SrCas13a-3 protein and gene editing system and application thereof

Abstract

The invention relates to a SrCas13a-3 protein as well as a gene editing system and application thereof. The amino acid sequence of the SrCas13a-3 protein is shown as SEQ ID NO. 1. The invention provides endonuclease SrCas13a-3 identified by utilizing metagenomics in combination with experiments, and the endonuclease SrCas13a-3 is a new member with low CRISPR / Cas13a family homology. The invention also establishes a nucleic acid visual detection technology based on CRISPR / SrCas13a-3 system mediation, and the nucleic acid visual detection technology has a wide application prospect in the field of nucleic acid detection.

Description

A SrCas13a-3 protein, its gene editing system, and its applications Technical Field

[0001] This invention relates to the field of gene editing technology, specifically to a SrCas13a-3 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 that can precisely modify 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 problem plaguing the entire field of biology. Then, CRISPR-Cas9 emerged, changing the status quo 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 CRISPR-Cas13a system utilizes guide RNA (crRNA) to target single-stranded RNA (ssRNA), amplifying nucleic acid signals through its unique collateral cleavage activity. It is widely used in pathogen detection, gene expression regulation, and molecular diagnostics. Although Cas13a and Cas12a both belong to the CRISPR-Cas system, they are of type VI-A (targeting RNA) and type VA (targeting DNA), respectively, and differ fundamentally in their mechanisms and applications.

[0004] Discovering new Cas13a enzymes not only contributes to a deeper understanding of the CRISPR-Cas system's operational mechanisms but also provides more efficient and precise tools for fields such as gene editing and disease diagnosis. Cas13a exhibits stronger paracleavage activity (cleaving any surrounding RNA) up to the aM level (10-18M). It does not require PAM sequence restrictions, offering greater targeting freedom. The RNA cleavage generates a large number of signal fragments, making it easier to design fluorescent / test strip sensors (such as SHERLOCK technology). Multiplex detection capabilities allow for the simultaneous design of multiple crRNAs targeting different RNAs, enabling single-tube multiplex detection. Summary of the Invention

[0005] Based on the above description, the present invention provides a SrCas13a-3 protein, its gene editing system, and its applications, aiming to provide a novel SrCas13a-3 protein.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: The present invention provides a SrCas13a-3 protein, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] The present invention also provides a fusion protein, comprising: a SrCas13a-3 protein with an amino acid sequence as 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.

[0008] The present invention also provides a nucleotide for encoding the SrCas13a-3 protein as described above, or for encoding a protein as described above.

[0009] The present invention also provides a carrier comprising nucleotides as described above.

[0010] The present invention also provides a CRISPR-Cas system comprising the SrCas13a-3 protein as described above and at least one gRNA; the gRNA being capable of binding the SrCas13a-3 protein as described above.

[0011] The present invention also provides a host cell comprising the SrCas13a-3 protein as described above, or the fusion protein as described above, or the nucleotide as described above, or the vector as described above, or the CRISPR-Cas system as described above.

[0012] The present invention also provides a method for editing target nucleic acid, the method comprising contacting the target nucleic acid with the aforementioned SrCas13a-3 protein, or a fusion protein as described above, or a polynucleotide as described above, or a vector as described above, or a CRISPR-Cas system as described above, or a host cell as described above, the method being a method for non-disease diagnosis or treatment purposes.

[0013] The present invention also provides the use of the aforementioned SrCas13a-3 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 in editing target nucleic acids.

[0014] The present invention also provides the use of the SrCas13a-3 protein as described above, or the fusion protein as described above, or the polynucleotide as described above, or the vector as described above, or the CRISPR-Cas system as described above, or the host cell as described above in the preparation of a kit, wherein the use is for purposes other than disease diagnosis or treatment.

[0015] The present invention also proposes a visual nucleic acid detection kit, the kit comprising a single-stranded DNA fluorescence-quenched reporter gene and a gRNA paired with a target nucleic acid, and further comprising the aforementioned SrCas13a-3 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.

[0016] Compared with the prior art, the technical solution of this application has the following beneficial technical effects: 1. The present invention provides a nuclease SrCas13a-3 identified by metagenomics combined with experiments, which is a new member of the CRISPR / Cas13a family with low homology.

[0017] 2. This invention also establishes a nucleic acid visualization detection technology based on the CRISPR / SrCas13a-3 system, which has broad application prospects in the field of nucleic acid detection. Attached Figure Description

[0018] Figure 1 is a phylogenetic tree diagram of the SrCas13a-3 protein provided in Example 1 of this invention; Figure 2 is a sequence homology analysis diagram of the SrCas13a-3 protein provided in Example 1 of this invention; Figure 3 is a schematic diagram of the locus comparing the amino acid sequence of the SrCas13a-3 protein provided in Example 1 of this invention with the amino acid sequence domains of known Cas13a proteins; Figure 4 is a diagram showing the conservation comparison results of the amino acid sequence of the SrCas13a-3 protein provided in Example 1 of this invention with the amino acid sequence of known Cas13a proteins; Figure 5 is a diagram showing the protein expression of the SrCas13a-3 supernatant and inclusion bodies provided in Example 2 of this invention detected by SDS-PAGE polyacrylamide gel electrophoresis; Figure 6 is a diagram showing the protein expression of the purified SrCas13a-3 protein provided in Example 2 of this invention detected by SDS-PAGE polyacrylamide gel electrophoresis; Figure 7 is a diagram showing the detection results of the cleavage activity of the SrCas13a-3 protein at different concentrations provided in Example 3 of this invention. Detailed Implementation

[0019] To facilitate understanding of this application, a more complete description will be provided below with reference to the accompanying drawings, which illustrate embodiments of the present application. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that the disclosure of this application will be thorough and complete.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0021] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0022] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0023] Example 1: Discovering Novel Gene-Editing Endonucleases Using Bioinformatics Methods. A bioinformatics strategy was employed to perform deep mining 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 process was as follows: For all contig sequences in the target database, the minced software was used to search for and locate CRISPR arrays. Then, the prodigal software was used to predict proteins expressed near the CRISPR array. Redundancy was removed from all predicted proteins using CD-hit software, followed by protein clustering analysis using mega software. Finally, CRISPR-Cas similarity protein identification and classification were performed using hmmer software, ultimately yielding a novel, previously unknown bacterial protein.

[0024] Phylogenetic analysis revealed that the novel bacterial protein resides on different CRISPR-Cas13a phylogenetic branches (as shown in Figure 1), suggesting it may be a new member of the Cas13a family. The inventors then performed homology analysis between the amino acid sequences of SrCas13a-3 and the known LwCas13a and LubCas13a proteins (as shown in Figure 2). The amino acid sequence of SrCas13a-3 showed 25% and 25.64% conservation of the reported LwCas13a and LubCas13a sequences, respectively (as shown in Figure 2), further confirming its status as a new member of the Cas13a family. The amino acid sequence of the SrCas13a-3 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.

[0025]

[0026] Amino acid multiple sequence alignments were performed on the HEPN1-I, HEPN1-II, and HEPN2 domains of SrCa13a-3 with those of the known LwCas13a and LubCas13a proteins. As shown in Figures 3 and 4, significant differences in amino acid sequence similarity were found between the SrCas13a-3 protein domains and the known Cas13a proteins. Therefore, further experiments are needed to determine whether it possesses targeted nucleic acid cleavage activity.

[0027] Example 2 Evaluation of Expression and Purification Conditions for the Gene Editing Endonuclease SrCas13a-3 In this example, the DNA sequence encoding SrCas13a-3 was synthesized after codon optimization in *E. coli*, ligated into the pET-28a prokaryotic expression vector, and transformed into *E. coli* strain BL21. After identifying positive clones, the culture was expanded to an OD600 value of 0.6-0.8, and IPTG-induced expression was performed. The bacteria were collected at 4℃ and 7500 rpm for 15 min. After discarding the supernatant and washing the precipitate, the protein was sonicated and purified by affinity chromatography to obtain the target protein. A certain amount of gradient-purified protein was taken and its purity and molecular weight were determined by SDS-PAGE polyacrylamide gel electrophoresis. The induction time was set to 18 h, the induction temperature to 16℃, and the IPTG induction concentration to 0.2 mM. A certain amount of broken bacterial cells, supernatant, and inclusion bodies were taken for SDS-PAGE polyacrylamide gel electrophoresis to detect the protein expression level (as shown in Figure 5).

[0028] NI-FF (IMAC) purification, washing (20 mM Tris, 300 mM NaCl, 20 mM imidazole, 5% glycerol, pH 7.0 @25℃), elution (20 mM Tris, 300 mM NaCl, 500 mM imidazole, 5% glycerol, pH 7.0 @25℃). The eluted target protein was collected, its conductivity reduced, and purified by Q-HP column purification, washing (20 mM Tris, 200 mM NaCl, 1 mM DTT, 5% glycerol, pH 7.0 @25℃), elution (20 mM Tris, 600 mM NaCl, 1 mM DTT, 5% glycerol, pH 7.0 @25℃). Gel size exclusion (20 mM Tris, 150 mM NaCl, 1 mM DTT, 5% glycerol, pH 7.0 @25℃). Under these conditions, a single protein band with high expression level was obtained (as shown in Figure 6).

[0029] Example 3: Establishing a CRISPR-SrCas13a-3 system-mediated rapid nucleic acid visualization detection technology to further evaluate whether the SrCas13a-3 protein possesses trans cleavage activity. Guide RNA that can pair with the target nucleic acid guides the SrCas13a-3 endonuclease to recognize and bind to the target nucleic acid; subsequently, its trans cleavage activity against any single-stranded nucleic acid is activated, thereby cleaving the RNA fluorescence-quenched reporter gene (ssRNA-FQ) in the reaction system; the trans cleavage function of the candidate gene editing endonuclease can be determined by the activated fluorescence intensity, background noise, and visual color changes.

[0030] To verify the effect of different target guide RNAs on activity, this example selected African swine fever virus P72 as the target single-stranded RNA (ssRNA). SrCas13a-3 and the known LwaCas13a protein were purified from prokaryotic expression. The following reaction system was then used: SrCas13a-3, 600 ng guide RNA, 2 μL 10×FO Buffer, 20 μM RNA fluorescence-quenched reporter gene, and 600 ng of PCR amplification of the target product. The negative control was no target added, incubated at 37℃ for 15 min. The trans-cleavage activity of the predicted proteins in vitro was determined by observing fluorescence intensity and background noise under a blue light detector. The results are shown in Figure 7, where 1 is the original solution, 2 is a 2-fold dilution, 3 is a 4-fold dilution, 4 is the positive control, 5 is the original solution negative control, 6 is a 2-fold dilution negative control, 7 is a 4-fold dilution negative control, and 8 is a blank negative control; the darker the color, the stronger the fluorescence and the better the cleavage activity. Therefore, the CRISPR / SrCas13a-3 system is suitable for nucleic acid fluorescence visualization detection.

[0031] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0032] In summary, the technical solution of this application has the following beneficial technical effects: 1. This invention provides a nuclease SrCas13a-3 identified by metagenomics combined with experiments, which is a new member of the CRISPR / Cas13a family with low homology.

[0033] 2. This invention also establishes a nucleic acid visualization detection technology based on the CRISPR / SrCas13a-3 system, which has broad application prospects in the field of nucleic acid detection.

Claims

1. A SrCas13a-3 protein, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

1.

2. A fusion protein, characterized in that, include: The SrCas13a-3 protein with the amino acid sequence shown in SEQ ID NO.1; or, a protein with more than 80% sequence identity compared to the amino acid sequence shown in SEQ ID NO.1, and which substantially retains the biological function derived from the sequence; or, a protein with one or more amino acid substitutions, deletions or additions compared to the amino acid sequence shown in SEQ ID NO.1, and which substantially retains the biological function derived from the sequence.

3. A nucleotide, characterized in that, The nucleotide is used to encode the SrCas13a-3 protein as described in claim 1, or to encode the protein as described in claim 2.

4. A carrier, characterized in that, Includes the nucleotides as described in claim 3.

5. A CRISPR-Cas system, characterized in that, The system comprises the SrCas13a-3 protein as described in claim 1 and at least one gRNA; the gRNA is capable of binding to the SrCas13a-3 protein as described in claim 1.

6. A host cell, characterized in that, It comprises the SrCas13a-3 protein as described in claim 1, or the fusion protein as described in claim 2, or the nucleotide as described in claim 3, or the vector as described in claim 4, or the CRISPR-Cas system as described in claim 5.

7. A method for editing target nucleic acids, characterized in that, The method comprises contacting a target nucleic acid with the SrCas13a-3 protein as described in claim 1, or the fusion protein as described in claim 2, or the polynucleotide as described in claim 3, or the vector as described in claim 4, or the CRISPR-Cas system as described in claim 5, or the host cell as described in claim 6, wherein the method is for non-disease diagnosis or treatment purposes.

8. The use of a SrCas13a-3 protein as described in claim 1, or a fusion protein as described in claim 2, or a polynucleotide as described in claim 3, or a vector as described in claim 4, or a CRISPR-Cas system as described in claim 5, or a host cell as described in claim 6, in editing target nucleic acids.

9. The use of a SrCas13a-3 protein as claimed in claim 1, or a fusion protein as claimed in claim 2, or a polynucleotide as claimed in claim 3, or a vector as claimed in claim 4, or a CRISPR-Cas system as claimed in claim 5, or a host cell as claimed in claim 6, in a preparation kit, wherein the use is for purposes other than disease diagnosis or treatment.

10. A visual nucleic acid detection kit, characterized in that, The kit includes a single-stranded DNA fluorescence-quenched reporter gene and a gRNA paired with a target nucleic acid, and also contains the SrCas13a-3 protein as described in claim 1, or the fusion protein as described in claim 2, or the polynucleotide as described in claim 3, or the vector as described in claim 4, or the CRISPR-Cas system as described in claim 5, or the host cell as described in claim 6.

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