SrCas12a-2 protein, gene editing system and application thereof

Abstract

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

Description

Technical Field

[0001] This invention belongs to the field of gene editing technology, specifically relating to a SrCas12a-2 protein, its gene editing system, and its applications. Background Technology

[0002] Gene editing technology, used for knock-in, knock-out, and site-directed mutation of target genes, is a genetic engineering technique capable of precisely modifying specific fragments. For decades, biologists have continuously upgraded their gene editing tools. Previous gene editing tools, such as zinc finger enzymes and TALENs, were complex to operate and not easy to master. Therefore, the lack of simple and easy-to-use gene editing tools has been a major challenge for the entire field of biology. Then, CRISPR-Cas9 emerged, changing this situation with its much simpler operation. The CRISPR / Cas system consists of a short RNA fragment and a highly efficient nuclease (Cas nuclease). Unlike TALENs and ZFNs, which rely on protein-target gene recognition, CRISPR-Cas uses a complex formed between the sgRNA and the target gene to edit a specific gene sequence. The CRISPR-Cas system comprises two parts: the CRISPR locus and the Cas gene (CRISPR-associated gene).

[0003] The Cas12a enzyme produces a 5' sticky end for cleavage, unlike the blunt end produced by Cas9. This cleavage pattern has unique advantages in gene editing. Currently, scientists have isolated and identified different Cas12a enzymes from various microorganisms, such as LbCas12a and FnCas12a. These enzymes have shown different application potentials and advantages in gene editing, disease diagnosis, and other fields. Based on this, discovering new Cas12a enzymes will not only help to deepen the understanding of 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 novel SrCas12a-2 protein, its gene editing system, and its applications.

[0005] One of the objectives of this invention is to provide a novel SrCas12a-2 protein, the amino acid sequence of which is shown in SEQ ID NO.1.

[0006] The second objective of this invention is to provide a fusion protein, which includes the following proteins:

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

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

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

[0010] A third objective of this invention is to provide a polynucleotide that encodes the aforementioned SrCas12a-2 protein or the aforementioned fusion protein.

[0011] The fourth objective of this invention is to provide a carrier comprising the aforementioned polynucleotides.

[0012] The fifth objective of this invention is to provide a CRISPRCas system comprising the aforementioned SrCas12a-2 protein and at least one gRNA;

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

[0014] The sixth objective of this invention is to provide a host cell comprising the aforementioned SrCas12a-2 protein, or the aforementioned fusion protein, or the aforementioned polynucleotide, or the aforementioned vector, or the aforementioned CRISPR-Cas system.

[0015] The seventh objective of this invention is to provide a method for editing target nucleic acids, the method comprising contacting the target nucleic acid with the aforementioned SrCas12a-2 protein, or the aforementioned fusion protein, or the aforementioned polynucleotide, or the aforementioned vector, or the aforementioned CRISPR-Cas system, or the aforementioned host cell, the method being a method not for 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 are used in editing target nucleic acids.

[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 the kit, wherein the use is for purposes other than disease diagnosis or treatment.

[0018] The present invention also provides a visual nucleic acid detection kit, the kit comprising 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, as well as a single-stranded DNA fluorescence-quenching reporter gene and gRNA paired with the target nucleic acid.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] This invention provides a novel protein, SrCas12a-2, identified using metagenomics combined with experiments. SrCas12a-2 is a new member of the CRISPR / Cas12a family with low homology. This invention also establishes a nucleic acid visualization detection technology based on the CRISPR / SrCas12a-2 system, which has broad application prospects in the field of nucleic acid detection. Attached Figure Description

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

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

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

[0024] Figure 4 The images show the protein expression levels detected by SDS-PAGE polypropylene gel electrophoresis of the supernatant and inclusion bodies in Example 2 of this invention (A), and the protein bands with higher expression levels obtained after purification (B).

[0025] Figure 5 This figure shows the trans-cleavage activity results of the newly discovered SrCas12a-2 protein at different concentrations.

[0026] Figure 6 The trans-cleavage activity was determined by the amplification curves of the qPCR instrument, where A represents the detection results at different concentration dilutions, and B represents the negative control results at the corresponding dilution concentrations. Detailed Implementation

[0027] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention.

[0028] Example 1

[0029] This embodiment provides a method for discovering novel SrCas12a-2 proteins based on bioinformatics methods, as detailed below:

[0030] Using bioinformatics strategies, we conducted in-depth analysis of bacterial-encoded proteins from massive metagenomic sequencing data in public databases such as the NCBI NR (Non-Redundant Protein Sequence Database) and the Global Microbial Genome Database. The general analysis workflow was as follows: for all contig sequences in the target database, we used Minced software to search for and locate CRISPR arrays; then, we used Prodigal software to predict proteins expressed near the CRISPR arrays; we used CD-HIT software to remove redundancy from all predicted proteins; we used Mega software for protein clustering analysis; and we used Hmmer software for CRISPR-Cas similarity protein identification and classification, ultimately obtaining a novel, previously unknown bacterial protein.

[0031] Phylogenetic analysis revealed that the new bacterial protein was located on different CRISPR-Cas12a phylogenetic branches. Figure 1 A) suggests it may be a new member of the Cas12a family. This is based on three-dimensional structural prediction (…). Figure 1 B) and genomic locus analysis ( Figure 2 The study found that it conformed to the characteristics of gene-editing nucleases, suggesting that it may possess gene-editing activity. Further multiple sequence alignment revealed that the amino acid sequence of SrCas12a-2 showed 33.63%, 35.58%, and 36.71% conservation compared to the known sequences of AsCas12a, LbCas12a, and FnCas12a, respectively. Figure 3 This further indicates that it is a new member of the Cas12a family. 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 endonuclease SrCas12a-2, as follows:

[0037] The DNA sequence encoding SrCas12a-2 was synthesized after codon optimization of *E. coli* and ligated into the pET-28a prokaryotic expression vector. This vector was then transformed into *E. coli* strain BL21. After identifying positive clones, the culture was expanded to an OD600 value of 0.6-0.8, followed by IPTG induction. The cells were collected at 4℃ and 7500 rpm for 15 min. After discarding the supernatant and washing the precipitate, the cells were sonicated and purified by affinity chromatography to obtain the target protein. A certain amount of the graded purified protein was analyzed by SDS-PAGE polyacrylamide gel electrophoresis to determine protein purity and molecular weight. The induction time was set to 16 h, the induction temperature to 16℃, and the IPTG induction concentration to 0.4 μM. A certain amount of the broken bacterial cells, supernatant, and inclusion bodies were analyzed by SDS-PAGE polyacrylamide gel electrophoresis to detect protein expression levels. Figure 4 A).

[0038] Purification was performed using NI-FF (IMAC), followed by washing (50 mM KH₂PO₄, 300 mM NaCl, 20 mM imidazole, 5% glycerol, pH 7.0 @ 25℃), and elution (50 mM KH₂PO₄, 300 mM NaCl, 300 mM imidazole, 5% glycerol, pH 7.0, 25℃). The target protein obtained after elution was collected, its conductivity reduced, and purified using a Q-HP column. Washing was performed (50 mM KH₂PO₄, 50 mM NaCl, 2 mM DTT, 0.1 mM EDTA, 5% glycerol, pH 7.0, 25℃), and elution (50 mM KH₂PO₄, 200 mM NaCl, 2 mM DTT, 0.1 mM EDTA, 5% glycerol, pH 7.0, 25℃). Under these conditions, a single protein band with a high expression level could be obtained. Figure 4 B).

[0039] Example 3

[0040] This embodiment provides a method for establishing a rapid visual detection technique for nucleic acids mediated by the CRISPR-SrCas12a-2 system. The specific method is as follows:

[0041] Further evaluation was conducted to determine whether the SrCas12a-2 protein possesses trans cleavage activity. A guide RNA that can pair with the target nucleic acid was used to guide the SrCas12a-2 endonuclease to recognize and bind to the target nucleic acid; subsequently, its trans cleavage activity against any single-stranded nucleic acid was activated, thereby cleaving the single-stranded DNA fluorescence-quenched reporter gene (ssDNA-FQ) in the reaction system. The trans cleavage function of the candidate gene-editing endonuclease was determined by the activated fluorescence intensity, background noise, and visual color changes.

[0042] To verify the effect of different target guide RNAs on activity, this example selected African swine fever virus P27 as the target double-stranded DNA (dsDNA). SrCas12a-2 and the known LbCas12a protein were purified from prokaryotic expression. The following reaction system was then used: SrCas12a-2 protein diluted at different gradients, 210 ng guide RNA, 2 μL 10×9⁻² buffer, 10 μM single-stranded DNA fluorescence-quenched reporter gene, and 0.5 μL of PCR amplification target product. The negative control was a quantitative real-time PCR reaction without the target (37℃, 30 s, 30 cycles, fluorescence acquisition channel: ROX). The trans-cleavage activity of the predicted proteins in vitro was determined by observing fluorescence intensity and background noise under blue light. Figure 5 Furthermore, the darker the color, the stronger the fluorescence, and the better the cleavage activity. The trans-cleavage activity of the predicted gene-editing endonucleases was determined in vitro using qPCR amplification curves. Figure 6 A represents the test results for different concentration dilutions, while Figure 6 B represents the negative control result at the corresponding dilution concentration. This demonstrates that the CRISPR / SrCas12a-2 system is suitable for nucleic acid fluorescence visualization detection.

[0043] Unless otherwise specified, all raw materials used in this invention are existing substances that can be purchased directly from the market.

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

Claims

1. A method for editing target nucleic acids, characterized in that, The method involves contacting a target nucleic acid with a SrCas12a-2 protein, the amino acid sequence of which is shown in SEQ ID NO.

1. The method is not for disease diagnosis or treatment purposes.

2. Application of SrCas12a-2 protein in the preparation kit, wherein the amino acid sequence of the SrCas12a-2 protein is shown in SEQ ID NO.

1.

3. A visual nucleic acid detection kit, characterized in that, The kit contains SrCas12a-2 protein and a single-stranded DNA fluorescence-quenching reporter gene, and gRNA paired with the target nucleic acid; The amino acid sequence of the SrCas12a-2 protein is shown in SEQ ID NO.1.

Images