CN116179512B - Endonuclease with wide target recognition range and application thereof - Google Patents
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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Abstract
The invention discloses endonucleases with wide target recognition range and application thereof, in particular to endonucleases Gs12-16 (PAM=NYYV) and Gs12-18 (PAM=YTYV) (V=A/C/G) (Y=C/T) identified by utilizing a metagenomic binding experiment, which have the advantages of being different from the PAM sequence of the known LbCAs12a, so that the endonucleases have wider target coverage. The invention also establishes a CRISPR/Gs12-16 and Gs12-18 system-mediated nucleic acid visual detection technology, and has wide application prospect in the field of nucleic acid detection.
Description
Technical Field
The invention belongs to the technical field of genome editing, and particularly relates to newly identified endonucleases Gs12-16 and Gs12-18 and application thereof.
Background
The gene editing technology is used for knocking in, knocking out and site-directed mutagenesis of target genes, and is a genetic engineering technology capable of accurately modifying specific fragments. Gene editing tools in biologists' hands have been continuously upgraded for decades. The conventional zinc finger enzyme, TALEN and other gene editing tools are relatively complex to operate and are not easy to grasp. Therefore, the lack of simple and easy-to-use gene editing tools has been a major problem that plagues the whole biological field. After that, CRISPR-Cas9 was left out, which changed the state of the art by means of a simpler operation. The CRISPR/Cas system consists of a system of a small piece of RNA and a highly efficient nucleic acid cleaving enzyme (Cas nuclease), unlike TALENs technology and ZFNs technology which rely on recognition between a protein and a target gene, a complex is formed between sgRNA and the target gene, thus completing editing of a specific gene sequence. The CRISPR-Cas system comprises two parts, a CRISPR locus and a Cas gene (CRISPR associated gene).
Recent studies have specifically classified CRISPR-Cas systems into 2 major classes, 6 subtypes, and the representative of type ii systems is the CRISPR/Cas9 protein system, depending on the kind of Cas nuclease. For example, the gene editing system most commonly used today, CRISPR-SpCas9, recognizes sequences on the genome that wild-type SpCas9 recognizes, which require a short DNA sequence-a spacer precursor adjacent motif (protospacer adjacent motif, PAM), i.e. "NGG". However, when there is no identifiable PAM sequence near the genomic sequence we want to edit, spCas9 will not be able to identify the target genomic sequence we want to edit, i.e. it will not be able to perform subsequent gene editing tasks. In 2020, team Benjamin Kleinstiver found that in human cells, spRY prefers sites containing NRN PAM, while targeting recognition of sites containing NYN PAM is weak (n=arbitrary base, r=a or G, y=c or T). Immediately after 2022, a SpRY spCas9 without PAM sequence requirement was developed, which can cleave DNA on almost any sequence on the genome, and would expand the application of CRISPR-Cas9 gene editing technology on any DNA sequence.
Modifications to Cas9 are still ongoing, but there are many other types of nucleases that offer unique properties. For example, cas12 Sup>A from the V-Sup>A CRISPR-Cas system produces Sup>A 5 nucleotide protruding cohesive end while Cas9 produces Sup>A blunt end, only one rnSup>A in the effector of the natural CRISPR/Cas12 Sup>A system, cas9 needs to bind to the tracrrnSup>A-crrnSup>A, two RNAs. The types and functions of the V-type Cas12 system are most abundant and there are a variety of nuclease systems of smaller molecular size, i.e., cpf1 (Cas 12 a), C2C1 (Cas 12 b), C2 (Cas 13 a), C2C3 (Cas 12C), casY (Cas 12 d) and CasX (Cas 12 e). The AsCas12a accommodates one G at some positions of the PAM, and Cas12a (HkCas 12 a) from Helcococcus kunzii preferentially recognizes two adjacent C and standard PAM at the second and third PAM positions, forming one YYV PAM. In addition, cas12a (PiCas 12 a) from Prevotella ihumii can recognize not only PAM of TYV, but also guanine (e.g., TTGC and GGCC) at the second, third and/or fourth positions of PAM.
Although researchers have improved the gene editing efficiency of these systems by rational design and directed evolution methods, the Cas12 proteins that have been identified so far can only recognize T-rich PAM (T-rich PAM), which severely hampers the application of Cas12 family in vivo gene editing, in vitro clinical detection, and the like. Thus, there is a need to find a protein that has a broader range of PAM availability and even is free of PAM.
Disclosure of Invention
The invention discloses CRISPR/Cas endonuclease with wider target recognition range and application thereof, in particular to two types of endonuclease Gs12-16 (PAM=NYYV) and Gs12-18 (PAM=YTYV) (V=A/C/G) (Y=C/T) identified by utilizing a metagenomic binding experiment, and also establishes a nucleic acid visualization detection technology based on Gs12-16 and Gs12-18 protein mediation.
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. gs12-16 of the amino acid sequence shown in SEQ ID NO.1 or Gs12-18 protein of the amino acid sequence shown in SEQ ID NO. 3;
II. Proteins having more than 80% sequence similarity compared to the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.3, and substantially retaining their biological function derived from the sequence;
III, proteins having one or more amino acid substitutions, deletions or additions compared to the amino acid sequence shown in SEQ ID NO.1, and substantially retain their biological function from the sequence.
A fusion protein comprising the above endonuclease protein, and a polypeptide linked to the N-terminus or C-terminus of the protein.
A polynucleotide encoding the endonuclease or a polynucleotide encoding the fusion protein. A vector or host cell containing said polynucleotide.
A visual nucleic acid detection kit comprises the endonuclease, a single-stranded DNA fluorescence-quenching reporter gene and a guide RNA paired with target nucleic acid.
The technical scheme of the invention has the following main beneficial effects:
1. the invention provides novel CRISPR/Cas12a system family members Gs12-16 and Gs12-18 which are excavated by combining metagenomics and experimental means for the first time.
2. The invention discovers endonuclease Gs12-16 and Gs12-18 with wide PAM application range, and has the advantages that target DNA sites with PAM of NYYV or YTYV can be identified in genome, thus greatly expanding the coverage range of gene editing targets.
3. The invention provides CRISPR/Gs12-16 and Gs12-18 system mediated nucleic acid visualization detection for the first time.
Drawings
FIG. 1 shows analysis of the amino acid sequence similarity and phylogenetic tree analysis of guide RNA dependent endonucleases Gs12-16 and Gs12-18 predicted using a metagenomic approach. A. Analysis of the amino acid sequence similarity of the guide RNA-dependent endonuclease to the known endonucleases LbCas12a, fnCas12a and AsCas12 a; B. analysis of the guide RNA dependent endonuclease's evolutionary tree.
FIG. 2 shows the DR sequence pattern of endonuclease Gs12-16 and Gs12-18 loci, domains and guide RNAs. Schematic representation of Gs12-16 and Gs12-18 loci; B. the DR sequence secondary structural fold of the guide RNA was aligned with multiple sequences.
FIG. 3. Analysis of predicted Gs12-16 and Gs12-18 protein amino acid sequences conservatively with the amino acid sequences of known Cas12a proteins (AsCas 12a, lbCAs12a and FnCas12 a).
FIG. 4 shows the molecular weight, purity and purification conditions of Gs12-16 protein detected by SDS-PAGE polyacrylamide gel electrophoresis. Gs12-16 protein induction temperature; B. assessing Gs12-16 protein induction time; C. estimating the induction concentration of Gs12-16 protein IPTG; D. gs12-16 protein elution conditions were evaluated.
FIG. 5 shows the molecular weight, purity and purification conditions of Gs12-18 protein detected by SDS-PAGE polyacrylamide gel electrophoresis. Gs12-18 protein induction temperature; B. assessing Gs12-18 protein induction time; C. estimating the induction concentration of Gs12-18 protein IPTG; D. the elution conditions of Gs12-18 protein were evaluated.
FIG. 6 identification of characteristics of Gs12-16 and Gs12-18 recognizing PAM in bacteria using a PAM library subtraction experiment. Pam library subtraction experimental flow chart; PAM recognizable by Gs12-16 and Gs12-18.
FIG. 7 detection of double stranded DNA target cleavage activity by Gs12-16 and Gs12-18 by gel electrophoresis. The target is a duck novel reovirus NDRV S3 gene amplified fragment, and the identified target site PAM is TTTV or TTV.
FIG. 8 shows a flow chart for detection of nucleic acids based on the mediation of Gs12-16 and Gs12-18 endonucleases.
FIG. 9 compares trans-cleavage activity of the same crRNA of Gs12-16 and Gs12-18 as LbCAs12a for different target preferences. The target is an amplified fragment of an African swine fever virus NDRV S3 gene, and the identified target site PAM is TTTV or TTV. Trans-cleavage activity of crrnas of the same Gs12-16 and Gs12-18 as LbCas12a for different target preferences; B. the heat maps show the cleavage activity of Gs12-16 and Gs12-18 with LbCAs12 a.
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, in which the specific conditions are not noted in the following examples, is generally followed by conventional conditions, such as, for example, molecular cloning: 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, new bacterial proteins were found on different CRISPR-Cas12a phylogenetic branches (fig. 1B), presumably it is a new RNA-guided endonuclease. The present invention designates this type of protein from a new discovery in different bacteria as a Genie Scissor (GS) endonuclease. In order to facilitate subsequent researches, the inventor respectively names the novel unknown bacterial proteins as Gs12-16 and Gs12-18 based on bacterial species sources, the naming rule is "endonuclease+digital number", the amino acid sequence of the Gs12-16 protein is shown as SEQ ID NO.1, the encoding nucleotide sequence is shown as SEQ ID NO.2, the amino acid sequence of the Gs12-18 protein is shown as SEQ ID NO.3, and the encoding nucleotide sequence is shown as SEQ ID NO. 4.
Next, the inventors used the localization blast program to align the sequence similarity of the newly discovered bacterial proteins to the NCBI NR database. As a result, the amino acid sequence conservation of the novel Gs12-16 proteins with the known endonucleases LbCas12a, fnCas12a and AsCas12a was found to be 43.50%, 42.75%, 33.43%, respectively, and the amino acid sequence conservation of the novel Gs12-18 proteins with the known endonucleases LbCas12a, fnCas12a and AsCas12a was found to be 47.21%, 43.81%, 34.59%, respectively (fig. 1A).
Further, the inventors analyzed the loci of both proteins by using crispassfinder software. As a result, gs12-16 and Gs12-18 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 carry out hidden Markov model alignment analysis on domain sequences in a Pfam database, REC1domain (Alpha helical recognition lobe domain), ruvC Nuclease domain and NUC domain (nucleic domain) are obtained by analysis, and it is presumed that the two novel bacterial proteins may have nucleic acid cleavage activity; next, the inventors predicted and multi-sequence alignments of the DR sequence secondary structures of Gs12-16 and Gs12-18 via an online website RNAfold web server (http:// rnia. Tbi. Univie. Ac. At/cgi-bin/RNAWebsite/RNAfold. Cgi), and found that these two newly predicted bacterial proteins were similar to the DR secondary structure of the known Cas12a protein, but with one base difference (FIG. 2).
Finally, the inventors performed amino acid multisequence alignments of RuvC and Nuc domains of Gs12-16 and Gs12-18 proteins with known LbCas12a, fnCas12a and AsCas12a proteins, respectively. As shown in FIG. 3, the amino acid sequence similarity of the Gs12-16 and Gs12-18 protein domains to known Cas12a proteins was found to be greatly different, and thus it was highly desirable to determine whether it had nucleic acid-directed cleavage activity by further experimentation.
EXAMPLE 2 evaluation of purification conditions for expression of guide RNA dependent endonucleases Gs12-16 and Gs12-18
In the embodiment, DNA sequences for encoding Gs12-16 and Gs12-18 are synthesized after the codon optimization of escherichia coli, NLS nuclear positioning signals are respectively added to the C ends of the DNA sequences, the encoding sequences after the optimization of the Gs12-16 are shown as SEQ ID NO.5, and the encoding sequences after the optimization of the Gs12-18 are shown as SEQ ID NO. 6. Then connected into pET-28a prokaryotic expression vectors, respectively transformed into escherichia coli BL21 strain, and amplified and cultured to OD after positive clone identification 600 Cooling at 0.4-0.6,4 deg.C for 30min, performing IPTG induction expression, collecting bacteria at 9000rpm for 5min at 4 deg.C, removing supernatant, rinsing and precipitating, ultrasonic crushing, purifying by affinity chromatography to obtain target protein, taking a certain amount of gradient purified protein, determining protein purity and molecular weight by SDS-PAGE polypropylene gel electrophoresis, setting induction time to be 12h, 16h and 22h respectively, and setting induction temperature to be 16 deg.C, 25 deg.C and 37 deg.C, respectively, wherein IPTG induction concentration is 0.1 μM, 0.4 μM, 1 μM and 2 μM, and taking a certain amount of supernatant and inclusion body to perform SDS-PAGE polypropylene gel electrophoresis to detect protein expression.
As shown in FIGS. 4 and 5, the optimal induction conditions of IPTG of Gs12-16 and Gs12-18 proteins are induction time of 16h, induction temperature of 16 ℃ and concentration of 0.4 mu M, and under the conditions, protein bands with single bands and high expression quantity can be obtained.
Example 3 characterization of Gs12-16 and Gs12-18 to recognize PAM
Through bacterial PAM library subtraction experiments, PAM sequences recognized by Gs12-16 and Gs12-18 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: synthesis of DNA oligo sequence GGCCAGTGAATTCGAGCTCGGTACCCGGGNNNNNNNGAGAAGTCATTTA ATAAGGCCACTGTTAAAAAGCTTGGCGTAATCATGGTCATAGCTGTTT where N is a random deoxynucleotide. With Oligo-F: GGCCAGTGAATTCGAGCTCGG and Oligo-R: AAACAGCTATGACCATGATTACGCCAA the primer is amplified by PCR to giveThe recombinant vector is connected into pUC19 vector through homologous recombination, and plasmid is extracted after colibacillus is transformed 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 Gs12-16 and Gs12-18 proteins are respectively co-expressed with crRNA to form pACYC-Duet-1-Gs12-16-crRNA and pACYC-Duet-1-Gs12-18-crRNA, and the vectors are transformed into DE3 (BL 21) competence to prepare bacterial strains with stable expression. A stable transgenic bacterial strain constructed without the Gs12-16/18 protein expression vector pACYC-Duet-1 was used as a negative control. 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 is carried out, the experimental group and the control group are respectively subjected to second-generation high-throughput sequencing after the products are recovered, and the sequencing result is displayed by Weblogo3.0 analysis.
Identified PAM sequence characteristics recognized by Gs12-16 and Gs12-18 proteins: 16384 different types of PAM sequences contained in the initial vector library were counted and the number of times they appeared in the experimental and control groups in high throughput sequencing was normalized with the total number of all PAM sequences in each group. 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 a result, as shown in fig. 6, two CRISPR/Cas gene editing systems of loose PAM sequences, gs12-16 and Gs12-18, were found to cleave target DNA in sequences of PAM "NYYV" and "YTYV" (v=a/C/G) (y=c/T), respectively, in the genome.
EXAMPLE 4 evaluation of Gs12-16 and Gs12-18 proteins Loose type PAM target cleavage Capacity
This example tests the cleavage activity of Gs12-16 and Gs12-18 proteins on double-stranded DNA by in vitro experiments. Guide RNA paired with the target nucleic acid is used for guiding the recognition and binding of Gs12-16 and Gs12-18 proteins on the target nucleic acid, so that the cleavage activity of Gs12-16 and Gs12-18 proteins on the target nucleic acid is excited, double-stranded target nucleic acid in a system is cleaved, and agarose gel electrophoresis is carried out to observe the change of the size of a target band so as to identify the cleavage activity.
In this example, the target double-stranded DNA (dsDNA) was selected as the novel duck reovirus NDRV S3 gene, PAM was TTTG, TTA, TTC, respectively, and the sequence thereof: bolded markers are PAM, underlined are targeting sequences. The guide RNA sequence is: AAUUUCUACUAUUGUAGAUUAUAUCGUAACCAGGACUGAA(crRNA3)；AAUUUCUACUAUUGUAGAUUGAGAGACGAGGAUACGACGC(crRNA5)；AAUUUCUACUAUUGUAGAUUCAACCCGCUCUAGCAAUCUC(crRNA 7) (underlined region is the targeting region). And performing PCR amplification by using pUC57-NDRV-S3 plasmid as a template and NDRV-F TGTTGCACTCAGTGCTGTGG, NDRV-R AATTGACCAGTGATGCCAAC as a primer to obtain the NDRV double-stranded DNA. The in vitro cleavage reaction employs the following system: 10 XCutSmart Buffer 2. Mu.L, predicted Gs12-16 and Gs12-18 proteins, enhanced enCas12 a500ng, crRNA 500ng, NDRV target amplification product 2. Mu.L. Incubate at 37℃for 15min, respectively. After completion of the reaction, 0.5. 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. After the reaction, the target bands of the novel proteases Gs12-16 and Gs12-18 experimental group and control group predicted by the same reaction time are detected through 2% agarose gel electrophoresis, imaging is carried out under a UV gel irradiation instrument, and the cutting efficiency is analyzed through Image J software.
As a result, as shown in FIG. 7, gs12-16 and Gs12-18 proteins in the experimental group were able to cleave double-stranded DNA in the reaction solution within 15min, 1 or 2 distinct cleavage bands were present, 2% agarose gel electrophoresis was prepared for three crRNAs, and the cleavage efficiency was calculated using the following formula, as compared with the control group without guide RNA.
Wherein a represents a double-stranded DNA wild-type strand which is not cleaved by an endonuclease; b, c represent bands after cleavage by endonucleases.
The cutting efficiency is Gs12-16 respectively: 41.53%, 23.18%, 22.51%; gs12-18:14.93%, 19.36%, 13.76%; 62.64%, 50.83% and 11.48% of enhanced enCas12 a. It follows that bacterial proteins predicted by metagenomic strategies have nucleic acid-targeted cleavage activity as expected to be presumed.
Example 5 establishment of CRISPR-Gs12-16 and CRISPR-Gs12-18 System-mediated nucleic acid Rapid detection in situ visualization
It was further evaluated whether Gs12-16 and Gs12-18 proteins have trans-cleavage (trans-cleavage) activity. Guiding recognition and binding of endonucleases Gs12-16 and Gs12-18 to a target nucleic acid using a guide RNA that can be paired with 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.
In order to verify the effect of the activity of the guide RNAs corresponding to the different targets, the target double-stranded DNA (dsDNA) was selected as the duck novel reovirus NDRV S3 gene in this example, and PAM was "TTTV" or "TTV", respectively, with the sequence: TGTTGCACTCAGTGCTGTGGTGTTCTGTACTATGGTGCCCCTCCCTCTGATGGAAACTGTTTTCCACATCACAAGTGTCATCAACAGCAATATCGTACTGAGACTCCGCTCATGAGATATATTAAGGTGGGTCGCACTACAGAGCAACTGCTTGATCAATATGCCATTGCTCTGCATGTCATTGCAGATTACTATGACGAGGCGAGTAAGCAACCTCATGATATCGCTGAAACTGAGTCAATCGCACCATTTGATATCGTAACCAGGACTGAATCTATTCGCAGTGACCGTGCCGTTGACCCGGAATTCTGGACTTATCCGTTAGAGAGACGAGGATACGACGCGCGACATGAGATTGCTAGAGCGGGTTGGAAGATGATCGATGCTTCATCGCGGAGTCACACTCTTCCTGAATGTCTGGTGTCAAATATGCTACATACTAGGCATGTCTTCAGTCAAATGTTGACCACGACAACCATCTATGATGTCGCTGTCACGGGTAAAGCTGTTAAATTCAGCCCGATGGTAGCAACCATGCCAACTCGAGGAGATGGTGCTGTGGCTCTGTCAAGAGGTAACTTGGATCATGATGTCGAGGACTGTTGGATGGATGGTTTTGCATTCTCCCCCCTCATCGGCGGTGTTGGCATCACTGGTCAATT; the single-stranded DNA fluorescence-quenching reporter gene sequence is ROX-random-BHQ 2 (5’ROX/GTATCCAGTGCG/3’BHQ 2 ) 13 guide RNA sequences were designed, each: AAUUUCUACUAUUGUAGAUUCUCUGCAUGUCAUUGCAGAU(crRNA1)；AAUUUCUACUAUUGUAGAUUAGUCCUGGUUACGA UAUCAA(crRNA2)；AAUUUCUACUAUUGUAGAUUAUAUCGUAACCAGGACUGAA(crRNA3)；AAUUUCUACUAUUGUAGAUUGCAGUGACCGUGCCGUUGAC(crRNA4)；AAUUUCUACUAUUGUAGAUUGAGAGACGAGGAUACGACGC(crRNA5)；AAUUUCUACUAUUGUAGAUUCCCGUGACAGCGACAUCAUA(crRNA6)；AAUUUCUACUAUUGUAGAUUCAACCCGCUCUAGCAAUCUC(crRNA7)；AAUUUCUACUAUUGUAGAUUGAAGAUGAUCGAUGCUUCAU(crRNA8)；AAUUUCUACUAUUGUAGAUUCACAUCACAAGUGUCAUCAA(crRNA9)；AAUUUCUACUAUUGUAGAUUACUGAAGACAUGCCUAGUAU(crRNA10)；AAUUUCUACUAUUGUAGAUUACAGCUUUACCCGUGACAGC(crRNA11)；AAUUUCUACUAUUGUAGAUUCUGUUGAUGACACUUGUGAU(crRNA12)；AAUUUCUACUAUUGUAGAUUAUAUAUCUCAUGAGCGGAGU(crRNA 13), the underlined region is the targeting region. First, gs12-16 and Gs12-18 and known LbCAs12a proteins are purified by prokaryotic expression, guide RNA is transcribed in vitro, and NDRV-S3 target gene double-stranded DNA is amplified by PCR. The following reaction system was then used: gs12-16, gs12-18 or LbCAs12a protein 500ng, guide RNA500ng, 2. Mu.L 10 XCutSmart Buffer, 1. Mu.M single-stranded DNA fluorescence-quenching reporter and 2. Mu.L PCR amplified target product. Negative control was no target. Reacting for 15min at 37 ℃ and inactivating for 2min at 98 ℃. The in vitro trans-cleavage activity of the above predicted protein was judged by observing fluorescence intensity and background noise under blue light (fig. 8).
Fluorescence intensity was identified using image j software, and the resulting data input Graphpad prism showed protein cleavage activity by heat map, and the darker the color, the stronger the fluorescence and the better the cleavage activity. As shown in FIG. 9, from the fluorescence change of the reaction solution before and after cleavage, the newly discovered Gs12-16 and Gs12-18 proteins have the nucleic acid trans-cleavage activity with the known LbCAs12a protein; compared to the known LbCas12a, it was found that the same crRNA may have a site preference for different proteins.
The foregoing description is only illustrative of the preferred embodiment of the present invention, and is not to be construed as limiting the invention, but is to be construed as limiting the invention to any and all simple modifications, equivalent variations and adaptations of the embodiments described above, which are within the scope of the invention, may be made by those skilled in the art without departing from the scope of the invention.
Claims (6)
1. The endonuclease in the CRISPR/Cas system is characterized by comprising Gs12-16 with an amino acid sequence shown as SEQ ID NO.1 or Gs12-18 protein with an amino acid sequence shown as SEQ ID NO. 3.
2. A polynucleotide encoding the endonuclease of claim 1.
3. A vector comprising the polynucleotide of claim 2.
4. A host cell comprising the polynucleotide of claim 2 or the vector of claim 3.
5. Use of the endonuclease of claim 1, or the polynucleotide of claim 2, or the vector of claim 3, or the host cell of claim 4 for nucleic acid detection for non-diagnostic purposes.
6. 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.
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