Cas12a nuclease and application thereof

Abstract

The invention belongs to the technical field of biology, and provides Cas12a nuclease, a CRISPR / Cas system comprising the Cas12a nuclease and application of the CRISPR / Cas system, and the Cas12a nuclease has an amino acid sequence as shown in SEQ ID NO: 1. The invention also provides a related nucleic acid, an expression vector comprising the nucleic acid and a recombinant cell. The cleavage and accessory cleavage activities of Cas12a nuclease gene editing are both verified, and a CRISPR tool library available for in-vitro detection is expanded.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology. More specifically, a novel Cas12a nuclease, a CRISPR / Cas system including the same, and uses thereof are provided. Background Art

[0002] As an acquired immune mechanism of prokaryotes, the CRISPR / Cas system has RNA-mediated endonuclease activity. The earliest discovered system that can be used for gene editing is the Cas9 system mediated by crRNA and tracrRNA. In this system, certain Cas proteins, mainly Cas1 and Cas2, capture phage virus DNA or foreign plasmid DNA, insert it into its own direct repeat sequence to form a CRISPR sequence. The CRISPR sequence is transcribed into pre-crRNA, which is processed and modified into crRNA and forms an RNP with Cas9, having RNA-guided DNA endonuclease activity. When the bacterium is infected by the virus again, the invading DNA can be targeted and cleaved. This process also requires the participation of tracrRNA (Trans-activating crRNA) and the presence of a specifically recognized PAM. The CRISPR / Cas system has been widely used in gene editing because of its RNA-mediated endonuclease activity.

[0003] The CRISPR (clustered regularly interspaced short palindromic repeats) system can be divided into class 1 and class 2 according to homology. Class 1 includes type I, type III, and type IV, and class 2 includes type II, type V, and type VI. The most obvious feature of class 2 is that a complex is formed by a single Cas (CRISPR-associated protein) and crRNA (CRISPR RNA) to perform the targeted cleavage function, making the operation simpler. Although the class 2 system has only a single effector protein, significant differences have been found in the protein molecular weight, domains, crRNA, as well as PAM (Protospacer-adjacent motif) preference and nucleic acid cleavage mode of different types of effector proteins, which provides more flexible options for gene editing. For example, the well-known Cas9, Cas12, and Cas13a all belong to class 2. Compared with gene editing technologies such as ZFN (zinc-finger nucleases) and TALEN (transcription activator-like effector nucleases), the CRISPR / Cas system has obvious advantages such as good specificity, broader targeting, simple steps, the ability to edit multiple loci simultaneously, and low cost.

[0004] In 2015, it was first reported that the novel nuclease Cas12a could bind and cleave specific sites of target DNA under the guidance of single-stranded guide RNA, and the effectiveness of 8 Cas12a family proteins in genome editing in mammalian cells HEK293FT was verified. The shorter guide RNA backbone part (only crRNA) of Cas12a is easier to identify and predict, making the assembly of the Cas12a gene editing tool more convenient and efficient. Its recognition property of preferring T-rich PAM greatly enriches the site selection of the editing tool in genome editing.

[0005] In addition, it was found that after the Cas proteins of the Cas12 and Cas13 families exert their specific cleavage effects, they will activate their non-specific collateral cleavage activity. If short nucleotide molecules with fluorescence quenching are added to the reaction system, the activated Cas proteins will non-specifically cleave these short nucleotide molecules, releasing fluorescence signals to achieve the purpose of detecting the target sequence. Relying on its unique DNA or RNA specific cleavage and non-specific collateral cleavage activities, the CRISPR / Cas system has been successfully widely applied in nucleic acid detection.

[0006] From the initial demonstration of Cas9's gene-editing activity by Jennifer Anna Doudna's laboratory, to its application in mammalian cell gene editing by Zhang Feng's laboratory, to the nucleic acid detection methods developed using the accessory cleavage activity of Cas13a / Cas12a: SHERLOCK (Specific High-Sensitivity Enzymatic Reporter Unlocking) and DETECTR (Endonuclease Targeted CRISPR Trans Reporter), the CRISPR / Cas system has demonstrated significant commercial potential. Many research institutions and companies have begun applying for patent protection in gene-editing technologies, including CRISPR / Cas, and are striving to establish a monopoly in the industry. In particular, after the outbreak of the novel coronavirus in 2019, pathogen detection research based on nucleic acid detection using the CRISPR / Cas system has rapidly gained momentum. For example, Jennifer Anna Doudna's laboratory developed the DETECTR platform based on RPA (Recombinase Polymerase Amplification) and the ssDNA cleavage activity of Cas12a; Feng Zhang's laboratory developed the SHERLOCKv2 kit based on LAMP (loop-mediated isothermal amplification) and the ssRNA cleavage activity of lwaCas13a; and Tolo Biotech's HOLMES (one-HOur Low-cost Multipurpose highly Efficient System) method.

[0007] CRISPR technology has developed rapidly, enabling applications in gene editing in bacteria, archaea, and eukaryotic cells. However, it has also exposed numerous challenges. The CRISPR / Cas system relies heavily on the presence of a PAM sequence for target sequence recognition. This limitation significantly restricts its application in cell editing and nucleic acid detection. Furthermore, the issue of off-target effects, resulting from low specificity, large deletions, and complex gene rearrangements, requires further attention. Although engineered CRISPR / Cas systems have significantly improved their specificity and have been applied to gene therapy for conditions such as β-thalassemia and sickle cell anemia, their PAM limitations continue to constrain their application. Currently, commercialized Cas proteins include SpCas9, which is restricted by the 3'-terminal PAM NGG, and LbCas12a / AsCas12a, which is restricted by the 5'-terminal PAM TTTN. The Cas12a system's dependence on a T-rich PAM significantly limits its application in gene editing and nucleic acid detection.

[0008] For pathogen detection methods based on the CRISPR / Cas system, the target sequence must be conserved. Otherwise, when the pathogen mutates at this sequence, the detection method will become ineffective. For example, for the detection of the novel coronavirus, the available sites are very limited. According to the statistics of the National Center for Biotechnology Information, the novel coronavirus has generated more than 29,410 single nucleotide variants. Due to the differences in the cleavage efficiency of each Cas protein for different nucleotide sequences and the limitation of PAM, it may be difficult for a single Cas protein to achieve a high cleavage efficiency at the only available sites, thereby affecting the sensitivity of detection.

[0009] Commercial Cas12a (such as LbCas12a) has relatively strict PAM requirements, which limit the design of target sites, and its editing activity also needs to be further improved. On the other hand, in the application scenario of in vitro nucleic acid detection, according to the enzyme kinetics, in order to increase the reaction rate, enzymes with a higher optimal temperature for the enzymatic reaction are required. It is necessary to expand the existing Cas12a nuclease tool library to meet the diverse needs of gene editing and nucleic acid detection. Summary of the Invention

[0010] The object of the present invention is to provide a Cas12a nuclease, as well as a CRISPR / Cas system including the same and its uses.

[0011] Therefore, in the first aspect, the present invention provides a Cas12a nuclease having an amino acid sequence as shown in SEQ ID NO: 1.

[0012] In the second aspect, the present invention provides a CRISPR / Cas system comprising the Cas12a nuclease according to the first aspect of the present invention.

[0013] In the third aspect, the present invention provides an isolated nucleic acid encoding the nucleotide sequence of the Cas12a nuclease according to the first aspect of the present invention.

[0014] In the fourth aspect, the present invention provides an expression vector comprising the nucleic acid according to the third aspect of the present invention.

[0015] In the fifth aspect, the present invention provides a recombinant cell comprising the expression vector according to the fourth aspect of the present invention.

[0016] In the sixth aspect, the present invention provides a kit comprising the expression vector according to the fourth aspect of the present invention or the recombinant cell according to the fifth aspect of the present invention.

[0017] In a seventh aspect, the present invention provides the use of a Cas12a nuclease, a CRISPR / Cas system, a nucleic acid, an expression vector or a recombinant cell according to the present invention in gene editing.

[0018] The cleavage and collateral cleavage activities of the Cas12a nuclease gene editing of the present invention have been verified, expanding the available CRISPR tool library for in vitro detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The present invention will be described in detail with reference to the following drawings.

[0020] Figure 1 It is the PAM preference result of the chip detection system.

[0021] Figure 2 It is the in vitro dsDNA cleavage electrophoresis pattern of each Cas12a system. Among them, M represents DL2000; + represents Cas+crRNA+dsDNA; - represents dsDNA.

[0022] Figure 3(A) shows the evaluation of the collateral cleavage activity of each Cas12a at each TTTN site.

[0023] Figure 3(B) shows that the collateral cleavage activity of BEST11 is significant. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0024] As used herein, the terms "Crispr", "crispr" or "CRISPR" all refer to Clustered regularly interspaced short palindromic repeats. Whether the terms are in uppercase, lowercase or capitalized at the beginning, they are common expressions in the art. Correspondingly, there are different expressions in the CRISPR / Cas system due to the case of the letters. In addition, when representing bases, unless otherwise specified, the bases represented by the letters N and V have their usual meanings in the art, that is, N represents a random or arbitrary base A, T, C or G, and V represents a random or arbitrary base A, C or G.

[0025] The Cas9 enzyme cleaves at the target DNA site. Usually, the target site is determined in the following way: an RNA molecule called Crispr RNA (crRNA) uses a part of its sequence to base-pair and bind with an RNA molecule called tracrRNA, forming a chimeric RNA (tracrRNA / crRNA). Then, with the help of another part of the crRNA sequence, it base-pairs with the target DNA site. Thus, the chimeric RNA guides the Cas protein to bind to this target site for cleavage. This chimeric RNA is also called guide RNA. Different from the CRISPR / Cas9 system, the Cas12 (Cpf1) enzyme can process the crRNA precursor independently and then use the processed crRNA to specifically target and cleave DNA, without the need for ribonucleases and tracrRNA from the host cell.

[0026] The targeting specificity of Crispr is determined by two parts. One part is the base pairing between the RNA chimera and the target DNA, and the other part depends on the Cas protein and a short DNA sequence. This short DNA sequence is at the 3' end of the target DNA and is called the protospacer adjacent motif (PAM).

[0027] If the PAM sequence is strict (for example, it may be specific several bases), then the number of target sites that the Cas protein can edit is relatively small, thus limiting the application of the CRISPR / Cas system. Both SpCas9 and LbCpf1 have relatively strict PAM sequences, which restricts the design of the targeting sites. For example, the PAM sequence recognized by the SpCas9 nuclease is NGG, located at the 3' end of the targeting sequence, and it cleaves to form blunt ends 3 bp away from the PAM sequence. Due to its PAM sequence being only NGG, the application of this editing system is limited.

[0028] In this article, nucleic acid sequences include DNA or RNA. Mentioning a DNA sequence also includes mentioning its corresponding RNA sequence, and vice versa. Those skilled in the art know how to convert between the two and know whether to use a DNA sequence or an RNA sequence in a specific situation.

[0029] In this text, the mention of a nucleic acid sequence includes the mentioned sequence itself, its reverse complementary sequence, and the complementary double-stranded sequence formed by them. Those skilled in the art know how to obtain the reverse complementary sequence from a nucleic acid sequence. The function of a sequence referred to herein includes that the sequence itself has this function, or its reverse complementary sequence has this function. For example, the mention of a nucleic acid sequence encoding a protein such as the Cas12a nuclease includes the mention that this nucleic acid sequence encodes this protein, or its reverse complementary sequence encodes this protein. In applications, those skilled in the art will know to select the sequence itself or its reverse complementary sequence, or the double-stranded formed by them.

[0030] Therefore, in this text, as long as it does not conflict with the common knowledge in the art, the mention of nucleic acid is equivalent to the mention of any one or more of the corresponding DNA, RNA, DNA double-strand, RNA double-strand, and DNA-RNA double-strand.

[0031] The inventors discovered a new Cas12a protein in the Sedimentisphaera cyanobacteriorum strain L21-RPul-D3 and named it BEST11. This Cas12a protein has a wider PAM site than lbCas12a. When performing nucleic acid detection based on Cas12a, the design of the target site and the corresponding crRNA follows: (1) select a conserved sequence in the target gene, (2) then select possible target sites in the conserved sequence according to the PAM restriction of the Cas nuclease, and (3) design a corresponding crRNA for the target site. Subsequently, the enzymatic reaction activity of the Cas nuclease at different target sites is evaluated through a fluorescence reporting system. For the reaction system, the stability of the crRNA and the intensity of the fluorescence signal response are improved by adjusting the buffer system where the crRNA is located, the ionic strength, and the concentration of Mg 2+ ions.

[0032] Therefore, the present invention provides a Cas12a nuclease, and a CRISPR / Cas system including the same and its uses.

[0033] In a first aspect, the present invention provides a Cas12a nuclease having an amino acid sequence as shown in SEQ ID NO: 1.

[0034] In a second aspect, the present invention provides a CRISPR / Cas system including the Cas12a nuclease according to the first aspect of the present invention.

[0035] In some embodiments, the CRISPR / Cas system further includes at least one of the following:

[0036] crRNA, tracrRNA, or chimeric RNAs formed by crRNA and tracrRNA. These RNAs can help the CRISPR / Cas system perform gene editing functions.

[0037] In a third aspect, the present invention provides an isolated nucleic acid encoding a nucleotide sequence of the Cas12a nuclease according to the first aspect of the present invention.

[0038] In some embodiments, the nucleic acid further comprises crRNA, tracrRNA, or chimeric RNAs formed by crRNA and tracrRNA, or DNA sequences corresponding to the above sequences.

[0039] In some embodiments, the nucleic acid is DNA or RNA.

[0040] In a fourth aspect, the present invention provides an expression vector comprising the nucleic acid according to the third aspect of the present invention. By constructing the above nucleic acid with a vector, an expression vector is obtained, and these expression vectors can express the corresponding Cas12a nuclease in target cells, thereby performing corresponding gene editing in target cells. Commonly used vectors can be plasmids, lentiviruses, etc., for example, it can be pET 28a vector, pMD19 vector, etc.

[0041] In a fifth aspect, the present invention provides a recombinant cell comprising the expression vector according to the fourth aspect of the present invention. By introducing the expression vector into a cell, a recombinant cell is formed, and by using the expression vector to express the corresponding Cas12a nuclease, gene editing of the recombinant cell can be achieved. In some embodiments, the recombinant cell can be a eukaryotic cell, such as a plant cell or an animal cell.

[0042] In a sixth aspect, the present invention provides a kit comprising the expression vector according to the fourth aspect of the present invention or the recombinant cell according to the fifth aspect of the present invention.

[0043] In a seventh aspect, the present invention provides the use of the Cas12a nuclease, CRISPR / Cas system, nucleic acid, expression vector, or recombinant cell according to the present invention in gene editing, wherein the Cas12a nuclease is the Cas protease according to the first aspect of the present invention, the CRISPR / Cas system is the CRISPR / Cas system according to the second aspect of the present invention, the nucleic acid is the nucleic acid according to the third aspect of the present invention, the expression vector is the expression vector according to the fourth aspect of the present invention, and the recombinant cell is the recombinant cell according to the fifth aspect of the present invention. For example, the use can be cell gene editing, gene therapy, nucleic acid detection, high-throughput sequencing. In a preferred embodiment, the use is cell gene editing.

[0044] The implementation scheme of the present invention will be described in detail below in conjunction with embodiments. Those skilled in the art will understand that the following embodiments are illustrative only and should not be construed as limiting the scope of the present invention. For those not specifying specific techniques or conditions in the embodiments, the techniques or conditions described in the literature in the art or according to the product specifications are followed. For reagents or instruments not indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0045] Example 1: Experiment on Expressing and Purifying the Effector Protein of the Cas12a System

[0046] Before protein expression and purification, according to the protein sequence (as shown in SEQ ID NO: 1), the physicochemical properties of the protein, including isoelectric point, relative molecular mass, extinction coefficient, etc., were analyzed using the ProtParam tool provided by ExPasy (https: / / web.expasy.org / protparam / ) to adjust the purification process and buffer.

[0047] The plasmid was introduced into competent cells BL21(DE3) (Takara) by heat shock transformation, and 300 μL of antibiotic-free medium was added for culturing for 60 min. The plate was coated (LB plate, kanamycin resistance) and cultured overnight at 37 °C, and a single colony was selected for scale-up culture. The BL21(DE3) cells expressing the protein were cultured in LB medium (supplemented with 50 mg / L kanamycin) at 37 °C until the OD600 reached 0.6, and protein expression was induced by adding 0.5 mM isopropyl β-D-thiogalactoside (IPTG). The BL21(DE3) cells were further cultured overnight at 16 °C (low-temperature induction). The cells were collected by centrifugation at 6000 rpm for 10 min at 4 °C, and the collected cells were resuspended at a ratio of 1 g:20 mL of binding buffer (50 mM Tris-HCl, pH 7.8, 500 mM NaCl, 5 mM imidazole), and the cells were lysed by ultrasonic disruption. Lysozyme (10 mg / mL) and PMSF (0.1 M) were added according to a volume ratio of 1:100 before ultrasonic disruption. The sonicated cells were centrifuged at 12000 rpm for 60 min at 4 °C, and the supernatant was collected.

[0048] (1) Affinity chromatography. Since the target protein carries a His tag, the inventor first selected a Ni-NTA gravity column for affinity chromatography purification of the protein. Before use, the packing material should be washed three times with water and once with binding buffer. The packing material was combined with the cell supernatant for 30 min, with sufficient shaking every 5 min to allow as much of the target protein as possible to bind to the Ni on the packing material. The flow-through was collected. 5% elution buffer (50 mM Tris-HCl, pH 7.8, 500 mM NaCl, 500 mM imidazole) was used for washing impurities, and the impurity-washing components were collected. 50% elution buffer was used to elute the target protein. The target protein was collected. The packing material was rinsed with elution buffer, and the components were collected. Samples of all the collected components were taken for SDS-PAGE to confirm the purification efficiency and recovery efficiency of the target protein.

[0049] (2) Size-exclusion chromatography. The target protein fraction was concentrated to a volume <2 mL using a 50K ultrafiltration tube and filtered through a 0.56 μm filter membrane. The protein with different molecular weights was separated using AKTA (Cytiva) through HiLoad 16 / 600 Superdex 200 pg (Cytiva). The sample was loaded onto a 2 mL sample loop and passed through the chromatography column at a flow rate of 0.5 mL / min in a low-salt buffer (30 mM phosphate, pH 7.0, 150 mM NaCl, 0.4 mM DTT). The collection tray collected continuously. Samples of the collection tubes at all UV peaks were taken for SDS-PAGE electrophoresis to confirm the target protein fraction.

[0050] (3) Ion-exchange chromatography. The low-salt buffer was added to the target protein fraction to make it up to 20 mL. It was filtered through a 0.56 μm filter membrane, and the protein was separated and purified using AKTA through HiTrap Capto S ImpRes (Cytiva). The target protein was bound to the column and eluted with a gradient of 50% high-salt buffer (30 mM phosphate, pH 7.0, 1 M NaCl, 0.4 mM DTT). The collection tray collected continuously. Samples of each of the collected tubes were taken for SDS-PAGE electrophoresis to confirm the purification efficiency and recovery efficiency. For systems with poor purity (<90%), cation-exchange was used for further purification. The components in the main peak collection tube were concentrated to a volume <3 mL using a 50 kDa ultrafiltration tube and made up to 20 mL with low-salt buffer, and the above process was repeated to reduce the NaCl content in the target protein fraction. The protein was separated and purified using AKTA through HiTrap Capto Q ImpRes (Cytiva). The target protein was bound to the column and eluted with a gradient of 50% high-salt buffer. The collection tray collected continuously. Samples of each of the collected tubes were taken for SDS-PAGE electrophoresis to confirm the purification efficiency and recovery efficiency.

[0051] Concentrate the protein using a 50K ultrafiltration tube, measure the absorbance A280 with an enzyme-linked immunosorbent assay (ELISA) reader (1 Abs = 1 mg / mL), and divide by the extinction coefficient to obtain the true protein concentration. Add 70% sterilized glycerol at a volume ratio of 1:1 and store at -20 °C.

[0052] Example 2: Experiment for identifying the Cas12a PAM sequence

[0053] Use the DocMF method (Li et al., "DNB-based on-chip motif finding: A high-throughput method to profile different types of protein-DNA interactions." Science Advances 6.31 (2020): eabb3350.) published by the applicant to identify the PAM of the Cas12a nuclease. Bioinformatics analysis shows that the PAM sequence identified by BEST11 is TTN (as Figure 1 ).

[0054] Table 1: Nucleic acid sequences used in PAM identification.

[0055]

[0056] Example 3: Experiment on in vitro cleavage of the Cas12a system

[0057] Conduct the experiment on in vitro cleavage of the Cas12a system according to the following protocol:

[0058] 1. Synthesize crRNA in vitro and the double-stranded DNA substrate used in the in vitro cleavage experiment, and measure the DNA concentration not less than 40 ng / μL and the crRNA concentration not less than 800 ng / μL;

[0059] 2. Mix 2 μL of 10× NEB buffer 3.1, 2 μL of 10 - 30 μM Cas12a protein, and 1 - 2 μL of crRNA, add RNase-free water to 16 μL, shake well, incubate at room temperature for 15 min, and then add 4 μL of substrate DNA and incubate at 37 °C for 3 - 4 hours;

[0060] 3. Take 10 μL of the cleavage product and perform 1% agarose gel electrophoresis.

[0061] Agarose gel electrophoresis shows ( Figure 2 ), after BEST9 binds to the corresponding crRNA (the nucleic acid sequences used are shown in Table 1), it can cleave the target double-stranded DNA, and the fragment size of the cleavage product is consistent with the expectation.

[0062] The target double-stranded DNA sequence is shown in SEQ ID NO: 4.

[0063] Example 4: Experiment on collateral cleavage of the Cas12a system

[0064] (1) Preparation of crRNA

[0065] RNA was transcribed by MEGAshortscript TM T7 Transcription Kit, and the corresponding DNA template was synthesized by BGI Tech Solutions Co., Ltd. 2 pmol of double-stranded DNA template was added, and the transcription was carried out by incubating in a Bio-rad S1000TM polymerase chain reaction (PCR) instrument at 37 °C for 12 hours. RNA was purified by saturated phenol, chloroform and isopropanol solutions. Then, it was quantified by Qubit TM RNA HS AssayKit.

[0066] (2) Preparation of cleavage substrates

[0067] (2.1) The cleavage substrate was amplified from the plasmid containing the cleavage substrate sequence by PCR for experimental use. The amplified product was identified by 1.5% agarose gel electrophoresis. According to the corresponding band size, the gel was cut out, and the cleavage substrate with a higher concentration was obtained by gel recovery and purification through Qubit TM dsDNA HS AssayKit. Among them, the cleavage substrate sequence is shown in SEQ ID NO: 5, and its PCR primer sequences COVID-2019primerF are shown in SEQ ID NO: 6, and COVID-2019primerR are shown in SEQ ID NO: 7.

[0068] (2.2) The AAVS1 substrate was prepared in the same way as (2.1). Among them, the AAVS1 substrate sequence is shown in SEQ ID NO: 8, and its PCR primer sequences AAVS1-F are shown in SEQ ID NO: 9, and AAVS1-R are shown in SEQ ID NO: 10.

[0069] (3) Cleavage experiment and results

[0070] The purified transcribed crRNA was diluted with 20 mM sodium acetate and 40 U / μL RNase inhibitor plus nuclease-free water to a final concentration of 300 nM crRNA, with the final concentrations of sodium acetate and RNase inhibitor being 1 mM and 4 U / μL, respectively. Then, each Cas12a was diluted with 10X NEBuffer 2.1, 70% sterile glycerol, 50 mM DTT and nuclease-free water to a final concentration of 1 μM. The final concentrations of NEBuffer 2.1, glycerol, and DTT were 1X, 35%, and 0.5 mM, respectively. The 1 μM Cas protein was mixed with 1X NEBuffer 2.1 in equal volume at a 1:1 ratio and reacted with 600 nM crRNA at room temperature for 10 minutes to form a total volume of 4 μL RNP (RNA-protein complex). Then, 2 μL of CRISPR reporter buffer 2.1 (prepared as shown in Table 2 below) and 1 μL of 10 ng / μL cleavage substrate were added, and nuclease-free water was added to make the final system volume 10 μL, and then the digestion reaction was carried out. Incubate at 45 °C for 40 minutes and take pictures once per minute with a qPCR instrument.

[0071] The FAM-reporter sequence is 5’-FAM-AAAAAA-BHQ1 (SEQ ID NO: 11)

[0072] Table 2 Preparation table of CRISPR reporter buffer 2.1

[0073]

[0074] (4) Activity evaluation of Cas12a

[0075] The optimized reaction system in 4.1 - 4.4 was used for the activity evaluation of this Cas12a, as shown in Figure 3. Significant collateral cleavage activity was observed in BEST11, which can be applied to nucleic acid detection.

[0076] Table 3 crRNA sequences used in Example 4

[0077]

Claims

1. A Cas12a nuclease having an amino acid sequence as shown in SEQ ID NO:

1.

2. A CRISPR / Cas system comprising the Cas12a nuclease according to claim 1.

3. The CRISPR / Cas system according to claim 2, wherein the CRISPR / Cas system further comprises at least one of the following: crRNA, tracrRNA, or a chimeric RNA formed by crRNA and tracrRNA.

4. An isolated nucleic acid having a nucleotide sequence encoding the Cas12a nuclease according to claim 1.

5. The nucleic acid according to claim 4, wherein the nucleotide further comprises crRNA, tracrRNA, or a chimeric RNA formed by crRNA and tracrRNA, or a DNA sequence corresponding to the above sequence.

6. The nucleic acid according to claim 4 or 5, wherein the nucleic acid is DNA or RNA.

7. An expression vector comprising the nucleic acid according to any one of claims 4-6.

8. A recombinant cell comprising the expression vector according to claim 7.

9. A kit comprising the expression vector according to claim 7 or the recombinant cell according to claim 8.

10. Use of the Cas12a nuclease according to claim 1, the CRISPR / Cas system according to claim 2 or 3, the nucleic acid according to any one of claims 4-6, the expression vector according to claim 7, or the recombinant cell according to claim 8 in gene editing, preferably, the use is cell gene editing, nucleic acid detection, high-throughput sequencing.

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