCGTTCTGAAGAAGAAGAAAGTTAATA-3'.Using pmd-18T-P72 plasmid as template, and P72-F:5'-CTGTAACGCAGCACAGCTGA-3' and P72-R:5'-CCATGGTTTATCCCAGGAGT-3' as primers, PCR amplification was performed to obtain P72 double-stranded DNA.

[0045] Table 1. Guide RNA sequences for evaluating endonucleases

[0046]

[0047]

[0048]

[0049] The in vitro trans-cutting reaction was performed using the following system: 600 ng nuclease, 2 μL 10×CutSmart Buffer, 400 ng each of the three classic crRNAs (PAM: TTTG, TTTA, TTTC), 300 ng of target DNA, 1 μM ssDNA-FQ reporter gene (sequence: 5'ROX / GTATCCAGTGCG / 3'BHQ2, the same applies below), and DEPC-treated sterile water to a total volume of 20 μL. The nuclease, 10×CutSmart Buffer, ssDNA-FQ reporter gene, and DEPC-treated sterile water were mixed thoroughly and divided into multiple equal systems. Different classic crRNAs were added to each system. For each classic crRNA, a double-stranded target experimental group, a positive experimental group, and a negative control group were set up. The ASFV P72 gene was selected as the target double-stranded DNA. The target of the positive experimental group was the ssDNA corresponding to the crRNA (i.e., the downstream primer for constructing the crRNA), and the target of the negative control group was DEPC-treated sterile water. The reaction was incubated at 37°C for 15 min, followed by inactivation at 98°C for 15 min. Results were observed using a blue light spectrometer and a gel electrophoresis apparatus.

[0050] The results are as follows Figure 5 As shown, the Gs12-15 and Gs12-17 nuclease experimental groups exhibited significantly higher fluorescence intensity compared to the negative control group, and both demonstrated trans-cleavage activity in recognizing classic PAMs (TTTG, TTTA, TTTC). Therefore, both novel nucleases can be applied in the field of nucleic acid detection.

[0051] To further demonstrate the reliability of the PAM library reduction experiment results, a novel nuclease was tested in vitro for double-stranded DNA cleavage, with LbCas12a serving as the reference group. The in vitro cis-cleavage reaction system consisted of: 2 μL of 10×CutSmart Buffer, 500 ng of nuclease, 400 ng of crRNA, 300 ng of ASFV P72 target amplification product, and finally, DEPC-treated sterile water to a final volume of 20 μL. The 10×CutSmart Buffer, nuclease, ASFV P72 target amplification product, and DEPC-treated sterile water were thoroughly mixed and evenly distributed to each system. The experimental groups received the corresponding amount of crRNA, while the negative control group received no crRNA. The mixtures were incubated at 37°C for 40 min. After the reaction, 1 μL of proteinase K was added to each system, and the mixture was incubated at 55°C for 10 min. A 10 μL sample was taken and detected by 2% agarose gel electrophoresis. Imaging was performed under a gel imaging system to observe the target band cutting of the experimental group and the control group of the novel nuclease with the same reaction time. The gray value was analyzed by ImageJ software to calculate the cutting efficiency.

[0052] The formula for calculating cutting efficiency is as follows:

[0053] In the formula, 'a' represents the wild-type double-stranded DNA band that has not been cleaved by nucleases; 'b' and 'c' represent the bands that have been cleaved by nucleases.

[0054] The results showed that both Gs12-15 and Gs12-17 exhibited certain cis-cleavage activity against non-classical PAMs. Ten crRNAs of the Gs12-17 nuclease showed cis-cleavage activity against dsDNA targets, and also showed high cis-cleavage activity against TTCA, TCTG, TCCG, and CATC non-classical PAMs. Figure 6 A); LbCas12a has 23 crRNAs that exhibit cis-cleavage activity against dsDNA targets, and also shows high cis-cleavage activity against multiple non-classical PAMs such as TCCG and GGCT. Figure 6 B); Gs12-15 nuclease has four crRNAs that exhibit cis-cleavage activity against dsDNA targets, and also shows high cis-cleavage activity against non-classical PAM targets such as TTCA. Figure 6 C). In summary, the cis-cleavage activity of the two novel nucleases is generally weaker than that of LbCas12a, but they still have the ability to recognize non-classical PAM targets.

[0055] To verify whether Gs12-15 and Gs12-17 nucleases have a broader ability to recognize PAM sites in nucleic acid detection, crRNA was designed targeting the PAM site of the conserved sequence of the P72 gene for detection. Guide RNA paired with the target nucleic acid guides the endonuclease to recognize and bind to the target nucleic acid, thereby activating the nuclease's "trans-cleavage" activity against any single-stranded nucleic acid, cleaving the single-stranded DNA fluorescence-quenched reporter gene (ssDNA-FQ) in the reaction system. The trans-cleavage function of the candidate bacterial protein can be further determined by the excitation fluorescence intensity, background noise, and visual color change. The in vitro trans-cleavage reaction used the following system: 500 ng nuclease, 2 μL 10×CutSmart Buffer, 1 μM ssDNA-FQ reporter gene, 400 ng crRNA, 300 ng P72 target, and DEPC-treated sterile water to a total volume of 20 μL. Nuclease, 10×CutSmartBuffer, ssDNA-FQ reporter gene, and DEPC-treated sterile water were mixed thoroughly and divided into multiple systems. Different non-classical crRNAs were added to each system. The experimental group was supplemented with ASFV P72 double-stranded DNA target, while the negative control group was supplemented with DEPC-treated sterile water. The reaction was incubated at 37℃ for 15 min, followed by inactivation at 98℃ for 15 min. Results were observed using a blue light analyzer and a gel electrophoresis apparatus. The trans-digestion results were measured using a microplate reader. The specific steps were as follows: 80 μL of DEPC-treated sterile water was added to each well of the microplate, and the digested sample was added to the reaction wells of the microplate. The mixture was thoroughly mixed to a total sample volume of 100 μL. The fluorescence intensity was measured using a multi-functional microplate reader, and the process was repeated three times. Data were collected, and bar charts were generated using GraphPad Prism 9.0 software.

[0056] The results showed that Gs12-15 and Gs12-17 exhibited trans-cleavage activity against non-classical PAMs, and some non-classical crRNAs had low background, making them suitable for nucleic acid detection. Compared with the negative control group, 21 crRNAs from Gs12-17 showed trans-cleavage activity, and the fluorescence intensity of non-classical PAMs AGTT and AACC was stronger. Figure 7 A); 15 crRNAs from Gs12-15 exhibited trans-cleavage activity. Figure 7 B). In summary, among the two nucleases, Gs12-17 has a wider range of non-classical PAM trans-cleavage activity, but its overall fluorescence intensity is weaker than that of Gs12-15. Gs12-15 has higher application value in nucleic acid detection.

[0057] Example 4. Evaluation of the in vitro cleavage activity temperature range of the novel nuclease

[0058] To evaluate the temperature range of cis-cleavage reactions mediated by a novel nuclease. The dsDNA of the P72 gene was used as the target, and the classic crRNA (with TTTA PAM) was employed. The following reaction system was used: 500 ng nuclease, 400 ng crRNA, 2 μL 10×CutSmart Buffer, 300 ng P72 PCR amplification product, and DEPC-treated sterile water to a final volume of 20 μL. The 10×CutSmart Buffer, nuclease, ASFV P72 target amplification product, and DEPC-treated sterile water were thoroughly mixed and evenly distributed to each system. The experimental group received crRNA, while the negative control group did not. Reactions were performed at 16℃, 25℃, 37℃, 45℃, 55℃, and 60℃ for 40 min each. After the reaction, 1 μL of proteinase K was added, and the reaction was terminated by incubation at 55℃ for 10 min. The predicted differences in target bands between the experimental and control groups were observed by 2% agarose gel electrophoresis under UV illumination.

[0059] The results showed that the cis-cutting temperature range for Gs12-15 was 25℃-45℃. Figure 8 A), the cis-cutting temperature range for Gs12-17 is 16℃-45℃. Figure 8 B). Therefore, Gs12-17 has a wide temperature range for cis-cutting activity.

[0060] To evaluate the temperature range of trans-cutting in a novel nuclease-mediated nucleic acid detection technology. The dsDNA of the P72 gene was used as the target, and the crRNA was the classic crRNA (with TTTA as the PAM). The reaction system was as follows: 500 ng nuclease, 400 ng crRNA, 2 μL 10×CutSmart Buffer, 1 μM ssDNA-FQ reporter gene, 300 ng P72 PCR amplification product, and DEPC-treated sterile water to a final volume of 20 μL. The 10×CutSmart Buffer, nuclease, crRNA, ssDNA-FQ reporter gene, and DEPC-treated sterile water were mixed thoroughly and evenly distributed to each system. The experimental group received the P72 target amplification product, while the negative control group did not. Reactions were performed in metal baths at 16℃, 25℃, 37℃, 45℃, 50℃, and 60℃ for 15 min, followed by inactivation at 98℃ for 15 min. Fluorescence intensity was observed under blue and ultraviolet light, and the excitation of fluorescence in the reaction products was measured using a microplate reader. This was repeated three times. Data were collected and bar charts were generated using GraphPad Prism 9.0 software.

[0061] The results showed that the active temperature range for Gs12-15 trans-cutting was 25℃-45℃. Figure 9A), the trans-cutting activity temperature range of Gs12-17 is 37℃-45℃. Figure 9 C), and the background levels of both negative control groups were low ( Figure 9 (B, 9D). Based on the exploration of the trans-cleavage temperature range, the novel nuclease can exert trans-cleavage activity in different temperature ranges, providing a wider temperature range for nucleic acid detection.

Claims

1. An endonuclease in a CRISPR / Cas system, characterized in that The proteins include: I. Gs12-15 nuclease of the amino acid sequence shown in SEQ ID NO. 1; Gs12-17 nuclease of the amino acid sequence shown in SEQ ID NO. 2; II. A protein having more than 80% sequence identity compared with the amino acid sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2, and substantially retaining the biological function derived from the sequence; III. A protein having substitution, deletion or addition of one or more amino acids compared with the amino acid sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2, and substantially retaining the biological function derived from the sequence.

2. A fusion protein, characterized in that, The protein of claim 1 and other modified moieties are included.

3. An isolated polynucleotide, characterized in that, The polynucleotide is a polynucleotide encoding the endonuclease of claim 1, or a polynucleotide encoding the fusion protein of claim 2.

4. Vector, characterized in that, The vector comprises the polynucleotide of claim 3.

5. A CRISPR / Cas gene editing system, characterized in that, The endonuclease of claim 1, the fusion protein of claim 2, the polynucleotide of claim 3, or the vector of claim 4 are included.

6. The CRISPR / Cas gene editing system of claim 5, wherein, The direct repeat sequence capable of binding the endonuclease of claim 1 and the guide sequence capable of targeting the target sequence are also included.

7. Use of the endonuclease of claim 1, the fusion protein of claim 2, the polynucleotide of claim 3, or the vector of claim 4 in nucleic acid detection.

8. A visual nucleic acid detection kit, characterized by, The endonuclease of claim 1, the single-stranded DNA fluorescence-quenching reporter gene, and the guide RNA paired with the target nucleic acid are included.

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