SrCas12a-2 protein as well as gene editing system and application thereof

Abstract

The invention belongs to the technical field of nucleic acid editing, a SrCas12a-2 protein is identified by utilizing metagenomics in combination with experiments, the specific amino acid sequence of the SrCas12a-2 protein is shown as SEQ ID NO.1, the SrCas12a-2 protein is a novel Cas12a enzyme, and the discovery of the novel enzyme expands the CRISPR / Cas12a system family. The invention also establishes a nucleic acid visual detection technology based on CRISPR / SrCas12a-2 system mediation, and the nucleic acid visual detection technology has a wide application prospect in the field of nucleic acid detection.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene editing, and specifically relates to a SrCas12a-2 protein and a gene editing system and application thereof. Background Art

[0002] Gene editing technology is used to knock in, knock out and site-directed mutagenesis of target genes. It is a genetic engineering technology that can precisely modify specific fragments. For decades, gene editing tools in the hands of biologists have been constantly upgraded. Previous gene editing tools such as zinc finger enzymes and TALENs are relatively complicated to operate and not easy to master. Therefore, the lack of simple and easy-to-use gene editing tools has always been a major problem that has plagued the entire field of biology. Later, CRISPR-Cas9 came out of nowhere, and it changed the status quo with its simpler operation. The CRISPR / Cas system consists of a short piece of RNA and an efficient nucleic acid cutting enzyme (Cas nuclease). Unlike TALENs technology and ZFNs technology, this technology relies on the recognition between proteins and target genes, but instead forms a complex between sgRNA and target genes to complete the editing of specific gene sequences. The CRISPR-Cas system consists of two parts: the CRISPR locus and the Cas gene (CRISPR-associated gene).

[0003] The cutting method of Cas12a enzyme to produce 5' sticky ends is different from the flat ends produced by Cas9. This cutting method has unique advantages in gene editing. At present, scientists have isolated and identified different Cas12a enzymes from a variety of microorganisms, such as LbCas12a, FnCas12a, etc. These enzymes have shown different application potentials and advantages in the fields of gene editing, disease diagnosis, etc. Based on this, the discovery of new Cas12a enzymes will not only help to deeply understand the operating mechanism of the CRISPR-Cas system, but also provide more efficient and accurate tools for gene editing, disease diagnosis and other fields. Summary of the invention

[0004] In view of this, the present invention provides a new SrCas12a-2 protein and its gene editing system and application.

[0005] One of the objects of the present invention is to provide a new SrCas12a-2 protein, the amino acid sequence of the SrCas12a-2 protein is shown in SEQ ID NO.1.

[0006] The second object of the present invention is to provide a fusion protein, wherein the fusion protein comprises the following proteins:

[0007] The amino acid sequence of the SrCas12a-2 protein is shown in SEQ ID NO.1;

[0008] Or a protein having more than 80% sequence identity with the amino acid sequence shown in SEQ ID NO.1 and substantially retaining the biological function derived from the sequence;

[0009] Or a protein having one or more amino acid substitutions, deletions or additions compared to the amino acid sequence shown in SEQ ID NO.1, and substantially retaining the biological function derived from the sequence.

[0010] A third object of the present invention is to provide a polynucleotide encoding the above-mentioned SrCas12a-2 protein, or encoding the above-mentioned fusion protein.

[0011] A fourth object of the present invention is to provide a vector comprising the above-mentioned polynucleotide.

[0012] A fifth object of the present invention is to provide a CRISPR Cas system, comprising the above-mentioned SrCas12a-2 protein and at least one gRNA;

[0013] The gRNA can bind to the above-mentioned SrCas12a-2 protein.

[0014] The sixth object of the present invention is to provide a host cell, which comprises the above-mentioned SrCas12a-2 protein, or the above-mentioned fusion protein, or the above-mentioned polynucleotide, or the above-mentioned vector, or the above-mentioned CRISPR-Cas system.

[0015] The seventh object of the present invention is to provide a method for editing a target nucleic acid, the method comprising contacting the target nucleic acid with the above-mentioned SrCas12a-2 protein, or the above-mentioned fusion protein, or the above-mentioned polynucleotide, or the above-mentioned vector, or the above-mentioned CRISPR-Cas system, or the above-mentioned host cell, and the method is a method for non-disease diagnosis or treatment purposes.

[0016] Furthermore, the above-mentioned SrCas12a-2 protein, or the above-mentioned fusion protein, or the above-mentioned polynucleotide, or the above-mentioned vector, or the above-mentioned CRISPR-Cas system, or the above-mentioned host cell is used in editing the target nucleic acid.

[0017] Furthermore, the use of the above-mentioned SrCas12a-2 protein, or the above-mentioned fusion protein, or the above-mentioned polynucleotide, or the above-mentioned vector, or the above-mentioned CRISPR-Cas system, or the above-mentioned host cell in the preparation of a kit is for non-disease diagnosis or treatment purposes.

[0018] The present invention also provides a visual nucleic acid detection kit, which comprises the above-mentioned SrCas12a-2 protein, or the above-mentioned fusion protein, or the above-mentioned polynucleotide, or the above-mentioned vector, or the above-mentioned CRISPR-Cas system, or the above-mentioned host cell, and a single-stranded DNA fluorescence-quenching reporter gene, and a gRNA paired with a target nucleic acid.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention provides a new protein SrCas12a-2 identified by metagenomics combined with experiments, which is a new member of the CRISPR / Cas12a family with low homology; the present invention also establishes a nucleic acid visualization detection technology mediated by the CRISPR / SrCas12a-2 system, which has broad application prospects in the field of nucleic acid detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram (A) of the newly discovered SrCas12a-2 protein located on different CRISPR-Cas12a system evolutionary branches, and three-dimensional structure prediction (B).

[0022] Figure 2 This is a genomic site analysis diagram of the newly discovered SrCas12a-2 protein of the present invention.

[0023] Figure 3 This is a sequence difference diagram between the newly discovered SrCas12a-2 protein of the present invention and the known reported AsCas12a, LbCas12a, and FnCas12a.

[0024] Figure 4 The protein expression level of the supernatant and inclusion bodies detected by SDS-PAGE polypropylene gel electrophoresis in Example 2 of the present invention (A) and the protein band with higher expression level obtained after purification (B) are shown.

[0025] Figure 5 This is a graph showing the trans-cleavage activity results of different concentrations of the newly discovered SrCas12a-2 protein of the present invention.

[0026] Figure 6 The trans-cleavage activity was determined by the qPCR amplification curve, where A was the test result at different dilution concentrations, and B was the negative control result at the corresponding dilution concentration. DETAILED DESCRIPTION

[0027] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0028] Example 1

[0029] This embodiment provides a method for mining a new SrCas12a-2 protein based on a bioinformatics method, as follows:

[0030] Using bioinformatics strategies, we conducted in-depth mining of bacterial encoded proteins based on massive metagenomic sequencing data in public databases such as the NCBI nr (Non-Redundant Protein Sequence Database) non-redundant protein library and the Global Microbial Genome Database. The general analysis process is as follows: for all contig sequences in the target database, we use the minced software to search and locate the CRISPR array, then use the prodigal software to predict the proteins expressed adjacent to the CRISPR array, use the CD-hit software to remove redundancy from all predicted proteins, use the mega software to perform protein clustering analysis, and use the hmmer software to identify and classify CRISPR-Cas similarity proteins, and finally obtain a new unknown bacterial protein.

[0031] Through phylogenetic tree analysis, it was found that the new bacterial protein is located on different CRISPR-Cas12a phylogenetic branches ( Figure 1 A), it is speculated that it may be a new member of the Cas12a family. Figure 1 B) and genomic loci analysis ( Figure 2 ), and found that it meets the characteristics of gene editing nucleases, and it is speculated that it may have gene editing activity. Then, through multiple sequence alignment, the protein amino acid sequence of SrCas12a-2 is 33.63%, 35.58%, and 36.71% conservative with the known reported sequences of AsCas12a, LbCas12a, and FnCas12a, respectively ( Figure 3 ), further indicating that it is a new Cas12a family member. The amino acid sequence of the SrCas12a-2 protein is shown in SEQ ID NO.1, its encoding nucleotide sequence is shown in SEQ ID NO.2, and its prokaryotic expression vector sequence is shown in SEQ ID NO.3.

[0032]

[0033]

[0034]

[0035] Example 2

[0036] This embodiment provides a method for evaluating the expression and purification conditions of the gene editing nuclease SrCas12a-2, as follows:

[0037] The DNA sequence encoding SrCas12a-2 was synthesized after codon optimization in Escherichia coli, connected to the pET-28a prokaryotic expression vector, and transformed into Escherichia coli BL21 strains. After identifying the positive clones, the culture was expanded to an OD600 value of 0.6-0.8, and IPTG was induced for expression. The bacteria were collected at 4°C and 7500rpm for 15 minutes. The supernatant was discarded and the precipitate was rinsed. After ultrasonic disruption, the target protein was purified by affinity chromatography. A certain amount of gradient purified protein was taken to determine the protein purity and molecular weight by SDS-PAGE polypropylene gel electrophoresis, and the induction time was set to 16h, the induction temperature was 16°C, and the IPTG induction concentration was 0.4μM. A certain amount of broken bacteria, supernatant and inclusion body were taken for SDS-PAGE polypropylene gel electrophoresis to detect protein expression ( Figure 4 A).

[0038] NI-FF (IMAC) purification, washing (50mM KH2PO4, 300mM NaCl, 20mM imidazole, 5% glycerol, pH7.0@25℃), elution (50mM KH2PO4, 300mM NaCl, 300mM imidazole, 5% glycerol, pH7.0, 25℃). The target protein obtained by elution was collected, the conductivity was reduced, and the Q-HP column was purified, washing (50mM KH2PO4, 50mM NaCl, 2mM DTT, 0.1mM EDTA, 5% glycerol, pH7.0, 25℃), elution (50mM KH2PO4, 200mM NaCl, 2mM DTT, 0.1mM EDTA, 5% glycerol, pH7.0, 25℃). Under this condition, a single protein band with a high expression level can be obtained ( Figure 4 B).

[0039] Example 3

[0040] This embodiment provides a method for establishing a rapid visualization detection technology of nucleic acids mediated by a CRISPR-SrCas12a-2 system, and the specific method is as follows:

[0041] Further evaluate whether the SrCas12a-2 protein has trans cleavage activity. Use a guide RNA that can pair with the target nucleic acid to guide the endonuclease SrCas12a-2 to recognize and bind to the target nucleic acid; then stimulate its "trans cleavage" activity on any single-stranded nucleic acid, thereby cutting the single-stranded DNA fluorescence-quenching reporter gene (ssDNA-FQ) in the reaction system; the trans cleavage function of the candidate gene editing endonuclease can be judged by the excited fluorescence intensity, background noise and naked eye color change.

[0042] In order to verify the influence of the activity of the guide RNA corresponding to different targets, the target double-stranded DNA (dsDNA) was selected as African swine fever P27 in this example, and SrCas12a-2 and the known LbCas12a protein were purified by prokaryotic expression. Then the following reaction system was used: SrCas12a-2 protein was diluted in different gradients, guide RNA 210ng, 2μL 10×9-2Buffer, 10μM single-stranded DNA fluorescence-quenched reporter gene and 0.5μL PCR amplification target product. The negative control was a fluorescence quantitative PCR reaction without adding a target (37°C, 30s, 30cycles, fluorescence collection channel: ROX). The trans-cleavage activity of the above-mentioned predicted protein in vitro was judged by observing the fluorescence intensity and background noise under blue light ( Figure 5 ). The darker the color, the stronger the fluorescence, and the better the cutting activity. The qPCR amplification curve determines the trans-cutting activity of the above-mentioned predicted gene editing nuclease in vitro, where Figure 6 A is the test result of different dilution concentrations, and Figure 6 B is the negative control result at the corresponding dilution concentration. It can be seen that the CRISPR / SrCas12a-2 system is suitable for nucleic acid fluorescence visualization detection.

[0043] The specific raw materials not described in the present invention are all existing materials and can be directly purchased from the market.

[0044] The above are only preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A SrCas12a-2 protein, characterized in that The amino acid sequence of the SrCas12a-2 protein is shown in SEQ ID NO.

1.

2. A fusion protein, characterized in that: The fusion protein includes the following proteins: The amino acid sequence of the SrCas12a-2 protein is shown in SEQ ID NO.1; Or a protein having more than 80% sequence identity with the amino acid sequence shown in SEQ ID NO.1 and substantially retaining the biological function derived from the sequence; Or a protein having one or more amino acid substitutions, deletions or additions compared to the amino acid sequence shown in SEQ ID NO.1, and substantially retaining the biological function derived from the sequence.

3. A polynucleotide, characterized in that The polynucleotide encodes the SrCas12a-2 protein according to claim 1, or encodes the fusion protein according to claim 2.

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

5. A CRISPR-Cas system, characterized in that: The system comprises the SrCas12a-2 protein of claim 1 and at least one gRNA; The gRNA is capable of binding to the SrCas12a-2 protein of claim 1.

6. A host cell, characterized in that The host cell comprises the SrCas12a-2 protein of claim 1, or the fusion protein of claim 2, or the polynucleotide of claim 3, or the vector of claim 4, or the CRISPR-Cas system of claim 5.

7. A method for editing a target nucleic acid, characterized in that: The method comprises contacting the target nucleic acid with the SrCas12a-2 protein of claim 1, or the fusion protein of claim 2, or the polynucleotide of claim 3, or the vector of claim 4, or the CRISPR-Cas system of claim 5, or the host cell of claim 6, and the method is a method for non-disease diagnosis or treatment purposes.

8. Use of the SrCas12a-2 protein according to claim 1, or the fusion protein according to claim 2, or the polynucleotide according to claim 3, or the vector according to claim 4, or the CRISPR-Cas system according to claim 5, or the host cell according to claim 6 in editing a target nucleic acid.

9. The SrCas12a-2 protein according to claim 1, or the fusion protein according to claim 2, or the polynucleotide according to claim 3, or the vector according to claim 4, or the CRISPR-Cas system according to claim 5, or the host cell according to claim 6 in the preparation of a kit, wherein the application is for non-disease diagnosis or treatment purposes.

10. A visual nucleic acid detection kit, characterized in that: The kit comprises the SrCas12a-2 protein of claim 1, or the fusion protein of claim 2, or the polynucleotide of claim 3, or the vector of claim 4, or the CRISPR-Cas system of claim 5, or the host cell of claim 6, and a single-stranded DNA fluorescence-quenching reporter gene, and a gRNA paired with a target nucleic acid.

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