CN116410955B - Two novel endonucleases and application thereof in nucleic acid detection - Google Patents
Two novel endonucleases and application thereof in nucleic acid detection Download PDFInfo
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Abstract
The invention discloses novel endonucleases of two CRISPR/Cas systems and application thereof, in particular to novel CRISPR/Cas12a system family novel members Gs12-3 and Gs12-5 which are excavated by combining metagenomics and experimental means for the first time, especially the newly discovered Gs12-3 has the genome editing capability of covering a wider range of target sites compared with the known Cas12a protein, and a CRISPR/Gs12-3 system-based nucleic acid visualization detection technology is also established.
Description
Technical Field
The invention belongs to the technical field of genome editing, and particularly relates to endonucleases Gs12-3 and Gs12-5 of a newly identified CRISPR/Cas system and application thereof.
Background
CRISPR (Clustered regularly interspaced short palindromic repeats) is a repetitive sequence within the genome of prokaryotes, an acquired immune system that is resistant to invasion by archaea and bacteria by foreign genetic material such as viruses. Some bacteria, after being invaded by a virus, are able to store a small piece of the viral gene in their DNA, a storage space called CRISPR. When the virus invades again, the bacteria can recognize the virus according to the written fragments, and cut off the DNA of the virus to disable the virus.
CRISPR/Cas9 systems are the most commonly used type II CRISPR systems, and the target site for Cas9 binding must contain a proto-spacer adjacent motif (PAM) that is recognized by protein-DNA interactions prior to single guide RNA (sgRNA) binding. The PAM requirement has great influence on realizing accurate editing, and cannot be subjected to accurate gene editing when no proper PAM exists, so that the PAM recognition sequence is the SpCas9 nuclease with high activity of NGG, and the PAM recognition sequence is applied to gene editing of eukaryotes at the earliest, and besides, the V-type PAM recognition sequence is the TTTN Cas12a protein. Cas12a has several advantages over Cas9, such as shorter guide RNAs, easier delivery into cells; the sticky tail end is generated after cutting, which is more beneficial to accurate identification and editing of genome; the cutting sites are far away from the recognition sites, so that the aim of continuous and repeated editing can be fulfilled. However, most mammalian genes are GC-rich, meaning that it is easy to find the necessary GG to localize Cas9 to one particular location. In contrast, it is difficult to find the necessary TT to localize Cas12 to one specific location, which limits the applicability of Cas12 precise gene editing methods. The existing researches show that diversified CRISPR-Cas systems exist in about 40% and 90% sequenced bacterial and archaea genomes, but compared with wide-range gene resources, the CRISPR-Cas members which are identified at present are very limited, new CRISPR-Cas12 members are further mined, and the new genome editing system which is simple, efficient, accurate and wide in application range is always an important direction of the efforts of researchers.
Cas12 has a unique attribute that is suitable for another application beyond genome editing: nonspecific cleavage of single-stranded DNA. This ability allows cleavage of specific single stranded DNA probes (fluorescent and quenching labels) for nucleic acid detection. Amplified double-stranded DNA can be used for cutting double-stranded template DNA and fluorescent labeled probes by Cas12a under the guidance of crRNA, fluorescent signals are released after the probes are hydrolyzed, and whether the DNA template exists or not can be known by detecting the fluorescent signals. The nucleic acid visual detection technology based on the Cas12a protein is the most promising diagnostic means at present, and has the advantages of simple and convenient operation, short time consumption, low price and the like.
Porcine circovirus type 2 (Porcine circovirus type, pcv 2) is one of the major pathogens that severely threatens the global pig farming industry. Infection with PCV2 not only causes various swine diseases, but also induces sustained suppression of host immunity, which is extremely susceptible to co-infection with other pathogens to cause serious conditions. At present, antibiotics have little therapeutic effect on the antibiotics, so early detection of viral nucleic acid is very critical for epidemic control. The existing detection methods comprise conventional fluorescent quantitative PCR, a loop-mediated isothermal amplification (LAMP) technology for rapid nucleic acid detection, a Recombinase Polymerase Amplification (RPA) method and the like, but the conventional detection methods generally require expensive instruments, are complex in operation and high in cost, and the isothermal amplification method is easy to cause false positive in the high-speed amplification process. The CRISPR/Cas12a system adds specific cleavage detection on the basis of a constant-temperature amplification method, so that the sensitivity of the detection method can be improved and false positives can be avoided. Thus, a CRISPR/Cas12a system-mediated low-cost, onsite PCV2 nucleic acid detection method would be beneficial for the prevention and control of this disease.
Therefore, there is still a need in the art to find a novel CRISPR/Cas gene editing system with high editing activity, simple PAM sequence, wide genome coverage and high specificity, and simultaneously to implement a method for rapidly detecting PCV 2-related diseases by using the LAMP technology and the novel CRISPR/Cas gene editing system technology.
Disclosure of Invention
In order to solve the problems, two novel endonucleases Gs12-3 and Gs12-5 of a CRISPR/Cas system are developed for the first time, and a nucleic acid visualization detection technology based on Gs12-3 protein mediation is also established.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
endonucleases in a CRISPR/Cas system, comprising the following proteins:
I. gs12-3 protein with an amino acid sequence shown in SEQ ID NO. 1;
II. Gs12-5 protein with an amino acid sequence shown in SEQ ID NO. 3;
III, proteins having more than 80% sequence identity compared to the amino acid sequence shown in SEQ ID NO.1 or 3, and substantially retaining the biological function of their derived sequence;
IV, a protein having one or more amino acid substitutions, deletions or additions as compared to the amino acid sequence shown in SEQ ID NO.1 or 3, and substantially retains its biological function derived from the sequence.
A fusion protein comprising the endonuclease described above, and a polypeptide linked to the N-terminus or the C-terminus of the protein.
A polynucleotide or a vector containing the polynucleotide, wherein the polynucleotide is a polynucleotide encoding the endonuclease or a polynucleotide encoding the fusion protein.
A visual nucleic acid detection kit comprises the endonuclease (preferably Gs12-3 protein), a single-stranded DNA fluorescence-quenching reporter gene and a guide RNA paired with target nucleic acid.
A kit for visually detecting porcine circovirus type 2 comprises LAMP amplification primers, the endonuclease (preferably Gs12-3 protein) described above, a single-stranded DNA fluorescent-quenching reporter gene, and a guide RNA paired with a target nucleic acid. Preferably, the LAMP amplification primers have the sequence shown in SEQ ID NOS.7-11 and the guide RNA has the sequence AAUUUCUACUAUUGUAGAUUUAGUCUCAGCCACAG CUGAU.
The technical scheme of the invention has the following main beneficial effects:
1. the invention provides two novel CRISPR/Cas12a system family new members Gs12-3 and Gs12-5 which are excavated by combining metagenomics and experimental means for the first time.
2. The invention discovers that endonuclease Gs12-3 has the advantage of wider genome editing capability covering a target site range compared with the known Cas12a protein (PAM is TTTV).
3. The invention provides a CRISPR/Gs12-3 system mediated nucleic acid visualization detection technology for the first time.
Drawings
FIG. 1 analysis of phylogenetic tree of guide RNA dependent endonucleases Gs12-3 and Gs12-5 predicted using a metagenomic approach.
FIG. 2 shows the DR sequence pattern of endonucleases Gs12-3 and Gs12-5 loci, domains and guide RNAs. Schematic representation of Gs12-3 and Gs12-5 loci; B. the DR sequence secondary structural fold of the guide RNA was aligned with multiple sequences.
FIG. 3. Predicted amino acid sequences of Gs12-3 and Gs12-5 proteins are analyzed conservatively with the amino acid sequences of known Cas12a proteins (AsCas 12a, lbCAs12a and FnCas12 a).
FIG. 4 expression and purification of Gs12-3 protein. A. Assessing the effect of different induction times on Gs12-3 protein expression, B. Assessing the effect of different induction temperatures on Gs12-3 protein expression, C. Assessing the effect of different IPTG concentrations on Gs12-3 protein expression.
FIG. 5 Gs12-5 protein expression and purification. A. Assessing the effect of different induction times on Gs12-5 protein expression, B. Assessing the effect of different induction temperatures on Gs12-5 protein expression, C. Assessing the effect of different IPTG concentrations on Gs12-5 protein expression.
FIG. 6 gel electrophoresis to detect the in vitro cleavage of double-stranded DNA target activity by Gs12-3 and Gs12-5. The target is an amplified fragment of PCV2 ORF2 gene of the porcine circovirus, and the identified target site PAM is TTTV.
FIG. 7 identifies and compares the PAM recognition characteristics of Gs12-3 and Gs12-5. PAM library subtraction experiments were used in bacteria to identify characteristics of Gs12-3 and Gs12-5 that recognize PAM. A. Experimental flow chart, b.gs12-3 and Gs12-5 recognize PAM characteristics.
FIG. 8 gel electrophoresis to detect Gs12-3 in vitro cleavage of double-stranded DNA targets at different PAM sites. The target is an amplified fragment of PCV2 ORF2 gene.
FIG. 9 shows the trans-cleavage activity of Gs12-3 at different target sites containing the same PAM in linear double stranded DNA. The target is an amplified fragment of PCV2 ORF2 gene of the porcine circovirus, and the identified target site PAM is TTTV. A. And B, detecting results by a blue light instrument.
FIG. 10 shows the validation of Gs12-3 trans-cleavage activity at different target sites containing different PAMs in linear double stranded DNA. The target is an amplified fragment of PCV2 ORF2 gene.
FIG. 11 evaluation of sensitivity detection of crRNA of different PAMs in Gs12-3 trans-cleavage. The target is an amplified fragment of PCV2 ORF2 gene. And A, PCR amplification results, and B, blue light detection results.
FIG. 12. Evaluation of the optimal cleavage reaction temperature and reaction time for Gs12-3 system mediated nucleic acid detection techniques. A. And B, optimizing the enzyme digestion reaction temperature and the reaction time. The target is an amplified fragment of PCV2 ORF2 gene.
FIG. 13 shows a schematic diagram of an experimental procedure for visualized detection of PCV2 nucleic acids by combining LAMP+CRISPR/Gs 12-3.
FIG. 14 visualized detection of PCV2 nucleic acids by binding LAMP+CRISPR/Gs 12-3. And A, screening an amplification result by using the LAMP primer, and B, detecting the result by using a blue light instrument.
FIG. 15 sensitivity evaluation of LAMP-CRISPR/Gs12-3 detection system. LAMP amplification results, B blue light detection results.
FIG. 16-specificity evaluation of LAMP-CRISPR/Gs12-3 detection system. LAMP amplification results, B blue light detection results.
Detailed Description
Description of the terms
Unless defined otherwise, 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.
A family of Genie scissistor endonucleases, where Genie is the meaning of the Genie, represented as a bacterial source, and scissistor represents a gene scissor, indicating the gene editing function that it may exert. The chinese name corresponding to the Genie scissistor endonuclease is called "clever-cut" endonuclease.
The pre-spacer adjacent motif (protospacer adjacent motif, PAM) is a short DNA sequence (typically 2-6 base pairs long). Traditionally, PAM is thought to be necessary for Cas nuclease cleavage, typically 3-4 nucleotides downstream of the cleavage site. There are many different Cas endoenzymes that can be purified from different bacteria, and each enzyme may recognize a different PAM sequence.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by conventional conditions, such as, for example, molecular cloning by Sambrook et al: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer.
Example 1 mining novel guide RNA dependent endonucleases based on metagenomic methods
Based on the bioinformatics identification flow of the novel guide RNA dependent endonuclease built by the inventor, bacterial coding protein deep mining is carried out on massive metagenome sequencing data in public databases such as NCBI nr (Non-Redundant Protein Sequence Database) Non-redundant protein library, global microbial gene catalog database (GMGC) and the like. The general analysis flow is as follows: and searching and positioning CRISPR array by using mini software aiming at all contig sequences in a target database, predicting proteins expressed adjacently by the CRISPR array by using prodigal software, removing redundancy of all the predicted proteins by using CD-hit software, performing protein cluster analysis by using mega software, and identifying and classifying CRISPR-Cas similarity proteins by using hmmer software to finally obtain two new unknown bacterial proteins.
Through phylogenetic tree analysis, these two new bacterial proteins were found to be located on different CRISPR-Cas12a phylogenetic branches, respectively (fig. 1), presumably as new RNA-guided endonucleases. The present invention relates to the name of a new protein from different bacteria, namely Genie scissor endonuclease. In order to facilitate subsequent researches, the inventor respectively names two new unknown bacterial proteins as Gs12-3 and Gs12-5 based on bacterial species sources, the naming rule is "endonuclease+digital number", the amino acid sequence of the Gs12-3 protein is shown as SEQ ID NO.1, the nucleotide sequence of the encoding gene is shown as SEQ ID NO.2, the amino acid sequence of the Gs12-5 protein is shown as SEQ ID NO.3, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 4.
Next, the inventors aligned the sequence similarity of these 2 newly discovered bacterial proteins to the NCBI nr database using the localization blast program. As a result, novel Gs12-3 proteins were found to have 38.52%, 34.46% and 29.99% amino acid sequence conservation with the known endonucleases LbCAs12a, fnCas12a and AsCas12a, respectively, and then compared with NCBI nr database (https:// www.ncbi.nlm.nih.gov /), and were found to have a highest 38.96% similarity with the known types of Cas12a proteins. The amino acid sequence conservation of newly discovered Gs12-5 with reported LbCAs12a, fnCas12a and AsCas12a is 32.46%, 30.77% and 27.71%, respectively; when aligned with the NCBI nr database, it was found to be only 37.94% at maximum similar to the known other types of Cas12a proteins. As can be seen, the newly discovered bacterial proteins, gs12-3, gs12-5, all have less than 40% amino acid sequence similarity to known Cas12 a.
Further, the inventors analyzed the loci of such proteins by using crispassfinder software. As a result, gs12-3, gs12-5 were found to have CRISPR array sequences comprising multiple repeat and spacer sequences, as well as Cas4, cas1 and Cas2 proteins. By using hmmer software to perform a hidden Markov model alignment analysis with domain sequences in the Pfam database, analysis results in REC1 domain (Alpha helical recognition lobe domain), ruvC Nuclease domain and NUC domain (nucleic domain), and it is speculated that these two new bacterial proteins may have nucleic acid cleavage activity; next, the inventors performed a predictive and multisequence alignment on the DR sequence secondary structures of Gs12-3, gs12-5, respectively, via RNAfold web server (http:// rnia. Tbi. Univie. Ac. At/cgi-bin/RNAWebsite/RNAfold. Cgi) on-line website, and as a result found that the newly predicted bacterial proteins were similar to the DR secondary structure of the known Cas12a protein, with one base difference for Gs12-5 (FIG. 2).
Finally, the inventors performed amino acid multisequence alignments of RuvC and Nuc domains of Gs12-3, gs12-5 with known LbCas12a, fnCas12a and AsCas12a proteins, respectively. As shown in FIG. 3, the amino acid sequence similarity of the Gs12-3, gs12-5 protein domains to known Cas12a proteins was found to be greatly different, and thus it was highly desirable to determine whether they have nucleic acid-directed cleavage activity by further experimentation.
EXAMPLE 2 evaluation of in vitro nucleic acid cleavage Activity of guide RNA dependent endonucleases Gs12-3 and Gs12-5
This example tests the cleavage activity of Gs12-3 and Gs12-5 proteins on double stranded DNA by in vitro experiments. Firstly, DNA sequences encoding Gs12-3 and Gs12-5 are synthesized after the codon optimization of escherichia coli, NLS nuclear localization signals are respectively added at the C ends of the DNA sequences, and the DNA sequences are shown as SEQ ID NO.5 and SEQ ID NO. 6. Then connecting the recombinant DNA into pET-28a prokaryotic expression vectors, respectively converting the recombinant DNA into escherichia coli BL21 strains, identifying positive clones, performing IPTG induction expression, and purifying by affinity chromatography to obtain target proteins. The temperature, duration and final concentration of IPTG affecting the induction of Gs12-3 and Gs12-5 protein expression were searched. The optimal time (12 h, 16h and 20 h) for inducing protein expression is identified under the condition that the temperature and the final concentration of IPTG are unchanged, and then the optimal culture temperature (16 ℃, 25 ℃ and 37 ℃) for inducing protein expression is identified under the condition that the time and the final concentration of IPTG are fixed; the optimal final IPTG concentrations (0.2 mM, 0.5mM, and 1.0 mM) for induction of protein expression were identified under fixed conditions of final temperature and incubation time. Detection was by SDS-PAGE electrophoresis. As a result, it was found that the optimal expression condition of Gs12-3 was 16℃for 16 hours, and the final concentration of IPTG was 0.5mM (FIG. 4); the optimal expression conditions for Gs12-5 were 16℃and 16h, with a final IPTG concentration of 0.5mM (FIG. 5).
Second, the cleavage activity of Gs12-3 and Gs12-5 proteins on double-stranded DNA was tested by in vitro experiments and compared to LbCAs12a and enasCas12a protein cleavage activity. The guide RNA paired with the target nucleic acid is used to guide the recognition and binding of Gs12-3 and Gs12-5 proteins on the target nucleic acid, so that the cleavage activity of Gs12-3 and Gs12-5 proteins on the target nucleic acid is excited, and double-stranded target nucleic acid in the system is cleaved. Then agarose gel electrophoresis is performed to observe the size change of the target band to identify the enzyme cleavage activity. In this example, the target double-stranded DNA (dsDNA) was selected as PCV2 ORF2 gene, PAM was TTTV or TTV, and a plurality of guide RNAs (crRNAs) recognizing the PAM sites at different positions were designed-1, crrna-2, crrna-3), PCV2 ORF2 gene sequence: bolded markers are PAM, underlined are targeting sequences.
The guide RNA sequences are shown in the following table:
| guide RNA | Sequences (5 '-3') (underlined regions are targeting regions) |
| crRNA-1 | AAUUUCUACUAUUGUAGAUUUUGUUUGGUUGGAAGUAAUC |
| crRNA-2 | AAUUUCUACUAUUGUAGAUUUUUUGUUGUUUGGUUGGAAG |
| crRNA-3 | AAUUUCUACUAUUGUAGAUUUAGUCUCAGCCACAGCUGAU |
And performing PCR amplification by taking the ORF2 plasmid as a template and taking the ORF2-F GAACCACAGTCAAAACGCCC, ORF2-R TTAAGGGTTAAGTGGGGGGT as a primer to obtain the ORF2 gene double-stranded DNA.
The in vitro cleavage reaction employs the following system: 10 XCutSmart Buffer 2. Mu.L, predicted Gs12-3 and Gs12-5 proteins and LbCAs12a and enasCas12a proteins 500ng, guide RNA500ng, ORF2 target amplification product 2. Mu.L. After the reaction was completed, 1. Mu.L of proteinase K was added to the mixture and the mixture was incubated at 37℃for 20min and 55℃for 10min, respectively, to terminate the reaction. The control group was not supplemented with guide RNA. And detecting the reaction by 1% agarose gel electrophoresis, and carrying out imaging observation under a glue irradiation instrument to distinguish target bands of the predicted novel proteases Gs12-3 and Gs12-5 and the known efficient proteases LbCAs12a and enaSCas12a in an experimental group and a control group.
As shown in FIG. 6, compared with the control group without the guide RNA, gs12-3 and enasCas12a proteins in the experimental group can cut double-stranded DNA in the reaction solution, 2 obvious cutting bands exist, and the cutting efficiency is higher. Gs12-5 and LbCAs12a proteins in the experimental group were less efficient in cleavage, and the cleavage bands were not apparent. As can be seen, bacterial proteins predicted by metagenomic strategies have as expected predicted nucleic acid-targeted cleavage activity, with Gs12-3 having higher nucleic acid-targeted cleavage activity and Gs12-5 being weak nucleic acid-targeted cleavage activity.
Example 3 characterization of PAM sequence characteristics of the Gs12-3 and Gs12-5 proteins
Through bacterial PAM library subtraction experiments, PAM sequences recognized by Gs12-3 and Gs12-5 proteins which have low homology and in vitro target nucleic acid cleavage activity are identified. The construction flow of the random mixed PAM vector library is as follows: the DNA oligo sequence GGCCAGTGAATTCGAGCTCGGTACCCGGGNNNNNNNGAGAAGTCATTTAA TAAGGCCACTGTTAAAAAGCTTGGCGTAATCATGGTCATAGCTGTTT was synthesized, where N is a random deoxynucleotide. With Oligo-F: GGCCAGTGAATTCGAGCTCGG and Oligo-R: AAACAGCTATGACCATGATTACGCCAA the primers are amplified by PCR, then connected into pUC19 vector by homologous recombination, transformed into Escherichia coli, and extracted to form random mixed PAM vector library. The guide RNA sequences used were: AAUUUCUACUAUUGUAGAUUGAGAAGUCAUUUAAUAAGGCCACU(underlined regions are targeting recognition sequences).
Bacterial PAM library subtraction experiments: the constructed predicted vectors pACYC-Duet-1-Gs12-3-crRNA, which co-express Gs12-3 and Gs12-5 proteins and crRNA, are transformed into DE3 (BL 21) competence to prepare a stably expressed bacterial strain. The stable transgenic bacterial strains constructed by the expression vectors pACYC-Duet-1-Gs12-3 and pACYC-Duet-1-Gs12-5 without crRNA are used as negative controls. 100ng of PAM library plasmids were respectively transferred into bacterial strains stably expressed, screened by plates with double resistance to ampicillin and chloramphenicol, and colonies on the plates were scraped off after 16h for plasmid extraction. 100ng of the extracted plasmid was used as a template, and the library was used to sequence the primers Seq-F: GGCCAGTGAATTCGAGCTCGG and PAM-Seq-R: CAATTTCACACAGGAAACAGCTATGACC PCR amplification was performed, and after recovery of the products, the experimental and control groups were subjected to second generation high throughput sequencing, respectively, and the sequencing results were shown by Weblogo3.0 analysis (FIG. 7A).
Identification of PAM sequence characteristics recognized by Gs12-3 and Gs12-5 proteins: the number of times of occurrence of different types of PAM sequences contained in the initial vector library in the experimental group and the control group in high-throughput sequencing is counted respectively, and the total number of all PAM sequences in each group is standardized. The calculation mode for each PAM consumption change is log 2 (control normalized value/experimental normalized value) when the value is greater than 3.5, the PAM is considered to be significantly consumed. The frequency of occurrence of bases at each position of the PAM sequence that is significantly depleted is then visualized using weblog 3.0. As shown in fig. 7B, the Gs12-3 protein was found to have the ability to recognize a broader range of PAM in vitro target cleavage, with a target recognition range that is much greater than the currently known Cas12a protein with PAM "TTTV", and the Gs12-5 target recognition range was consistent with the currently known Cas12a protein with PAM "TTTV".
In order to prove the reliability of the bacterial PAM library subtraction experimental result, the experimental result is verified by an in vitro enzyme digestion double-stranded DNA experiment of Gs12-3 protein. In this example, the target double-stranded DNA (dsDNA) was selected as PCV2 ORF2 gene, PAM was "VTV", V was A, G or C, and a plurality of different guide RNAs (crRNA), crRNA-ATA, crRNA-ATC, crRNA-ATG, crRNA-CTA, crRNA-CTC, crRNA-CTG, crRNA-GTA, crRNA-GTC and crRNA-GTG were designed. The PCV2 ORF2 gene sequence is: bolded markers are PAM, underlined are targeting sequences.
The guide RNA sequences are shown in the following table:
and performing PCR amplification by taking the ORF2 plasmid as a template and taking the ORF2-F GAACCACAGTCAAAACGCCC, ORF2-R TTAAGGGTTAAGTGGGGGGT as a primer to obtain the ORF2 double-stranded DNA.
The in vitro cleavage reaction employs the following system: 10 XCutSmart Buffer 2. Mu.L, predicted Gs12-3 protein 500ng, guide RNA500ng, ORF2 target amplification product 2. Mu.L. Incubate at 37℃for 20min. After completion of the reaction, 1. Mu.L of proteinase K was added, and the reaction was terminated by incubation at 55℃for 10 min. The control group was not supplemented with guide RNA. The target bands of the novel protease Gs12-3 experimental group and the control group are distinguished by 1% agarose gel electrophoresis detection after reaction and imaging observation and prediction under a gum-irradiating instrument.
As a result, as shown in FIG. 8, the Gs12-3 protein in the experimental group was able to cleave double-stranded DNA of different PAM in the reaction solution, compared with the control group without the guide RNA. Wherein 2 obvious cutting bands exist in crRNA-ATG, crRNA-CTA, crRNA-CTC and crRNA-GTC in the experimental group, and the cutting efficiency is higher. The results demonstrate that Gs12-3 can recognize non-classical PAM "ATG", "CTA", "CTC", "GTC" etc., further proves that the identification result of the bacterial PAM library subtraction experiment is reliable.
Example 4 establishment of CRISPR/Gs12-3 System mediated nucleic acid Rapid detection in situ visualization
It was further evaluated whether the Gs12-3 protein has trans-cleavage (trans-cleavage) activity. As shown in FIG. 9A, guide RNA paired with the target nucleic acid can be used to guide recognition and binding of endonuclease Gs12-3 to the target nucleic acid; then exciting the trans-cleavage activity of the single-stranded DNA to any single-stranded nucleic acid, so as to cleave the single-stranded DNA fluorescence-quenching reporter gene (ssDNA-FQ) in the reaction system; the trans-cleavage function of the candidate bacterial protein can be further judged by the intensity of the excited fluorescence, background noise and macroscopic color change.
The target double-stranded DNA (dsDNA) selected in this example was the PCV2 ORF2 gene,a plurality of different guide RNAs (crRNAs), crRNA-1, crRNA-2, crRNA-3, were designed and their sequences were identical to those of example 2. The single-stranded DNA fluorescence-quenching reporter gene sequence is ROX-random-BHQ 2 (5'ROX/GTATCCAGTGCG/3'BHQ 2 ) First, 3 proteins (Gs 12-3, lbCAs12a and enasCas2 a) were purified by prokaryotic expression, guide RNA was transcribed in vitro and ORF2 target gene double-stranded DNA was amplified by PCR. The following reaction system was then used: gs12-3, lbCAs12a or enasCas2a protein 500ng, guide RNA500ng,10 XCutSmart Buffer 2. Mu.L, 0.75. Mu.M Single-stranded DNA fluorescence-quenching reporter (ROX-random-BHQ) 2 ). 2 mu L of double-stranded DNA of the ORF2 gene amplified fragment is added into the experimental group, 2 mu L of complementary primer single-stranded DNA of the corresponding crRNA is added into the positive control group, and no target is added into the negative control group. The reaction was carried out at 37℃for 10min and at 98℃for 2min for inactivation. The color change, fluorescence intensity and background noise were observed under blue light to judge the trans-cleavage activity of the three proteins in vitro.
As shown in fig. 9B, the newly discovered Gs12-3 protein was substantially identical to the known LbCas12a and enacas 12a protein nucleic acid trans-cleavage activities from the changes in color and fluorescence of the reaction solution before and after cleavage; the Gs12-3, lbCAs12a and enasCas12a proteins all recognize traditional PAM "TTTV". Thus, the Gs12-3 protein has high trans-cleavage activity and relatively low background fluorescence signal, which indicates that the Gs12-3 protein is very suitable for in-situ visual nucleic acid detection experiments. Thus, a new technology for detecting the porcine circovirus type 2 virus nucleic acid based on Gs12-3 system mediation is successfully established.
To verify whether the Gs12-3 protein has the ability to recognize a wider range of PAM in nucleic acid detection, crRNA was designed for detection against the PAM site of the conserved sequence of ORF2 gene. The sequences of crRNA-ATA, crRNA-ATC, crRNA-ATG, crRNA-CTA, crRNA-CTC, crRNA-CTG, crRNA-GTA, crRNA-GTC, and crRNA-GTG are the same as in example 3.
The following system reaction is adopted: gs12-3, lbCAs12a or enasCas12a protein 500ng, guide RNA500ng for different PAM, 10×CutSmart Buffer 2. Mu.L, 0.75. Mu.M single-stranded DNA fluorescence-quenching reporter (ROX-random-BHQ) 2 ) And 2. Mu.L of ORF2 target amplification product. Double addition of 2. Mu.L of ORF2 gene amplification fragment to the experimental group2 mu L of complementary primer single-stranded DNA of the corresponding crRNA is added into the strand DNA, and the negative control is not added with a target. Respectively reacting at 37 ℃ for 10min and inactivating at 98 ℃ for 2min. By observing the fluorescence intensity, background noise, and the like under blue light. As shown in fig. 10, gs12-3 can recognize non-classical PAM "CTA", while LbCas12a and enacas 12a cannot recognize, which proves that Gs12-3 has the ability to recognize PAM in a wider range and can be used for nucleic acid detection, and further proves that the identification result of bacterial PAM library subtraction experiment is reliable.
The sensitivity of crrnas of different PAMs in Gs12-3 system mediated nucleic acid detection techniques was then assessed. Calculation of PCV2-ORF2 target Gene template copy number 4.98X10 11 The copies/. Mu.L was diluted to 4.98X10 s in this order 10 COPIES/. Mu.L to 4.98X10 -1 PCR amplification was performed with copies/. Mu.L, and as shown in FIG. 11A, the amplification was as low as 4.98X10 3 COPies/. Mu.L. Different concentrations of amplification products were used to assess the sensitivity of different PAM crrnas in Gs12-3 system-mediated nucleic acid detection techniques. The following system reaction is adopted: gs12-3 protein 500ng, guide RNA for different PAM 500ng,10 XCutSmart Buffer 2. Mu.L, 0.75. Mu.M Single-stranded DNA fluorescence-quenching reporter (ROX-random-BHQ) 2 ) And 2 μl of ORF2 target amplification product at different concentrations. Negative control was no target. Respectively reacting at 37 ℃ for 10min and inactivating at 98 ℃ for 2min. By observing the fluorescence intensity, background noise, and the like under blue light. As shown in FIG. 11B, the sensitivity of the crRNA cleavage reaction was high for "TTT", "CTA" and "CTC", and it was found that 4.98X10 4 The copies/. Mu.L was consistent with that identified by the Gs12-3 bacterial PAM library subtraction experiment.
The temperature of the reaction optimal for the cleavage reaction of Gs12-3 system-mediated nucleic acid detection technique was then assessed and compared to the reaction temperature of LbCAs12 a. Double-stranded DNA (dsDNA) was used as a target of PCV2 ORF2 gene as a site for nucleic acid detection, and guide RNA was crRNA-3, the sequence of which was the same as that of example 2. The following system reactions were carried out: gs12-3 or LbCAs12a protein 500ng, guide RNA500ng,10 XCutSmart Buffer 2. Mu.L, 0.75. Mu.M Single-stranded DNA fluorescence-quenching reporter (ROX-random-BHQ) 2 ) And 2. Mu.L of ORF2 PCR amplified target product. Negative control was no target. Respectively atThe reaction was carried out at 16℃at 25℃at 37℃at 45℃at 55℃at 65℃at 72℃for 15min and at 98℃for 2min of inactivation. By observing the fluorescence intensity, background noise, and the like under blue light and ultraviolet light. As shown in FIG. 12A, gs12-3 protein can react at 16-55 deg.C, lbCAs12A protein at 16-45 deg.C, and the temperature tolerance range of Gs12-3 is wider.
Finally, the reaction time of Gs12-3 system mediated nucleic acid detection technique was evaluated and compared with that of LbCAs12 a. Double-stranded DNA (dsDNA) was used as a target of PCV2 ORF2 gene as a site for nucleic acid detection, and guide RNA was crRNA-3, the sequence of which was the same as that of example 2. The system reaction: gs12-3 or LbCAs12a protein 500ng, guide RNA500ng,10 XCutSmart Buffer 2. Mu.L, 0.75. Mu.M Single-stranded DNA fluorescence-quenching reporter (ROX-random-BHQ) 2 ) And 2. Mu.L of ORF2 PCR amplified target product. Negative control was no target. The reaction time is 30s, 1min, 2min, 3min, 4min, 5min, and 10min respectively, and inactivating at 98deg.C for 2min. By observing the fluorescence intensity, background noise, and the like under blue light and ultraviolet light. As shown in FIG. 12B, the Gs12-3 and LbCAs12a proteins can react within 3min to generate stronger fluorescent signals, which proves that Gs12-3 has the advantage of high reaction efficiency and can generate fluorescent reaction in shorter time.
Example 5 LAMP-CRISPR/Gs12-3 visual detection kit and method for PCV2 nucleic acid
The Gs12-3 system was further used in combination with the LAMP method for visualization of PCV2 nucleic acid in situ. The detection flow is as shown in fig. 13: collecting clinical samples, rapidly extracting nucleic acid, performing LAMP amplification, performing Gs12-3 enzyme digestion reaction detection, and observing by blue light.
Multiple sequence comparisons were made in DNAman according to the different genotypes of PCV2 ORF2 gene sequences (JQ 002672, KC620515, KF035059, KF850461, KF951567, KF951570, KM624031, KP768481, KR 559695) to obtain the conserved sequences, ACCTATGACCCCTATGTAAACTACTCCTCCCGCCATACCATAACCCAGCCCTTCTCCTACCACTCCCGCTACTTTACCCCCAAACCTGTCCTAGATTCCACTATTGATTACTTCCAACCAAACAACAAAAGAAATCAGCTGTGGCTGAGACTACAAACTGCTGGAAATGTAGACCACGTAGGCCTCGGCACTGCGTTCGAAAACAGTATATACGACCAGGAATACAATATCCGTGTAACCATGTATGTACAATTCAGAGAATTTAATCTTAAAGACCCCCCACTTAACC, 3 sets of LAMP amplifications were designed according to the PCV2 ORF2 gene conserved sequences, and the primers were as shown in the following table:
the LAMP reaction system is shown in the following Table
The composition of the primer mixture system is shown in the following table
| System composition | Volume of |
| F3(5μM) | 1μL |
| B3(5μM) | 1μL |
| LF(10μM) | 2μL |
| LB(10μM) | 2μL |
| FIP(40μM) | 8μL |
| BIP(40μM) | 8μL |
PCV2-ORF2 standard plasmid copy number 1.38X10 11 The copies/. Mu.L was diluted to 1.38X10 in sequence 10 copies/. Mu.L to 1.38X10 -1 cobies/. Mu.L; 1.38X10 were taken 4 copies/. Mu.L and 1.38X10 1 LAMP primer screening is carried out by cobies/. Mu.L, an LAMP amplification system is established according to the table, and the negative control is RNase H-free 2 O, the reaction was carried out on ice. After the preparation of the reaction system, vortex vibration and uniform mixing are carried out, then instantaneous centrifugation is carried out, and the reaction is carried out for 10min at 37 ℃ and then for 40min at 65 ℃. After the reaction, the mixture was centrifuged instantaneously, and 5. Mu.L of the mixture was added to 1. Mu.L of 6 XLoading Buffer, and the reaction result was observed by 2% agarose gel electrophoresis. Meanwhile, the LAMP reaction product was added to the nucleic acid detection reaction mediated by Gs12-3 system, and the guide RNA used in this example was crRNA-3, the sequence of which was the same as that in example 2. The system reaction: gs12-3 protein 500ng, guide RNA500ng,10×CutSmart Buffer 2. Mu.L, 0.75. Mu.M Single-stranded DNA fluorescence-quenching reporter (ROX-random-BHQ) 2 ) And 2. Mu.L of LAMP amplification target product. Inactivating at 37deg.C for 10min and 98deg.C for 2min. By observing the fluorescence intensity, background noise, etc. under blue light and ultraviolet light. As shown in FIG. 14, three LAMP primers were able to amplify the target, wherein the third primer set was highly sensitive and free of negative contamination, and the third LAMP primer set was used in the subsequent experiments.
Next, the sensitivity of the LAMP-CRISPR/Gs12-3 based PCV2 detection system was evaluated, and the standard plasmid PCV2-ORF2 (copy number 1X 10) -1 ～1×10 3 copies/. Mu.L) as different targets by LAMP method, and the negative control is RNase H-free 2 O. After the reaction, 5. Mu.L of the sample was added to 1. Mu.L of 6×loading Buffer by instantaneous centrifugation, followed by 2% agarose gelAnd (5) observing the reaction result by electrophoresis. Meanwhile, LAMP amplification products of different reactions are added into a Gs12-3 system-mediated nucleic acid detection reaction, the reaction is carried out for 10min at 37 ℃, and the inactivation is carried out for 2min at 98 ℃. After the reaction, fluorescence intensity, background noise and the like are observed under blue light and ultraviolet light. As a result, as shown in FIG. 15, it was found that the LAMP-amplified copy number was 1X 10 0 ～1×10 3 The copies/. Mu.L of plasmid. Copy number of 1X 10 0 ～1×10 3 The fluorescence signal generated by the copies/. Mu.L plasmid group is very significantly different compared to the NC group without the target sequence. The results showed that the LAMP-CRISPR/Gs12-3 detection sensitivity was 1 copies/. Mu.L.
Finally, the specificity of the LAMP-CRISPR/Gs12-3 based PCV2 detection system was evaluated. The nucleic acid detection reaction was performed with PCV2 using other porcine-derived viruses as targets in order to verify the specificity of the LAMP-CRISPR/Gs12-3 detection system for PCV2 detection. In the LAMP amplification system, different groups of target DNAs are respectively TGEV, JEV, ASFV, PEDV, PCV2, PDCoV and PRRSV virus cDNA, and no RNase H exists 2 O served as a negative control. After the reaction, the mixture was centrifuged instantaneously, and 5. Mu.L of the mixture was added to 1. Mu.L of 6 XLoading Buffer, and the reaction result was observed by 2% agarose gel electrophoresis. Meanwhile, LAMP amplification products of different reactions are added into a Gs12-3 system-mediated nucleic acid detection reaction, the reaction is carried out for 10min at 37 ℃, and the inactivation is carried out for 2min at 98 ℃. After the reaction, fluorescence intensity, background noise and the like are observed under blue light and ultraviolet light. As a result, as shown in FIG. 16, the target band was amplified only when the target DNA was PCV2, and the specific band could not be amplified by other viral targets. Meanwhile, only when the target DNA is PCV2, the generated fluorescence signal value has extremely obvious difference compared with the NC group, obvious red fluorescence is visible under blue light, and in other groups, the fluorescence signal is not different compared with the NC group because the target DNA in the system is not matched with crRNA. The results show that the LAMP-CRISPR/Gs12-3 detection system has high specificity for PCV 2.
Claims (7)
- An endonuclease in a crispr/Cas system, comprising the following proteins:I. gs12-3 protein with an amino acid sequence shown in SEQ ID NO. 1;II. Gs12-5 protein with the amino acid sequence shown in SEQ ID NO. 3.
- 2. An isolated polynucleotide, wherein the polynucleotide is a polynucleotide encoding the endonuclease of claim 1.
- 3. A vector comprising the polynucleotide of claim 2.
- 4. A visual nucleic acid detection kit comprising the endonuclease of claim 1, a single-stranded DNA fluorescence-quenching reporter, and a guide RNA paired with a target nucleic acid.
- 5. A kit for visually detecting porcine circovirus type 2, which is characterized by comprising LAMP amplification primers, endonuclease according to claim 1, single-stranded DNA fluorescence-quenching reporter gene and guide RNA paired with target nucleic acid.
- 6. The kit according to claim 4 or 5, wherein the endonuclease has an amino acid sequence shown in SEQ ID NO. 1.
- 7. The kit of claim 6, wherein the LAMP amplification primers have the sequence shown in SEQ ID NOS.7-11 and the guide RNA has the sequence of AAUUUCUACUAUUGUAGAUUUAGUCUCAGCCACAGCUGAU.
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