Cas12a nuclease and applications thereof

Abstract

The application belongs to the technical field of biotechnology, and provides a Cas12a nuclease, a CRISPR / Cas system comprising the same and application thereof, wherein the Cas12a nuclease has an amino acid sequence as shown in SEQ ID NO:1. The application further provides related nucleic acids, expression vectors comprising the nucleic acids and recombinant cells. The genome editing activity of the Cas12a nuclease in mammalian cells has been verified, which provides more tools and effective site selection for the application direction of in vivo gene editing.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to providing a novel Cas12a nuclease, a CRISPR / Cas system including the same, and its uses. Background Technology

[0002] The CRISPR / Cas system, as an acquired immune mechanism in prokaryotes, possesses RNA-mediated endonuclease activity. The earliest discovered system for gene editing was the Cas9 system, mediated by crRNA and tracrRNA. This system uses certain Cas proteins, primarily Cas1 and Cas2, to capture bacteriophage viral DNA or exogenous plasmid DNA, inserting it into its own orthogonal repeat sequence to form a CRISPR sequence. The CRISPR sequence is transcribed into pre-crRNA, which is then processed and modified into crRNA to form an RNP with Cas9, possessing RNA-directed DNA endonuclease activity. When bacteria are reinfected by the virus, 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 is widely used in gene editing due to its RNA-mediated endonuclease activity.

[0003] The CRISPR (clustered regularly interspaced short palindromic repeats) system can be divided into class 1 and class 2 based on homology. Class 1 includes type I, type III, and type IV, while class 2 includes type II, type V, and type VI. The most distinctive feature of class 2 is that it consists of a single Cas (CRISPR-associated protein) protein forming a complex with crRNA (CRISPR RNA) to perform targeted cleavage, making it simpler to operate. Although the class 2 system only has a single effector protein, the different types of effector proteins discovered have significant differences in protein molecular weight, domains, crRNA, PAM (Protospacer-adjacent motif) preference, and nucleic acid cleavage modes, providing more flexible options for gene editing. For example, the well-known Cas9, Cas12, and Cas13a all belong to class 2. Compared to gene editing technologies such as ZFN (zinc-finger nucleases) and TALEN (transcription activator-like effector nucleases), the CRISPR / Cas system has significant advantages such as good specificity, broader targeting, simpler procedures, simultaneous editing of multiple sites, and lower cost.

[0004] In 2015, the novel Cas12a nuclease was first reported to bind to and cleave target DNA at specific sites under the guidance of single-stranded guide RNA. The effectiveness of eight Cas12a family proteins in genome editing in mammalian HEK293FT cells was also verified. The shorter guide RNA backbone (crRNA only) compared to Cas9 makes Cas12a gene editing tools easier to identify and predict, resulting in more convenient and efficient assembly. Its preference for T-rich PAM recognition greatly enriches the site selection capabilities of editing tools in genome editing. Subsequently, more types of Cas12a tools have been explored and used. Researchers have found that the VA Cas12a system is conserved, with different source systems showing some similarity in the sequence, secondary structure, and PAM selection of mature crRNA. This characteristic gives most Cas12a genome editing tools an "inherent advantage" in T-rich nucleic acid sequence target site selection. Of course, to adapt to different application scenarios, a series of new Cas12a proteins and variants have emerged, exhibiting more lenient PAM recognition and fewer off-target events.

[0005] Furthermore, research has revealed that Cas12 and Cas13 family proteins, after performing specific cleavage, activate their non-specific collateral cleavage activity. If fluorescence-quenched short nucleotide molecules are added to the reaction system, the activated Cas proteins will non-specifically cleave these short nucleotide molecules, releasing a fluorescent signal to detect the target sequence. The CRISPR / Cas system, with its unique DNA or RNA specific cleavage and non-specific collateral cleavage activity, has been successfully and widely applied in nucleic acid detection.

[0006] From Jennifer Anna Doudna's lab first demonstrating the gene-editing activity of Cas9, to Zhang Feng's lab applying it to mammalian cell gene editing, and then to the nucleic acid detection methods developed using the auxiliary cleavage activity of Cas13a / Cas12a: SHERLOCK (Specific High-Sensitivity Enzymatic Reporter UnLOCKing) and DETECTR (Endonuclease Targeted CRISPR Trans Reporter), the CRISPR / Cas system has shown extremely high commercial application potential. Many research institutions and companies have begun to apply for patent protection in the field of gene-editing technology, including CRISPR / Cas, creating an industry monopoly.

[0007] CRISPR technology has developed rapidly and can be applied to gene editing in bacteria, archaea, and eukaryotic cells, but it has also exposed many problems. The CRISPR / Cas system's recognition of target sequences is heavily dependent on the presence of PAM sequences, and the limitations of PAM significantly restrict its application in cell editing and nucleic acid detection. Secondly, off-target effects due to low specificity, as well as large fragment deletions and complex gene recombination, require further attention. Although engineered CRISPR / Cas systems have significantly improved specificity and have been applied to gene therapy, such as for β-thalassemia and sickle cell anemia, the limitations of PAM still restrict their applicability. Currently, commercially available Cas proteins mainly include SpCas9, which is restricted by the 3' PAM NGG, and LbCas12a / AsCas12a, which is restricted by the 5' PAM TTTN. The T-rich PAM dependence of the Cas12a system greatly limits its application in gene editing and nucleic acid detection.

[0008] Commercial Cas12a (such as LbCas12a) has stringent PAM requirements, which limits target site design, and its editing activity needs further improvement. On the other hand, in in vitro nucleic acid detection applications, based on enzyme reaction kinetics, enzymes with higher optimal reaction temperatures are needed to improve reaction speed. Therefore, the existing Cas12a nuclease tool library needs to be expanded to meet diverse gene editing and nucleic acid detection needs. Summary of the Invention

[0009] The purpose of this invention is to provide a Cas12a nuclease, a CRISPR / Cas system including the same, and its uses.

[0010] Therefore, in a first aspect, the present invention provides a Cas12a nuclease having an amino acid sequence as shown in SEQ ID NO: 1.

[0011] In a second aspect, the present invention provides a CRISPR / Cas system comprising the Cas12a nuclease described in the first aspect of the present invention.

[0012] In a third aspect, the present invention provides an isolated nucleic acid that encodes the nucleotide sequence of the Cas12a nuclease according to the first aspect of the present invention.

[0013] In a fourth aspect, the present invention provides an expression vector comprising the nucleic acid described in the third aspect of the present invention.

[0014] In a fifth aspect, the present invention provides a recombinant cell comprising the expression vector described in the fourth aspect of the present invention.

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

[0016] 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 invention in gene editing.

[0017] The genome editing activity of the Cas12a nuclease of this invention in mammalian cells has been verified, providing more tools and effective site selection for in vivo gene editing applications. Attached Figure Description

[0018] The present invention will be described in detail with reference to the following figures.

[0019] Figure 1This is an electrophoresis image of T7E1 in mammalian cell genome editing (AAVS1) using the BEST7 system, where M represents DL2000; + indicates that T7E1 has been added; and - indicates that T7E1 has not been added.

[0020] Figure 2 The BEST7 PAM preference (5'TTTN) is described.

[0021] Figure 3 The in vitro cutting efficiency of BEST7 and LbaCas12a is compared.

[0022] Figure 4 The accessory cleavage activity of BEST7 and LbaCas12a is described.

[0023] Figure 5 The targeting regions of BEST7 and SpCas9 in the HBG promoter are described.

[0024] Figure 6A and Figure 6B This is a peak diagram of SpCas9 and BEST7 after editing at the HBG site. Detailed Implementation

[0025] In this paper, the terms "Crispr," "crispr," or "CRISPR" all refer to clustered regularly interspaced short palindromic repeats. Whether the terminology is uppercase, lowercase, or begins with a capital letter, it represents the commonly used terminology in the field. Correspondingly, the CRISPR / Cas system uses different terminology due to capitalization. Furthermore, when representing bases, unless otherwise specified, the letters N and V represent bases with their usual meanings in the field: N represents a random or arbitrary base A, T, C, or G, and V represents a random or arbitrary base A, C, or G.

[0026] The Cas9 enzyme cleaves target DNA at specific sites, typically by using a CRISPR RNA (crRNA) molecule to bind a portion of its sequence to a tracrRNA molecule, forming a chimeric RNA (tracrRNA / crRNA). The tracrRNA then guides the Cas protein to the target DNA site for cleavage via a portion of its sequence. This chimeric RNA is also known as a guide RNA. Unlike the CRISPR / Cas9 system, the Cas12 (Cpf1) enzyme can independently process the crRNA precursor and then specifically target and cleave DNA using the resulting crRNA, without requiring ribonucleases or tracrRNA from the host cell.

[0027] The targeting specificity of Caspr is determined by two parts: one part is the base pairing between the RNA chimera and the target DNA, and the other part relies on the Cas protein and a short DNA sequence located at the 3' end of the target DNA, called the protospacer adjacent motif (PAM).

[0028] If the PAM sequence is restrictive (e.g., it may consist of only a few specific bases), the number of target sites that Cas proteins can edit is relatively small, thus limiting the application of the CRISPR / Cas system. Both SpCas9 and LbCpf1 have relatively restrictive PAM sequences, which limits the design of target sites. For example, the PAM sequence recognized by the SpCas9 nuclease is NGG, located at the 3' end of the target sequence, and is cleaved 3 bp from the PAM sequence to form a blunt end. Because its PAM sequence is only NGG, the application of this editing system is limited.

[0029] In this document, nucleic acid sequences include DNA or RNA, and reference to a DNA sequence also includes reference to its corresponding RNA sequence, and vice versa. Those skilled in the art will understand how to convert between the two and will know whether to use a DNA sequence or an RNA sequence in a specific context.

[0030] In this document, references to nucleic acid sequences include the sequence itself, its inverse complementary sequence, and the complementary double-stranded sequence formed by them. Those skilled in the art will understand how to derive the inverse complementary sequence from a nucleic acid sequence. The function of a sequence referred to herein includes the sequence itself having that function, or its inverse complementary sequence having that function. For example, referring to a nucleic acid sequence encoding a protein such as the Cas12a nuclease includes referring to the nucleic acid sequence encoding that protein, or its inverse complementary sequence encoding that protein. In application, those skilled in the art will understand the importance of selecting the sequence itself, its inverse complementary sequence, or the double-stranded sequence formed by them.

[0031] Therefore, in this article, as long as it does not contradict common sense in the field, mentioning nucleic acid sequences is equivalent to mentioning any one or more of the corresponding DNA, RNA, DNA double strand, RNA double strand, and DNA-RNA double strand.

[0032] The inventors discovered a novel Cas12a protein in the Clostridium xylanovorans HESP1 strain and named it BEST7. After optimizing the new Cas12a protein based on human codons, a genome editing plasmid was constructed. Efficient systemic delivery was achieved via liposome transfection. The editing activity of the new system was verified at the AAVS1 site of the human genome's "Safe harbor gene," and effective editing sites were screened, laying the foundation for future tool or system modifications based on the new system.

[0033] Therefore, the present invention provides a Cas12a nuclease, a CRISPR / Cas system including the same, and its uses.

[0034] In a first aspect, the present invention provides a Cas12a nuclease having an amino acid sequence as shown in SEQ ID NO: 1.

[0035] In a second aspect, the present invention provides a CRISPR / Cas system comprising the Cas12a nuclease described in the first aspect of the present invention.

[0036] In some embodiments, the CRISPR / Cas system further includes at least one of the following:

[0037] crRNA, tracrRNA, or chimeric RNA formed from crRNA and tracrRNA. These RNAs help the CRISPR / Cas system perform gene editing functions.

[0038] In a third aspect, the present invention provides an isolated nucleic acid that encodes the nucleotide sequence of the Cas12a nuclease according to the first aspect of the present invention.

[0039] In some implementations, the nucleic acid may further include crRNA, tracrRNA, or a chimeric RNA formed from crRNA or tracrRNA, or a DNA sequence corresponding to the above sequence.

[0040] In some implementations, the nucleic acid is DNA or RNA.

[0041] In a fourth aspect, the present invention provides an expression vector comprising the nucleic acid described in the third aspect of the invention. The above-described nucleic acid is used to construct an expression vector, which can express the corresponding Cas12a nuclease in target cells, thereby enabling corresponding gene editing in the target cells. Commonly used vectors can be plasmids, lentiviruses, etc., such as pET 28a vector, pMD19 vector, etc.

[0042] In a fifth aspect, the present invention provides a recombinant cell comprising the expression vector described in the fourth aspect of the invention. By introducing the expression vector into the cell to form a recombinant cell, and by expressing the corresponding Cas12a nuclease using the expression vector, gene editing of the recombinant cell can be achieved. In some embodiments, the recombinant cell may be a eukaryotic cell, such as a plant cell or an animal cell.

[0043] In a sixth aspect, the present invention provides a kit comprising the expression vector according to the fourth aspect of the invention or the recombinant cells according to the fifth aspect of the invention.

[0044] 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 described in the first aspect of the present invention, the CRISPR / Cas system is the CRISPR / Cas system described in the second aspect of the present invention, the nucleic acid is the nucleic acid described in the third aspect of the present invention, the expression vector is the expression vector described in the fourth aspect of the present invention, and the recombinant cell is the recombinant cell described in the fifth aspect of the present invention. For example, the use may be cell gene editing, gene therapy, nucleic acid detection, or high-throughput sequencing. In a preferred embodiment, the use is cell gene editing.

[0045] The embodiments of the present invention will be described in detail below with reference to examples. Those skilled in the art will understand that the following examples are merely illustrative and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not specified in the examples, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0046] Example 1: Expression and purification of various Cas12a system effector proteins

[0047] Before protein expression and purification, the physicochemical properties of the protein, including isoelectric point, relative molecular mass, and extinction coefficient, were analyzed using the ProtParam tool provided by ExPasy (https: / / web.expasy.org / protparam / ) based on the protein sequence (as shown in Table 1) in order to adjust the purification process and buffer.

[0048] The plasmid was introduced into competent BL21(DE3)(Takara) cells using a heat shock transformation method, and the cells were cultured in 300 μL of antibiotic-free medium for 60 min. The cells were then plated (LB plate, kanamycin resistant) and cultured overnight at 37°C. Single colonies were selected for expansion. 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. Protein expression was induced by adding 0.5 mM isopropyl β-D-thiogalactopyranoside (IPTG). BL21(DE3) cells were then cultured overnight at 16°C (low-temperature induction). Collect bacterial cells by centrifugation at 6000 rpm and 4°C for 10 min. Resuspend the collected bacterial cells in a 1 g:20 mL binding buffer (50 mM Tris-HCl, pH 7.8, 500 mM NaCl, 5 mM imidazole). Lyse the cells by sonication. Before sonication, add lysozyme (10 mg / mL) and PMSF (0.1 M) at a volume ratio of 1:100. Centrifuge the sonicated bacterial cells at 12000 rpm and 4°C for 60 min, and collect the supernatant.

[0049] (1) Affinity chromatography. Because the target protein carries a His tag, the inventors first selected a Ni-NTA gravity column for affinity chromatography to purify the protein. Before use, the packing material should be washed three times with water and once with binding buffer. The packing material is bound to the bacterial supernatant for 30 min, with thorough shaking every 5 min to allow as much of the target protein as possible to bind to the Ni on the packing material. Flow-through is collected. The material is washed with 5% elution buffer (50 mM Tris-HCl, pH 7.8, 500 mM NaCl, 500 mM imidazole), and the washed components are collected. The target protein is eluted with 50% elution buffer. The target protein is collected. The packing material is rinsed with elution buffer, and the fractions are collected. All collected fractions are sampled and subjected to SDS-PAGE to confirm the purification and recovery efficiency of the target protein.

[0050] (2) Molecular sieve 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. Proteins of different molecular weights were separated using AKTA (Cytiva) via a HiLoad 16 / 600 Superdex 200 pg (Cytiva) filter. Samples were loaded onto a 2 mL loop and passed through the chromatography column at a flow rate of 0.5 mL / min in low-salt buffer (30 mM phosphate, pH 7.0, 150 mM NaCl, 0.4 mM DTT). Continuous collection was performed. Samples from all UV peak collection tubes were taken for SDS-PAGE electrophoresis to confirm the target protein fraction.

[0051] (3) Ion exchange chromatography. Add low-salt buffer to 20 mL to obtain the target protein fraction. Filter using a 0.56 μm filter membrane and purify the protein using AKTA via HiTrap Capto SP ImpRes (Cytiva). Attach the target protein to the column and elute with a gradient of 50% high-salt buffer (30 mM phosphate, pH 7.0, 1 M NaCl, 0.4 mM DTT). Collect continuously. Perform SDS-PAGE electrophoresis on samples from each collected tube to confirm purification and recovery efficiency. For systems with poor purity (<90%), further purification is performed using cation exchange. Concentrate the main peak fraction in the collection tube to a volume <3 mL using a 50 kDa ultrafiltration tube, add low-salt buffer to 20 mL, and repeat the above process to reduce the NaCl content in the target protein fraction. Purify the protein using AKTA via HiTrap Capto Q ImpRes (Cytiva). Attach the target protein to the column and elute with a gradient of 50% high-salt buffer. Collect continuously. The collected samples were subjected to SDS-PAGE electrophoresis to confirm the purification and recovery efficiency.

[0052] Protein was concentrated using 50K ultrafiltration tubes, and the absorbance (A280) was measured using a microplate reader (1 Abs = 1 mg / mL). The absorbance was then divided by the extinction coefficient to obtain the true protein concentration. 70% sterile glycerol was added at a 1:1 volume ratio and stored at -20℃.

[0053] Table 1: Sequences of proteins expressed and purified in Example 1

[0054] name SEQ ID NO: Wild-type BEST7 sequence 1 BEST7 gene coding sequence 2

[0055] Example 2: Extracorporeal Dissection using the Cas12a System

[0056] The following protocol was used to conduct an in vitro dissection experiment of the Cas12a system:

[0057] 1. Synthesize crRNA in vitro and use double-stranded DNA substrates for in vitro cleavage experiments. The DNA concentration should be no less than 40 ng / μL and the crRNA concentration should be no less than 800 ng / μL.

[0058] 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 a final volume of 16 μL, vortex to mix, incubate at room temperature for 15 min, add 4 μL of substrate DNA, and incubate at 37°C for 3-4 hours.

[0059] 3. Take 10 μL of the cleavage product and perform 1% agarose gel electrophoresis.

[0060] The target double-stranded DNA sequence is shown in SEQ ID NO: 3.

[0061] Example 3: Mammalian Cell Genome Editing Experiment using the Cas12a System

[0062] (1) Human cell culture

[0063] The human embryonic kidney cell-derived cell line HEK293T was selected for in vivo editing activity testing.

[0064] The culture conditions were as follows: DMEM medium (high glucose, Gibco) containing 10% fetal bovine serum (FBS, Gibco), 1% non-essential amino acids (NEAA, Gibco), and 1% glutamine (GlutaMAX, Gibco), at 37°C and 5% CO2 concentration.

[0065] (2) Plasmid preparation

[0066] For editing HEK293T cells, the inventors used the endogenous gene AAVS1 (gene bank ID: AC005782.1) for targeted cleavage verification.

[0067] The nucleotide sequence of the target region of AAVS1 is shown in SEQ ID NO: 4.

[0068] Based on the T-rich PAM characteristics of the Cas12a family, three target sites were designed for this gene (see Table 2). The crRNA scaffold information corresponding to each protein is shown in Table 3. Gene editing plasmids for the corresponding proteins were designed and synthesized. The specific nucleotide sequences are shown in Table 7 below. All nucleotide sequences were synthesized by Beijing Liuhe BGI Genomics Co., Ltd.

[0069] Table 2 Target Site Information

[0070] site SEQ ID NO: AAVS1-g1 5 AAVS1-g2 6 AAVS1-g3 7

[0071] Table 3. crRNA scaffolds used in the experiment

[0072] SEQ ID NO: BEST7 crRNA 8 PAM TTTN

[0073] The following steps were used to complete the intracellular plasmid delivery (transfection), genome editing, and activity verification experiments:

[0074] (a) Take 15 mL of LB liquid medium (sterilized by high temperature and autoclave in advance, at room temperature), add 15 μL of 1000X Amp antibiotic, use a 10 μL pipette tip to pick up the stab strain (Beijing Liuhe Huada) that preserves the target plasmid, put it into the medium, and incubate at 37℃ and 200 rpm for 12-16 h.

[0075] (b) Centrifuge the cultured and amplified bacterial solution at 8000 rpm for 3 min and discard the culture medium;

[0076] (c) Extract the target plasmid using an endotoxin-free kit (Tiangen Biotech) according to the instructions;

[0077] (d) After extraction, the DNA concentration was quantified using Nanodrop (Thermo) and stored at -20℃.

[0078] (3) Plasmid transfection

[0079] (a) One day before transfection, the original culture medium of HEK293T cells (~90% confluence) in the seeding tray was aspirated using a pipette. Approximately 2 mL of pre-warmed 37°C DPBS (Gibco) was slowly added along the cell wall to wash the cell surface. Then, 1 mL of pre-warmed digestion buffer (TrypLE Express, Gibco) was added for digestion. After approximately 3 minutes, an appropriate amount of pre-warmed DMEM containing serum was added to terminate digestion. The cells were resuspended by pipetting. A small amount of the cell suspension was gently mixed with an equal proportion of trypan blue (Solarbio) by pipetting. Approximately 20 μL of the mixture was added to a Countstar cell counting chamber, and viable cell counts were performed using a Countstar Rigel S2 analyzer. Finally, the cells were seeded in 12-well cell culture plates, with approximately 0.5–1 × 10⁶ cells per well. 6 One cell;

[0080] (b) When the confluence of cells to be transfected reaches 50%–70%, the target plasmid can be transfected using the Lipofectamine 3000 kit (Invitrogen) according to the instructions (2 μg plasmid and 2.4 μL Lipofectamine 3000 Reagent per well). Change the culture medium as needed 6 hours after transfection.

[0081] (c) After transfection, cells need to be cultured for 2-3 days to allow for sufficient gene editing;

[0082] (d) After cell culture, transfection efficiency was calculated and cells were recovered. Cell digestion and resuspension were performed as described above (reagent amounts were added proportionally based on the cell culture area). Approximately 20 μL of cell suspension was added to a cell counting chamber, and the plasmid transfection efficiency was calculated using a cell analyzer and the green fluorescence (GFP) counting channel. Finally, the remaining cells were transferred to 1.5 mL centrifuge tubes, centrifuged at 12000 rpm for 1 min, and the supernatant was removed to harvest the cells.

[0083] (4) Identification of genome editing activity

[0084] After harvesting cells, preliminary detection and editing were performed using genome extraction and T7E1 restriction enzyme digestion experiments, as follows:

[0085] (a) Genomic DNA extraction: Genomic DNA was extracted using a genomic DNA extraction kit (Tiangen), and the concentration of genomic DNA (gDNA) was quantified using Nanodrop. The samples were stored at -20°C.

[0086] (b) Targeted Region PCR: Target regions were amplified from gDNA using a high-fidelity amplification enzyme (PrimeSTAR GXL DNA Polymerase, Takara). The primers are shown in Table 4 below. All deoxynucleotide sequences used were synthesized at the Shenzhen National Gene Bank Synthesis and Editing Platform. After observing a clear and single target band under a gel imaging system via 1% agarose gel electrophoresis (7.5V / cm, 30min), the gel was excised and purified using a PCR purification and gel extraction kit (NucleoSpinExtract, MN). The concentration was measured using Nanodrop.

[0087] (c) Denaturation and Annealing: The mutant DNA and control group reaction system were mixed as shown in Table 5, and subjected to heat denaturation and annealing. The above program settings for the PCR instrument (Bio-rad) are shown in Table 6.

[0088] (d) T7E1 digestion: Add 0.3 μL of T7EI nuclease to the reaction system in step (c), for a total of 20 μL. Perform the digestion reaction at 37℃ for 20 min.

[0089] (e) Activity detection: After the reaction is complete, add 4 μL of 6×Gel Loading Dye (NEB) and perform agarose gel electrophoresis to detect the bands (detailed steps are the same as the agarose gel electrophoresis operation described above).

[0090] Table 4 Primers for PCR amplification of the AAVS1 gene target region

[0091]

[0092] Table 5 Annealing Reaction System

[0093] Components Input DNA 200ng 10×TE7I Buffer 2μL ddH2O Add to 19.7 μL

[0094] Table 6. Denaturation and Annealing Conditions

[0095] temperature time 95℃ 3min -2℃ / min 10min 75℃ 30 seconds -6℃ / min 8min 27℃

[0096] Table 7. Sequences of gene-editing plasmids for mammalian cells in the new system.

[0097] sequence name SEQ ID NO: BEST7-AAVS1-G1 11 BEST7-AAVS1-G2 12 BEST7-AAVS1-G3 13

[0098] like Figure 1 As shown, the new BEST7 system has human cell editing activity, and it has genome editing activity at three sites of AAVS1 g1, g2, and g3.

[0099] Example 4: Experiment for identifying Cas12a PAM sequences

[0100] The PAM of each Cas12a nuclease was identified using the applicant's published DocMF method (Li et al., "DNB-based on-chip motiffinding: A high-throughput method to profile different types of protein-DNA interactions." Science Advances 6.31 (2020): eabb3350.). The specific DocMF experimental procedure included DNA library and DNB preparation, sequencing, and protein cleavage and imaging. The DNA library consisted of a 23 nt fixed sequence region and flanking 15 nt random base regions. The fixed sequence served as the gRNA target sequence, and the random base regions served as PAM recognition sites, as shown in Table 8. The PAM sequence of BEST7 was 5'TTTN. N is A, T, C, or G (e.g., ...). Figure 2 ).

[0101] Table 8. Nucleic acid sequences used in PAM identification

[0102]

[0103] Example 5: Efficiency of in vitro enzymatic digestion of Cas12a

[0104] 1. Preparation of cutting substrate

[0105] (1) Genomic DNA from 293T cells was amplified and cleaved using PCR for experimental use. The amplified products were identified by 1.5% agarose gel electrophoresis. Based on the corresponding band size, the gel was excised and analyzed using Qubit. TM The dsDNAHS Assay Kit was used for gel recovery and purification to obtain a high concentration of cleavage substrate. The cleavage substrate sequence is shown in SEQ ID NO:18, and its PCR primer sequences HBG primer F and HBG primer R are shown in SEQ ID NO:17.

[0106] Dilute with 10×NEBuffer2.1 (New England Biolabs, B7202) and enzyme-free water to a final concentration of 50 ng / μL. The final concentration of NEBuffer2.1 was 1×.

[0107] 2. Preparation of crRNA

[0108] Three gRNAs were designed, each targeting one of the three sites on the HBG gene, and named HBG-1, HBG-2, and HBG-3, as shown in Table 9. The RNAs were transmitted via MEGA shortscript. TM Transcription was performed using the T7 Transcription Kit, with the corresponding DNA template synthesized by BGI Genomics. 2 pmol of double-stranded DNA template was added, and transcription was performed using a Bio-rad S1000™ polymerase chain reaction (PCR) instrument at 37°C for 12 hours. RNA was purified using saturated phenol, chloroform, and isopropanol solutions. Subsequently, it was transcribed using a Qubit... TM Quantification was performed using the RNA HS AssayKit.

[0109] The final crRNA sequence information is shown in Table 9.

[0110] Table 9. crRNA sequences used in Example 5

[0111]

[0112] To compare the enzymatic cleavage activity of Cas12a in the in vitro environment, the applicant used... Lba Cas12a (Cpf1) (New England Biolabs, M0653T) was used as a positive control. The protein was diluted to a final concentration of 500 nM with 10× NEBuffer 2.1 and enzyme-free water. Transcribed and purified crRNA and 40 U / μL RNase inhibitor were also diluted to a final concentration of 500 nM with 10× NEBuffer 2.1 and enzyme-free water. The final concentration of NEBuffer 2.1 was 1×. 1 μL of the diluted Cas protein was mixed with 1 μL of crRNA, 1 μL of 10× NEB Buffer 2.1, and 71 μL of enzyme-free water (totaling 1 μL) and reacted at 37°C for 15 minutes to form RNPs. Then, 1 μL of 50 ng / μL HBG was added to cleave the substrate to a final volume of 10 μL, and the mixture was incubated at 45°C for 10 minutes. Add 1 μL of 10 mg / ml RNase A, incubate at 37°C for 10 min, and detect product bands by 1.5% agarose gel electrophoresis. Substrate band intensity analysis was performed using ImageLab software from the Bio-lab gel imaging system, referring to the formula: Quantitative percentage of DNA cleavage = 100 × (1-a) / (a+b+c), where a is the integral intensity of the undigested product band, and b and c are the integral intensities of each product band produced by cleavage. Figure 3 )

[0113] Among them, BEST7 showed higher cleavage activity than LbaCpf1 at all three target sites, thus the protein showed a certain increase in activity relative to LbaCas12a.

[0114] Example 6: Experiment on Cas12a system auxiliary cutting

[0115] The transcription recovery and substrate preparation of crRNA were performed in the same manner as in Example 5. The transcribed and purified crRNA and 40 U / μL of RNase inhibitor were diluted to a final concentration of 2 μM with 10× NEB Buffer 2.1 and enzyme-free water, where the final concentration of the RNase inhibitor was 4 U / μL and the final concentration of NEB Buffer 2.1 was 1×. Then, the Cas12a protein was diluted to a final concentration of 1 μM with 10× NEB Buffer 2.1, 50 mM DTT, and enzyme-free water, where the final concentrations of NEB Buffer 2.1 and DTT were 1× and 0.5 mM, respectively. 1 μL of the diluted Cas protein and 1 μL of crRNA were reacted in 1× NEB Buffer 2.1 at 37°C for 15 minutes to form 8 μL of LRNP. Next, 1 μL of 50 ng / μL cleavage substrate and 1 μL of CRISPR reporter buffer 2.1 (prepared as shown in Table 10 below) were added to a final volume of 10 μL. The mixture was incubated at 45°C for 30 minutes using a qPCR instrument. The detection mode was set to FAM fluorescence in the qPCR software, and the fluorescence value at the set wavelength was recorded every minute to plot a time-varying curve, thus reflecting the progress of the enzyme digestion reaction. (Figure 6)

[0116] Table 10 CRISPR reporter buffer 2.1 preparation table

[0117]

[0118] The FAM-reporter sequence is 5'-FAM-AAAAAA-BHQ1-3' (SEQ ID NO: 22).

[0119] Example 7: Activity of the Cas12a system at different temperatures

[0120] A 10 μL enzyme digestion reaction system was prepared in the same manner as in Example 5, and incubated at 37°C, 45°C, and 60°C for 30 minutes, respectively. The Cas12a protein reactivity at each temperature was compared by fluorescence value. Figure 4 As shown in the figure, the BEST7 protein exhibits double-strand recognition and paracleavage activity levels no lower than those of LbaCas12a at 60℃.

[0121] Example 8: Amplicon library construction and sequencing of Cas12a protein

[0122] Referring to the experiment in Example 3, the edited genome was used as a template to amplify the target site region. Editing efficiencies of SpCas9 and BEST7 were compared using amplicon library construction and sequencing. The edited plasmid sequences are shown in Table 11 below.

[0123] Table 11 Edit plasmid information

[0124] sequence name SEQ ID NO: SpCas9-H1 23 SpCas9-H3 24 SpCas9-H4 25 SpCas9-NC 26 BEST7-H1.1 27 BEST7-H3.1 28 BEST7-H3.3 29 BEST7-NC 30

[0125] Among them, the SpCas9 and BEST7 target sites (SpCas9-H1 / H3 / H4, BEST7-H1.1 / H3.1 / H3.3) are as follows: Figure 5 As shown in Table 12.

[0126] Table 12 Target HBG gene region sequences and target sites

[0127]

[0128] (1) Primers were generated for the above sites using Primer3 (https: / / primer3.ut.ee / ). The primer sequences are shown in Table 13. The primers for the above steps were synthesized by Beijing Liuhe BGI Genomics Co., Ltd.

[0129] Table 13 Primers for HBG promoter target site amplification

[0130]

[0131] (2) DNB library construction and sequencing: The purified amplification products obtained above were used for PCR-Free library construction. For detailed procedures, please refer to the instructions of the MGIEasy PCR-Free DNA Library Preparation Reagent Kit (1000013453). Sequencing was performed using MGISEQ-2000RS (PE100).

[0132] (3) The above amplicon sequencing data were analyzed using Crispresso2 (https: / / github.com / pinellolab / CRISPResso2), as shown in Figure 6.

[0133] In summary, the editing efficiency (excluding transfection efficiency) of SpCas9-H1 is 57%, SpCas9-H3 (53%), and SpCas9-H4 (46%); BEST7-H1.1 (19%), BEST7-H1.1 (39%), and BEST7-H3.3 (41%).

Claims

1. A Cas12a nuclease having the amino acid sequence 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 chimeric RNA formed from crRNA or tracrRNA.

4. An isolated nucleic acid having a nucleotide sequence encoding the Cas12a nuclease of 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 of claim 7.

9. A kit comprising the expression vector of claim 7 or the recombinant cells of claim 8.

10. Use of the Cas12a nuclease of claim 1, the CRISPR / Cas system of claim 2 or 3, the nucleic acid of any one of claims 4-6, the expression vector of claim 7, or the recombinant cell of claim 8 in gene editing.

11. The use according to claim 10, wherein the use is cell gene editing, nucleic acid detection, or high-throughput sequencing.

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