A ScCas12a protein and its applications

Abstract

This invention belongs to the field of gene editing technology, specifically disclosing a ScCas12a protein and its applications, wherein the protein is derived from... Sedimentisphaera cyanobacteriorum The ScCas12a protein of the strain has the amino acid sequence shown in SEQ ID NO.1. This invention also provides a mutant of the ScCas12a protein, ScCas12a-VV, with the amino acid sequence shown in SEQ ID NO.7. This mutant broadens the PAM recognition sequence from 5'-TTN-3' (N=A / T / C / G) to 5'-DTN-3' (D=A / T / G), achieving a gene editing efficiency of 65% in rice. This invention also provides a gene editing method and a nucleic acid detection system based on the aforementioned protein. The ScCas12a-VV mutant of the ScCas12a protein of this invention has advantages such as higher editing efficiency, a wider PAM recognition range, and higher nucleic acid detection sensitivity, and has broad application prospects in the fields of gene editing and molecular diagnostics.

Description

Technical Field

[0001] This invention relates to the field of gene editing technology, and in particular to a ScCas12a protein and its applications. Background Technology

[0002] The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system is an adaptive immune system widely found in bacterial and archaea genomes. CRISPR systems are mainly divided into Class 1 and Class 2 types. Class 2 CRISPR systems require only a single Cas effector protein to perform nucleic acid cleavage, and are therefore widely used for gene editing in eukaryotes. The Cas effector proteins in Class 2 CRISPR systems mainly include Type II Cas9, Type V Cas12, and Type VI Cas13.

[0003] Cas12a (also known as Cpf1) is a class of CRISPR effector proteins that have attracted much attention in recent years. Compared with Cas9, Cas12a has the following unique advantages: (1) Cas12a recognizes thymine (T)-rich protospace adjacent motif (PAM) sequences and has better targeting ability in genomic regions with high AT content; (2) Cas12a only needs mature crRNA to guide the protein to recognize the target site and does not require tracrRNA; (3) Cas12a produces sticky ends at the target site, which is conducive to homologous recombination repair-mediated gene knock-in; (4) Cas12a has a lower off-target rate; (5) Cas12a activates trans-cutting activity after cutting the target double-stranded DNA, non-specifically cutting the surrounding single-stranded DNA. This characteristic makes it an ideal tool for nucleic acid detection.

[0004] However, the existing Cas12a system still faces many technical bottlenecks in practical applications: First, the PAM recognition range of existing Cas12a effector proteins is relatively narrow, severely limiting the range of target sites they can target. The most commonly used LbCas12a and AsCas12a strictly recognize TTTV (V=A / C / G) or TTN PAM sequences, which limits Cas12a's gene targeting ability, allowing it to cover only about 1% of genomic targets. The number of sites conforming to TTN or TTTN sequences in the genome is limited, especially in genomic regions with high GC content (such as many plant and microbial genomes), where the number of target sites is further reduced. Although studies have attempted to broaden the PAM recognition range of Cas12a through protein engineering, existing PAM broadening strategies often result in reduced catalytic activity, and structural differences among different Cas12a homologs make it difficult to directly transplant known modification strategies. Therefore, identifying and developing novel Cas12a effector proteins with a wider PAM recognition range is a pressing technical problem to be solved in this field.

[0005] Secondly, the gene editing efficiency of existing Cas12a homologs in plants such as rice is generally low, limiting their application in plant genome editing and molecular breeding. The editing efficiency of LbCas12a and FnCas12a in plant cells is far lower than that of Cas9. Although some researchers have reported editing efficiency data for novel Cas12a homologs in plants in recent years, overall, Cas12a tools that combine high editing efficiency with a wide PAM recognition range are still lacking. Therefore, developing a Cas12a protein with high editing efficiency and a wider PAM range in plants is of great significance for plant gene function research and molecular breeding.

[0006] Furthermore, nucleic acid detection technologies based on the trans-cleavage activity of Cas12a have insufficient detection sensitivity and typically require a pre-amplification step of the target nucleic acid. Existing Cas12a nucleic acid detection systems generally only achieve detection sensitivity at the picomolar (pM) level, far from meeting the needs of clinical diagnosis and early disease screening. Therefore, researchers often need to combine Cas12a detection with nucleic acid amplification technologies such as PCR, RPA, or LAMP. This not only increases the number of steps and detection time but also introduces the risks of amplification bias and cross-contamination. Therefore, developing a Cas12a protein with higher trans-cleavage activity is of great significance for improving nucleic acid detection sensitivity and achieving pre-amplification-free detection.

[0007] In summary, there is an urgent need in this field to identify and develop a novel CRISPR / Cas12a protein that possesses a wider PAM recognition range, higher plant gene editing efficiency, and superior nucleic acid detection performance to overcome the aforementioned deficiencies of existing technologies. Summary of the Invention

[0008] To address the aforementioned technical problems, this invention provides a ScCas12a protein and its applications.

[0009] To achieve the above objectives, the present invention is implemented according to the following technical solution: The first technical solution provided by this invention is a ScCas12a protein, wherein the ScCas12a protein has one of the following amino acid sequences: (a) The amino acid sequence as shown in SEQ ID NO.1; (b) An amino acid sequence that has at least 95% sequence homology with SEQ ID NO.1 and has nucleic acid cleavage function; (c) An amino acid sequence that retains the same nucleic acid cleavage function as SEQ ID NO.1 after one or more amino acid substitutions, deletions or additions based on the amino acid sequence shown in SEQ ID NO.1.

[0010] Preferably, the ScCas12a protein is a ScCas12a-VV mutant protein, and its amino acid sequence is shown in SEQ ID NO.7.

[0011] It should be noted that: The protein of this invention can be derivatized. Generally, derivatization does not affect the protein's activity, such as its activity in forming a complex with sgRNA or its activity in cleaving nucleic acids under the guidance of sgRNA. Therefore, the protein of this invention can be linked to different molecular groups, such as nuclear localization signal (NLS) sequences, to allow the protein to enter the nucleus of eukaryotic cells; and to various tags, such as His tags, GST tags, or MBP tags, to facilitate the expression and purification of the protein. Preferably, the ScCas12a protein of this invention is linked to a His tag.

[0012] Typically, fusing one protein with another preserves the original protein's activity (such as the ability to bind to sgRNA to form a complex, or the ability to recognize and cleave specific nucleic acid sequences under sgRNA guidance). Therefore, the proteins of this invention are also intended to include such fused forms.

[0013] In particular, the protein of the present invention can be combined with various polypeptides having active structures, such as fusion of transcription activation domains and transcription repression domains, to give it the ability to activate or repress transcription; or combined with nucleotide deaminases to give it the activity of modifying the bases of the target sequence; so as to edit the target sequence with a single base.

[0014] The second technical solution provided by this invention is an isolated nucleic acid molecule, which contains a nucleotide sequence encoding the aforementioned ScCas12a protein, and the nucleic acid molecule has a nucleotide sequence of one of the following sequences: (a) A nucleotide sequence as shown in SEQ ID NO.2 or SEQ ID NO.5; (b) Codon-optimized nucleotide sequences as shown in SEQ ID NO.3 or SEQ ID NO.4; (c) A nucleotide sequence having one or more base substitutions, deletions or additions compared to SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO.5; or a nucleotide sequence having at least 70% sequence identity compared to SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO.5 and encoding a protein with nucleic acid cleavage function.

[0015] The third technical solution provided by the present invention is a recombinant vector, wherein the recombinant vector contains the above-mentioned nucleic acid molecules.

[0016] The fourth technical solution provided by the present invention is a recombinant cell, characterized in that the recombinant cell contains the above-mentioned recombinant vector, or the above-mentioned nucleic acid molecules are integrated into its genome.

[0017] The fifth technical solution provided by this invention is the application of the above-mentioned ScCas12a protein or the above-mentioned nucleic acid molecule in the preparation of gene editing tools or nucleic acid detection tools.

[0018] The sixth technical solution provided by the present invention is a gene editing method, which includes introducing the ScCas12a protein or its coding sequence into a target cell, so that the ScCas12a protein recognizes and cleaves the in situ adjacent motif of the target nucleic acid sequence under the guidance of guide RNA.

[0019] Furthermore, in the gene editing method described above, when the ScCas12a protein is the ScCas12a protein shown in SEQ ID NO.1, its recognized in-situ neighboring motif is 5'-TTN-3', where N is A / T / C / G; When the ScCas12a protein is the ScCas12a-VV mutant protein shown in SEQ ID NO.7, its recognized in situ neighboring motif is 5'-DTN-3', where D stands for A / T / G.

[0020] The seventh technical solution provided by this invention is a nucleic acid detection system based on CRISPR / Cas12a, the system comprising: The ScCas12a-VV mutant protein or its coding sequence; Guide RNA, the guide RNA comprising a guide sequence capable of hybridizing with a target nucleic acid sequence; A signal reporter molecule, wherein the signal reporter molecule is a single-stranded DNA with a fluorescent group and a quenching group; scaffold RNA; TCC-crRNA, wherein the TCC amplifier is a DNA oligonucleotide with a double stem-loop structure; The system utilizes the trans-cleavage activity activated by the ScCas12a-VV mutant protein after recognizing the target nucleic acid under the guidance of guide RNA, and the cleavage signal reporter molecule generates a detectable signal.

[0021] Compared with the prior art, the present invention has the following beneficial effects: 1) This invention provides a novel Cas12a effector protein, ScCas12a, derived from the strain Sedimentisphaera cyanobacteriorum. It has low sequence similarity to known Cas12a homologs, enriching the CRISPR / Cas12a effector protein tool library. ScCas12a recognizes the 5'-TTN-3' PAM sequence, has typical Cas12a nucleic acid cleavage activities (cis- and trans-cleavage), and has enabled gene editing in the rice genome.

[0022] 2) This invention also provides a ScCas12a-VV mutant protein with a wider PAM recognition range. By mutating lysine residues at positions 205 and 567 of ScCas12a to valine, the PAM recognition sequence was successfully broadened from 5'-TTN-3' to 5'-DTN-3' (D=A / T / G), expanding the PAM coverage from 3 (TTA / TTC / TTG) to 9 (ATA / ATC / ATG, TTA / TTC / TTG, GTA / GTC / GTG), significantly improving the target site coverage. The editing efficiency of ScCas12a-VV in rice protoplasts is 1.7 times that of ScCas12a, and the editing efficiency in stable transgenic rice reaches 65%, significantly superior to ScCas12a and several previously reported Cas12a homologs.

[0023] 3) This invention demonstrates the superior performance of the ScCas12a-VV mutant protein in nucleic acid detection. ScCas12a-VV maintains trans-cleavage activity over a wide temperature range of 35 ℃ to 50 ℃, with the highest activity at 37.5 ℃. Its in vitro nucleic acid detection activity is higher than that of LbCas12a and FnCas12a. The ScCas12a-VV mutant protein has good resolution for single base mismatches. ScCas12a-VV has nucleic acid detection activity under four PAM conditions: ATN, TTN, CTN, and GTN. The PAM recognition range is significantly better than that of LbCas12a (only TTN and CTN) and FnCas12a (only TTN).

[0024] 4) The nucleic acid detection system constructed in this invention, with its TNH amplifier, significantly amplifies the intensity of the fluorescence signal in the ScCas12a-VV in vitro detection system, thereby improving the sensitivity of the in vitro detection system. Attached Figure Description

[0025] Figure 1 The amino acid sequence alignment results of ScCas12a with other commonly used Cas12a proteins are shown.

[0026] Figure 2 The results of PAM preference analysis for ScCas12a are shown.

[0027] Figure 3 The results show the expression of the ScCas12a protein.

[0028] Figure 4 The results of ScCas12a protein purification.

[0029] Figure 5 The results show the in vitro cleavage target sequence of the ScCas12a protein.

[0030] Figure 6 Results of ScCas12a protein cleavage site detection.

[0031] Figure 7 The editing efficiency of ScCas12a protein in rice protoplasts.

[0032] Figure 8 The editing efficiency of ScCas12a protein in rice.

[0033] Figure 9 The results of PAM preference analysis for ScCas12a-VV protein.

[0034] Figure 10 The results show the in vitro cleavage target sequence of the ScCas12a-VV protein.

[0035] Figure 11The editing efficiency of ScCas12a-VV protein in rice protoplasts.

[0036] Figure 12 The editing efficiency of ScCas12a-VV protein in rice.

[0037] Figure 13 The trans-cleavage activity of ScCas12a-VV protein at different temperatures.

[0038] Figure 14 Comparison of trans-cleavage activities of different Cas12a proteins.

[0039] Figure 15 The trans-cleavage activity of ScCas12a-VV protein under different crRNA mismatches was measured.

[0040] Figure 16 The trans-cleavage activity of different Cas12a proteins under different PAM sequences was investigated.

[0041] Figure 17 Detection of long RNA samples by ScCas12a-VV.

[0042] Figure 18 To detect the increased sensitivity of TNH to the ScCas12a-VV in vitro nucleic acid detection system.

[0043] Figure 19 The results of ScCas12a-VV testing at different cDNA concentrations are shown. Detailed Implementation

[0044] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0045] The PCR and transformation methods used in the following examples are all conventional methods and techniques well known to those skilled in the art, and the reagents and materials used, unless otherwise specified, are all commercially available. Some of the reagents are from the following sources: DH5α competent cells were purchased from Qingke Biotechnology.

[0046] TransDB3.1 competent cells, BL21(DE3) competent cells, and pUC19 plasmid were all purchased from Beijing TransGen Biotech Co., Ltd.

[0047] Isopropyl sulfide-D-galactoside (IPTG) was purchased from Solarbio.

[0048] Protein purification consumables were purchased from Beyotime Corporation.

[0049] The 50 kDa ultrafiltration tube was purchased from Millipore.

[0050] Manitol, NaCl, MES, KCl, MgCl2·6H2O, PEG4000, Bovine Serum Albumin, and CaCl2·2H2O were all purchased from Sigma-Aldrich. LB liquid medium: 10 g tryptone, 5 g yeast extract, 10 g NaCl, add double-distilled water, bring the volume to 1 L, sterilize, and after the medium cools, add 1 mL of antibiotic at a concentration of 1 mg / mL to make the final concentration of antibiotic in LB liquid medium 25 μg / mL.

[0051] LB solid medium: Add 15 g of agar powder to 1 L of LB liquid medium, sterilize, and then add the corresponding antibiotic to a final concentration of 25 μg / mL.

[0052] SOB liquid medium: 20 g tryptone, 5 g yeast extract, 0.584 g NaCl, 0.186 g KCl, add double-distilled water, and bring the volume to 1 L.

[0053] 10% glycerol: 126 g glycerol, add double-distilled water, and bring the volume to 1 L.

[0054] Protein washing buffer: 3.152 g Tris-HCl (final concentration 20 mM), 29.22 g NaCl (final concentration 500 mM), 3.405 g imidazole (final concentration 50 mM). Adjust the pH to 7.4 with 1 M HCl, add nuclease-free water, bring the volume to 1 L, and store at 4°C.

[0055] Protein elution buffer: 3.152 g Tris-HCl (final concentration 20 mM), 29.22 g NaCl (final concentration 500 mM), 34.05 g imidazole (final concentration 500 mM). Adjust the pH to 7.4 with concentrated hydrochloric acid, add nuclease-free water, bring the volume to 1 L, and store at 4°C.

[0056] Protein preservation buffer: 3.152 g Tris-HCl (final concentration 20 mM), 11.688 g NaCl (final concentration 200 mM), 252 g glycerol, adjust pH to 7.4, add nuclease-free water, bring volume to 1 L, and store at 4°C.

[0057] Protein reaction buffer (2×): 0.788 g Tris-HCl (final concentration 5 mM), 2.922 g NaCl (final concentration 50 mM), 0.077125 g DTT (final concentration 0.5 mM), 1.90422 g MgCl2 (final concentration 20 mM). Adjust the pH to 7.4, add nuclease-free water, and bring the volume to 1 L. Store at 4°C.

[0058] MES (0.2 M): Weigh 3.9048 g of MES, bring the volume to 100 mL with sterile double-distilled water, adjust the pH to 5.7 with 1 M KOH, filter through a 0.22 μm filter membrane, and store at 4°C.

[0059] CaCl2·2H2O (1 M): Weigh 14.701 g of CaCl2·2H2O, bring the volume to 100 mL with sterile double-distilled water, filter through a 0.22 μm filter membrane, and store at 4℃.

[0060] KCl (2 M): Weigh 14.91 g of KCl, bring the volume to 100 mL with sterile double-distilled water, filter through a 0.22 μm filter membrane, and store at 4℃.

[0061] MgCl2·6H2O (0.5 M): Weigh 10.165 g of MgCl2·6H2O, bring the volume to 100 mL with sterile double-distilled water, filter through a 0.22 μm filter membrane, and store at 4 °C.

[0062] MMG: Weigh 9.11 g of Manitol, measure 3 mL of MgCl2·6H2O (0.5 M) and 1 mL of MES (0.2 M), mix them, and then make up to 100 mL with sterile double-distilled water. Filter the mixture through a 0.22 μm filter membrane and store at 4℃.

[0063] W5: Weigh 0.9 g of NaCl, 1.838 g of CaCl2·2H2O, measure 0.25 mL of KCl (2 M) and 1 mL of MES (0.2 M), mix them, and then make up to 100 mL with sterile double-distilled water. Filter the mixture through a 0.22 μm filter membrane and store at 4℃.

[0064] PEG4000: Weigh 40 g of PEG4000 and 3.644 g of Manitol, measure 10 mL of CaCl2·2H2O (1M), mix them, and then make up to 100 mL with sterile double-distilled water. Filter the mixture using a 0.22 μm filter membrane and store it at -20℃.

[0065] Enzyme Solution: Weigh 9.11 g of Manitol, 0.1 g of Bovine Serum Albumin, 5 mL of MES (0.2 M), and 1 mL of CaCl2·2H2O (1 M). Mix the contents and bring the volume to 100 mL with sterile double-distilled water. Adjust the pH to 5.7 with KOH and filter through a 0.22 μm filter membrane. Store at 4°C.

[0066] Annealing buffer (10×): 1.576 g Tris-HCl (final concentration 10 mM), 8.766 g NaCl (final concentration 150 mM), 0.476055 g MgCl2 (final concentration 20 mM), add nuclease-free water, bring the volume to 1 L, and store at 4℃.

[0067] Example 1: Identification of Cas12a protein (1) Using CRISPRCasFinder software, bacterial genome data in the NCBI database were scanned and identified, and a Cas enzyme from the strain Sedimentisphaera cyanobacteriorum was identified. Phylogenetic analysis and gene annotation showed that the Cas protein is a Cas12a protein, and its sequence similarity with previously reported Cas12a proteins is low. The similarity with commonly used LbCas12a, AsCas12a and FnCpf1 is 33.6%, 34.14% and 37.25%, respectively. Figure 1 Therefore, this invention names it the ScCas12a protein. The amino acid sequence, encoding nucleotide sequence, E. coli codon-optimized nucleotide sequence, and rice codon-optimized nucleotide sequence of the ScCas12a protein are detailed in the nucleotide and / or amino acid sequence listing (SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4, respectively).

[0068] ScCas12a amino acid sequence: (2) The domains of the ScCas12a protein were predicted and analyzed using the InterPro website (https: / / www.ebi.ac.uk / interpro / ). The results showed that the structure of the ScCas12a protein was as follows: RECⅠ domain (amino acids 51-295): KQLKEVIDSYHRDFIEQAFARQQTLSEEDFKQTYQLYKEAQKEKDGETLTKQYEHLRKKIAAMFSKATKEWAVMGENNELIGKNKESKLYQWLEKNYRAGRIEKEEFDHNAG LIEYFEKFSTYFVGFDKNRANMYSKEAKATAISFRTINENMVKHFDNCQRLEKIKSKYPDLAEELKDFEEFFKPSYFINCMNQSGIDYYNISAIGGKDEKDQKANMKINLFTQKNHLKGSDKPPFFAKLYKQI.

[0069] RuvCI domain (amino acids 906-1095): FNEQVNLYLKDNPDVNIIGIDRGERHLLYYTLINQNGEILQQGSLNRIGEEESRPTDYHRLLDEREKQRQQARETWKAVEGIK DLKAGYLSRVVHKLAGLMVQNNAIVVLEDLNKGFKRGRFAVEKQVYQNFEKALIQKLNYLVFKEVNSKDAPGHYLKAYQLTAPFISFEKLGTQSGFLFYVRAWNTSK.

[0070] NUC domain (amino acids 1096-1254): IDPATGFTDQIKPKYKNQKQAKDFMSSFDSVRYNRKENYFEFEADFEKLAQKPKGRTRWTICSYGQERYSYSPKERKFVKHNVTQNLAELFNSEGISFDSGQCFKDEILKVEDASFFKSIIFNLRLLLKLRHTCKNAEIERDFIISPVKGNNSSFFDSR.

[0071] RuvCⅡ domain (amino acids 1255-1309): IAEQENITSIPQNADANGAYNIALKGLMNLHNISKDGKAKLIKDEDWIEFVQKRK.

[0072] Analysis of the direct repeat sequence of the gRNA of ScCas12a protein revealed that the direct repeat sequence of ScCas12a protein is: ATCTACGACAGTAGAAATTTTATAAGGCCTTTAGAC (as shown in SEQ ID NO.5). The crRNA sequence of the ScCas12a protein is: AATTTCTACTGTCGTAGAT (as shown in SEQ ID NO.6).

[0073] Example 2: Identification of the PAM sequence recognized by the ScCas12a protein In this embodiment, the PAM sequence recognized by the ScCas12a protein was identified by an in vivo PAM library subtraction experiment in E. coli.

[0074] (1) PAM library construction: The pUC19 vector was reverse-amplified. The PCR reaction system was as follows: 1 μL of pUC19 plasmid (50 to 100 ng / μL), 2 μL each of pUC19-F (10 μmol / L) and pUC19-R (10 μmol / L), 25 μL of 2×PhantaMaxBuffer, 1 μL of dNTP, 1 μL of PhantaMax Super-Fidelity DNA Polymerase (1U / μL), and 18 μL of double-distilled water. The PCR amplification program was as follows: 94 ℃ denaturation for 3 min, 94 ℃ denaturation for 15 s, 56 ℃ annealing for 15 s, 72 ℃ extension for 1 min, for a total of 30 cycles, with a final extension at 72 ℃ for 5 min. The amplification primer sequences were as follows: pUC19-F:AGCTGTTTCCTGTGTGAAAATTGTT; pUC19-R: TTAAGCCAGCCCCGACACCCGCCA; The target sequence with 6 random bases at the 5' end was synthesized artificially, as shown below (the underlined part is the target sequence): Target-F: TGTCGGGGCTGGCTTAANNNNNN AAGTGATTGAAGCATACACAAGAA AGCTGTTTCCTGTGTGAAATTG (N=A / T / C / G); Target-R: CAATTTCACACAGGAAACAGCT TTCTTGTGTATGCTTCAATCACTT NNNNNNTTAAGCCAGCCCCGACA (N=A / T / C / G); Equal amounts of Target-F and Target-R primers were taken and added to an appropriate amount of double-distilled water to make the concentration of Target-F and Target-R in the system 0.5 μM. The mixture was reacted at 95 °C for 5 min and then placed at room temperature. The resulting fragments were ligated using the Gibson reaction, with a program of 50 °C for 5 min, 55 °C for 10 s, for a total of 6 cycles. After the reaction, the cells were transformed into DH5α competent cells and cultured overnight at 37 °C. The entire culture medium was then aspirated to extract plasmids, thus obtaining the PAM library.

[0075] (2) Construction of recombinant plasmid pET28a-CCDB: pET28a was reverse amplified using primers pET28a-F and pET28a-R. The PCR reaction system was as follows: 1 μL pET28a plasmid (50 to 100 ng / μL), 2 μL each of pET28a-F (10 μmol / L) and pET28a-R (10 μmol / L), 25 μL 2×PhantaMax Buffer, 1 μL dNTP, 1 μL PhantaMax Super-Fidelity DNA Polymerase (1 U / μL), and 18 μL double-distilled water. The PCR amplification program was: 94 °C denaturation for 3 min, 94 °C denaturation for 15 s, 56 °C annealing for 15 s, 72 °C extension for 3 min, for a total of 30 cycles, followed by a 5 min extension at 72 °C. The primer sequences are as follows: pET28a-F: TGTGCTTAGTGCATCTAACGAATCACCGATACGCGAGCGAAC; pET28a-R: GGCCTCGTGATACGCCTCTGCTAACCAGTAAGGCAACC.

[0076] The amplified product was ligated to the CCDB sequence using a Gibson reaction. The procedure was: 50 °C for 5 min, 55 °C for 10 s, for a total of 6 cycles. After the reaction, the cells were transfected into TransDB3.1 competent cells, and positive strains were picked to extract the plasmid, obtaining the pET28a-CCDB vector. Subsequently, the pET28a-CCDB plasmid was reverse amplified using pET28a-2F and pET28a-2R primers. The PCR reaction system was as follows: 1 μL of pET28a-CCDB plasmid (50 to 100 ng / μL), 2 μL each of pET28a-2F (10 μmol / L) and pET28a-2R (10 μmol / L), 25 μL of 2×PhantaMax Buffer, 1 μL of dNTP, 1 μL of PhantaMax Super-Fidelity DNA Polymerase (1 U / μL), and 18 μL of double-distilled water. The PCR amplification program was as follows: 94 °C denaturation for 3 min, 94 °C denaturation for 15 s, 56 °C annealing for 15 s, 72 °C extension for 3.5 min, for a total of 30 cycles, followed by a 5 min extension at 72 °C. The tag sequence was removed while retaining the NcoI and BamHI restriction sites. The primer sequences are as follows: pET28a-2F: GAAGGAGATATACCATGGACTGAGGATCCGGCTGCTAACAAAGCCC; pET28a-2R:CCATGGTATATCTCCTTCTTAAAGTTAAACAAAATTATTTCTAGAGGGGAATTGTTATCC; The amplified fragment was subjected to a Gibson reaction with a program of 50℃ for 5 min, 55℃ for 10 s, for a total of 6 cycles. After the reaction, the fragment was transferred into TransDB3.1 competent cells, and positive strains were picked to extract plasmids, thus obtaining the pET28a-CCDB tag-removed vector.

[0077] (3) Constructing the recombinant plasmid pET28a-ScCas12a-sgRNA The sgRNA sequence used in this embodiment is: GGTCTCACTCGTAATACGACTCACTATAGGG AATTTCT ACTGTCGTAGAT AAGTGATTGAAGCATACACAAGAA AATTTCTACTGTCGTAGAT CTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCGGTTGAGACC (The bolded sequence is the T7 promoter sequence, the italicized sequence is the ScCas12a crRNA sequence, and the underlined sequence is the target sequence). Artificially synthesized sgRNA primer sequences are as follows: sgRNA-F: GGTCTCACTCGTAATACGACTCACTATAGGGAATTTCTACTGTCGTAGATAAGTGATTGAAGCATACACAAGAAAATTTCTACTGTCGT; sgRNA-R: GGTCTCAACCGCAAAAAACCCCTCAAGACCCGTTTAGAGGCCCCAAGGGGTTATGCTAGATCTACGACAGTAGAAATTTTCTTGTGTAT; The complete sgRNA sequence was constructed by PCR. The PCR reaction system consisted of 2 μL each of sgRNA-F (10 μmol / L) and sgRNA-R (10 μmol / L), 25 μL of 2×PhantaMax Buffer, 1 μL of dNTP, 1 μL of PhantaMax Super-Fidelity DNA Polymerase (1 U / μL), and 19 μL of double-distilled water. The PCR amplification program was as follows: 94 °C denaturation for 3 min, 94 °C denaturation for 15 s, 56 °C annealing for 15 s, 72 °C extension for 5 s, for a total of 30 cycles, with a final extension of 5 min at 72 °C.

[0078] The sgRNA sequence was ligated and digested simultaneously with the pET28a-CCDB plasmid (with the tag sequence removed). The reaction mixture consisted of: 1.5 μL pET28a-CCDB plasmid (100 ng / μL), 1.5 μL sgRNA (100 ng / μL), 1.5 μL CutSmart buffer (NEB), 1.5 μL 10×ATP, 0.5 μL BsaI-HF v2 (NEB), 0.2 μL T4 DNA Ligase (TAKARA), and 8.3 μL double-distilled water. The reaction program was: 37 ℃ for 15 min, 37 ℃ for 5 min, 10 ℃ for 3 min, 20 ℃ for 5 min, for a total of 17 cycles, followed by a final 10 min at 37 ℃. After the reaction, the cells were transfected into DH5α competent cells and cultured overnight at 37 ℃. Positive strains were then picked, and the plasmid was extracted to obtain the pET28a-sgRNA plasmid.

[0079] The pET28a-sgRNA plasmid was double-digested using NcoI (Thermo Fisher) and BamHI (Thermo Fisher) enzymes. The double-digestion system consisted of 6 μL (100 ng / μL) of pET28a-sgRNA plasmid, 2 μL of 10×FastDigest buffer, 0.5 μL of NcoI (Thermo Fisher) enzyme, 0.5 μL of BamHI (Thermo Fisher) enzyme, 0.2 μL of FastAP (Thermo Fisher), and 10.8 μL of double-distilled water. The reaction program was 37 ℃ for 45 min, followed by 80 ℃ for 15 min. After digestion, the codon-optimized ScCas12a sequence of *E. coli* was constructed into the pET28a-sgRNA plasmid using a Gibson reaction. The Gibson reaction program was 50 ℃ for 5 min, followed by 55 ℃ for 10 min. The reaction was repeated for 6 cycles. After the reaction was completed, the cells were transferred into DH5α competent cells. Positive strains were picked and plasmids were extracted to obtain the pET28a-ScCas12a-sgRNA plasmid.

[0080] (3) Preparation of competent cells The pET28a-ScCas12a-sgRNA plasmid was transformed into BL21(DE3) competent cells. Positive strains were picked, diluted, and streaked in antibiotic-free solid medium, and incubated overnight at 37 °C. Single colonies were picked from the streaked plate and transferred to 3 mL of SOB liquid medium, and incubated at 37 °C for 4 h. 3 mL of the bacterial culture was then poured into 400 mL of SOB liquid medium and incubated at 37 °C until OD (October Expiratory Time) reached. 600 When the concentration reaches 0.6, remove the bacterial culture and shake it rapidly on ice for 10 min. After standing on ice for 10 min, transfer the bacterial culture to a centrifuge bottle and centrifuge at 1200 g for 10 min at 4 ℃. Wash with 10% glycerol, centrifuge at 1500 g for 15 min at 4 ℃, wash again with 10% glycerol, and aliquot into 1.5 mL centrifuge tubes. Store at -80 ℃ and name it BL21(DE3)-pET28a-ScCas12a-sgRNA.

[0081] (4) PAM library reduction The PAM library was transformed into BL21(DE3)-pET28a-ScCas12a-sgRNA competent cells via electroporation. The cells were cultured overnight at 37 °C in a solid medium containing kanamycin, ampicillin, and IPTG. All bacterial cells were collected, and plasmids were extracted to obtain the subtracted PAM library. A subtracted PAM next-generation sequencing library was constructed using PCR. The unsubtracted PAM library served as the control group, and the subtracted PAM library served as the experimental group. Statistical analysis was performed on the sequencing data. The frequency of each PAM sequence was calculated in both the experimental and control groups, and normalization was applied. For each PAM sequence, log2 (normalized value of control group / normalized value of experimental group) was calculated. A result greater than 3.5 indicated significant consumption of the PAM sequence. The Weblogo3 tool (https: / / weblogo.berkeley.edu / logo.cgi) was used to plot the significantly consumed PAM sequences. The results are shown below. Figure 2 As shown, the PAM sequence recognized by ScCas12a is 5'-TTN-3' (N=A / T / C / G).

[0082] Example 3: Expression and purification of ScCas12a protein (1) Construction of recombinant plasmid pET28a-HisScCas12a-sgRNA The pET28a-CCDB plasmid constructed in Example 2 was subjected to double digestion using BamHI (Thermo Fisher) enzyme and EcoRI (Thermo Fisher) enzyme. The double digestion system consisted of 6 μL (100 ng / μL) of pET28a-CCDB plasmid, 2 μL of 10×FastDigest buffer, 0.5 μL of BamHI (Thermo Fisher) enzyme, 0.5 μL of EcoRI (Thermo Fisher) enzyme, 0.2 μL of FastAP (Thermo Fisher) and 10.8 μL of double-distilled water. The reaction program was as follows: 37 °C for 20 min, followed by 80 °C for 10 min. The enzyme-digested plasmid fragments were subjected to a Gibson reaction with the codon-optimized ScCas12a sequence of E. coli. The Gibson program was 50 °C for 5 min, 55 °C for 10 s, for a total of 6 cycles. The Gibson reaction product was transformed into TransDB3.1 competent cells and cultured overnight at 37 °C. Positive strains were picked and plasmids were extracted to obtain the pET28a-HisScCas12a-CCDB plasmid.

[0083] Artificially synthesized sgRNA primer sequences are as follows: sgRNA-F: GGTCTCACTCGTAATACGACTCACTATAGGGAATTTCTACTGTCGTAGATAAGTGATTGAAGCATACACAAGAAAATTTCTACTGTCGT; sgRNA-R: GGTCTCAACCGCAAAAAACCCCTCAAGACCCGTTTAGAGGCCCCAAGGGGTTATGCTAGATCTACGACAGTAGAAATTTTCTTGTGTAT; The complete sgRNA sequence was constructed by PCR using primers F and R. The PCR reaction mixture consisted of 2 μL each of sgRNA-F (10 μmol / L) and sgRNA-R (10 μmol / L), 25 μL of 2×PhantaMax Buffer, 1 μL of dNTPs, 1 μL of PhantaMax Super-Fidelity DNA Polymerase (1 U / μL), and 19 μL of double-distilled water. The PCR amplification program was as follows: 94 °C denaturation for 3 min, 94 °C denaturation for 15 s, 56 °C annealing for 15 s, 72 °C extension for 5 s, for a total of 30 cycles, followed by a final extension at 72 °C for 5 min. The complete sgRNA sequence is as follows: GGTCTCACTCGTAATACGACTCACTATAGGG AATTTCTACTGTCGTAGAT AAGTGATTGAAGCATACA CAAGAA AATTTCTACTGTCGTAGAT CTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCGGTTGAGACC (The bolded sequence is the T7 promoter sequence, the italicized sequence is the ScCas12a crRNA sequence, and the underlined sequence is the target sequence). The sgRNA was ligated into the pET28a-HisScCas12a-CCDB plasmid using a cleavage-ligation reaction. The reaction mixture consisted of: 1.5 μL (100 ng / μL) pET28a-HisScCas12a-CCDB plasmid, 1.5 μL (100 ng / μL) sgRNA, 1.5 μL CutSmart buffer (NEB), 1.5 μL 10×ATP, 0.5 μL BsaI-HF v2 (NEB), 0.2 μL T4 DNA Ligase (TAKARA), and 8.3 μL double-distilled water. The reaction program was: 37 ℃ for 15 min, 37 ℃ for 5 min, 10 ℃ for 3 min, 20 ℃ for 5 min, for a total of 17 cycles, followed by 37 ℃ for 10 min. After the reaction, the cells were transfected into DH5α competent cells and incubated at 37 ℃. After overnight culture at ℃, positive strains were selected and plasmids were extracted to obtain pET28a-HisScCas12a-sgRNA plasmid.

[0084] (2) Expression and purification of HisScCas12a protein The pET28a-HisScCas12a-sgRNA plasmid was transformed into BL21(DE3) competent cells. Positive single colonies were picked and transferred to 2 mL of LB broth. After culturing at 37 °C for 12 h, an appropriate amount of bacterial culture (1:100) was transferred to 100 mL of LB broth containing kanamycin resistance and cultured at 37 °C until the bacterial culture reached OD. 600 The concentration was 0.6, and IPTG was added to a final concentration of 0.5 mM. The mixture was then transferred to a 18 ℃ incubation chamber for 16 h. The cultured cells were collected by centrifugation at 8000 g for 5 min. The cells were resuspended in protein wash buffer, sonicated to disrupt the cell structure, and then centrifuged at 8000 g for 5 min. The supernatant was transferred to an affinity chromatography column, and an appropriate amount of nickel beads (1:10) was added. The column was incubated at 4 ℃ for 3–4 h. After removing the protein wash buffer, protein elution buffer was added, and the column was incubated at 4 ℃ for 1 h. The eluent was collected in a 50 kDa ultrafiltration tube for protein concentration, and the buffer was replaced with protein storage buffer. The concentrated protein was stored at -80 ℃. The expressed protein product was an RNP complex of sgRNA and HisScCas12a.

[0085] 40 μL of the RNP complex was mixed with 10 μL of 5× protein loading buffer and incubated at 100 °C for 10 min. 10 μL of the incubation product was then subjected to SDS-polyacrylamide gel electrophoresis. The gel was then stained with Coomassie Brilliant Blue and Western blotting to verify protein expression and purification. The results are as follows: Figure 3 and Figure 4 As shown, the target protein was highly enriched after purification, while other proteins were relatively few.

[0086] Example 4: Validation of ScCas12a in vitro cleavage activity This embodiment utilizes the RNP complex from Example 3 to digest the linearized target fragment, verifying the in vitro nucleic acid cleavage activity of ScCas12a. The target sequence involved is located at 700 bp of the linear target fragment. After cleavage, it is expected that nucleic acid fragments of approximately 700 bp and 560 bp can be obtained. The specific implementation steps are as follows: (1) Construction of TTN target fragments Artificial primers TTN-F and TTN-R were synthesized. Equal amounts of TTN-F and TTN-R primers were taken and an appropriate amount of double-distilled water was added to make the concentration of TTN-F and TTN-R in the system 0.5 μM. The mixture was reacted at 95 ℃ for 5 min and then placed at room temperature to obtain the target fragment containing TTN. The primer sequences are as follows: TTN-F: TGTCGGGGCTGGCTTAATTN AAGTGATTGAAGCATACACAAGAA AGCTGTTTCCTGTGTGAAATTG (underlined part is the target sequence); TTN-R: CAATTTCACACAGGAAACAGCT TTCTTGTGTATGCTTCAATCACTT NAATTAAGCCAGCCCCGACA (underlined part is target sequence); The pUC19 vector was reverse-amplified. The PCR reaction system was as follows: 1 μL of pUC19 plasmid (50 to 100 ng / μL), 2 μL each of pUC19-F (10 μmol / L) and pUC19-R (10 μmol / L), 25 μL of 2×PhantaMax Buffer, 1 μL of dNTP, 1 μL of PhantaMax Super-Fidelity DNA Polymerase (1 U / μL), and 18 μL of double-distilled water. The PCR amplification program was: 94 °C denaturation for 3 min, 94 °C denaturation for 15 s, 56 °C annealing for 15 s, 72 °C extension for 1 min, for a total of 30 cycles, followed by a 5 min extension at 72 °C. The amplification primer sequences were: pUC19-F:AGCTGTTTCCTGTGTGAAAATTGTT; pUC19-R: TTAAGCCAGCCCCGACACCCGCCA; The target fragment containing TTN was subjected to a Gibson reaction with the inversely amplified pUC19 fragment. The reaction program was: 50℃ for 5 min, 55℃ for 10 s, for a total of 6 cycles. The TTN target fragment was then inserted into the pUC19 vector to obtain the TTN target library. The TTN target library was amplified by PCR using Vitro-F and Vitro-R primers to obtain a linearized target fragment of 1283 bp containing TTN. The primer sequences are as follows: Vitro-F: CTGTCATGCCATCCGTAAG; Vitro-R: AAGGCGGACAGGTATCCGGTAAG.

[0087] (2) ScCas12a extracorporeal cutting 500 ng of the linearized target fragment containing TTN, 4 μg of purified RNP complex, and 10 μL of protein reaction buffer were added. Double-distilled water was added to a final reaction volume of 20 μL. The mixture was incubated at 37 °C for 1 h. Then, 0.5 μL of proteinase K was added, and the reaction was continued at 56 °C for 20 min to digest the protein in the system. Finally, the reaction was stopped at 85 °C for 10 min to inactivate proteinase K, thus terminating the enzymatic reaction. The resulting product was electrophoresed on a 1.2% agarose gel to detect the in vitro protease activity of ScCas12a. Results are as follows: Figure 5 As shown, after treatment with ScCas12a protein, the linearized target fragment with TTN can be cleaved to obtain nucleic acid fragments of about 700 bp and 560 bp, indicating that ScCas12a has nucleic acid cleavage activity in vitro.

[0088] Example 5: Identification of the Cutting Location of ScCas12a In this embodiment, the ScCas12a protein is used to cleave the target DNA fragment in vitro, and the cleaved fragment is then subjected to Sanger sequencing to determine the location of the nucleic acid cleaved by ScCas12a.

[0089] The targeted DNA fragment used in this embodiment is: TGTCGGGGCTGGCTTAATTG AAGTGATTGAAGCATACACAAGAA AGCTGTTTCCTGTGTGAAATTG (underlined part is the target sequence); Artificial primers TTG-F and TTG-R were synthesized. Equal amounts of TTG-F and TTG-R primers were taken and an appropriate amount of double-distilled water was added to make the concentration of TTG-F and TTG-R in the system 0.5 μM. The reaction was carried out at 95 °C for 5 min and then placed at room temperature to obtain the target fragment containing TTG. The primer sequences are as follows: TTG-F: TGTCGGGGCTGGCTTAATTG AAGTGATTGAAGCATACACAAGAA AGCTGTTTCCTGTGTGAAATTG (underlined part is the target sequence); TTG-R: CAATTTCACACAGGAAACAGCT TTCTTGTGTATGCTTCAATCACTT CAATTAAGCCAGCCCCGACA (underlined part is the target sequence).

[0090] The pUC19 vector was reverse-amplified. The PCR reaction system was as follows: 1 μL of pUC19 plasmid (50 to 100 ng / μL), 2 μL each of pUC19-F (10 μmol / L) and pUC19-R (10 μmol / L), 25 μL of 2×PhantaMax Buffer, 1 μL of dNTP, 1 μL of PhantaMax Super-Fidelity DNA Polymerase (1 U / μL), and 18 μL of double-distilled water. The PCR amplification program was: 94 °C denaturation for 3 min, 94 °C denaturation for 15 s, 56 °C annealing for 15 s, 72 °C extension for 1 min, for a total of 30 cycles, followed by a 5 min extension at 72 °C. The amplification primer sequences were: pUC19-F:AGCTGTTTCCTGTGTGAAAATTGTT; pUC19-R: TTAAGCCAGCCCCGACACCCGCCA.

[0091] The target fragment containing TTG was subjected to a Gibson reaction with the inversely amplified pUC19 fragment. The reaction program was 50℃ for 5 min, 55℃ for 10 s, for a total of 6 cycles. The TTG target fragment was then inserted into the pUC19 vector to obtain the TTG target plasmid.

[0092] The reaction was performed as follows: 500 ng of TTG target plasmid, 4 μg of purified RNP complex, and 10 μL of 2× protein reaction buffer were added. Double-distilled water was added to a final reaction volume of 20 μL. The mixture was incubated at 37 °C for 1 h. Then, 0.5 μL of proteinase K was added, and the reaction was carried out at 56 °C for 20 min to digest the protein in the system. The proteinase K was then inactivated at 85 °C for 10 min to terminate the enzymatic reaction. An appropriate volume (1:1) of 2× Taq DNA Polymerase Mix (Novizan) was added, and the reaction was carried out at 72 °C for 30 min to complete the cleavage fragment. An A base was added to the 3' end of the product. The completed cleavage fragment was then purified and recovered.

[0093] Synthetically synthesized primer fragments containing T bases, the sequences of which are as follows: Adapter-F: CTTCGATAAGAACCGTGCTAACATGTACAGCAAAGAGGCAAAGGCCACTGCAATCAGCT; Adapter-R: GCTGATTGCAGTGGCCTTTGCCTCTTTGCTGTACATGTTAGCACGGTTCTTATCGAAG.

[0094] 10 μL each of Adapter-F and Adapter-R primers were denatured at 95 °C for 5 min and then placed at room temperature to obtain adapter fragments with a T base at the 3' end. The purified and recovered products were ligated to the adapter fragments using T4 ligase. The reaction mixture consisted of: 2 μL of 10×T4 Buffer, 1 μL of T4 ligase (TAKARA), 2 μL of the recovered fragment, 4 μL of the adapter fragment, and 11 μL of double-distilled water. The reaction was carried out at 16 °C for 1 h. The ligated fragments were then subjected to PCR using synthetic primers Extend and Vitro-F and Vitro-R. The Extend primer sequences are shown below: Extend: TGATCGAGTACTTTGAGAAGTTCTCTACCTATTTCGTTGGCTTCGATAAGAACCGTGCT.

[0095] The obtained product was subjected to Sanger sequencing, and the results are as follows: Figure 6 As shown, the results indicate that ScCas12a cleaves between the 18th and 19th nt and between the 22nd and 23rd nt of the non-complementary strand at the target site, producing sticky ends.

[0096] Example 6: Editing efficiency of ScCas12a protein in rice protoplasts This embodiment edits rice protoplasts, and the selected target is rice. BADH2 The gene contains GCACACCTGAAGGAAGACCAACCT. The specific steps are as follows: (1) The rice codon-optimized ScCas12a sequence (as shown in SEQ ID NO.4) was artificially synthesized. The primers ScCas12a-F and ScCas12a-R were artificially synthesized. The rice codon-optimized ScCas12a sequence was amplified by PCR and NLS sequence was added. The PCR amplification system was as follows: 1 μL of rice codon-optimized ScCas12a (50 to 100 ng / μL), 2 μL each of ScCas12a-F (10 μmol / L) and ScCas12a-R (10 μmol / L), 25 μL of 2×PhantaMax Buffer, 1 μL of dNTP, 1 μL of PhantaMax Super-Fidelity DNA Polymerase (1U / μL), and 18 μL of double-distilled water. The PCR amplification program was as follows: 94℃ denaturation for 3 min, 94℃ denaturation for 15 s, 56℃ annealing for 15 s, 72℃ extension for 2 min 30 s, for a total of 30 cycles, followed by a 72℃ extension for 5 min. The primer sequences for ScCas12a-F and ScCas12a-R are as follows: ScCas12a-F: GATCAACGGTACACGCGTatggctcctaagaagaagcggaaggttggtattcacggggtgcctgcggctAAGGAGTTCACCAACCAGTAC (lowercase bases are NLS sequences). ScCas12a-R: GAGCTCGAATTCACGCGTcttctttttcttagcctgtccggcctttttggtggcagcaggacgcttGAACTTCCTCTTCTGCAC (lowercase bases are NLS sequences).

[0097] The PCR amplification product was constructed into the pUC19-myc vector using the Gibson ligation method. The Gibson reaction program was as follows: incubation at 50 °C for 5 min, incubation at 55 °C for 10 s, for a total of 6 cycles. The Gibson reaction product was transformed into DH5α competent cells and cultured overnight at 37 °C. Positive strains were picked and plasmids were extracted to obtain the pUC19-ScCas12a-myc plasmid.

[0098] The pUC19-ScCas12a-myc plasmid was digested with NcoI (Thermo Fisher enzyme). The reaction mixture consisted of 6 μL (100 ng / μL) of pUC19-ScCas12a-myc plasmid, 2 μL of 10×FastDigest buffer, 0.5 μL of NcoI (Thermo Fisher enzyme), 0.2 μL of FastAP (Thermo Fisher), and 11.3 μL of double-distilled water. The reaction program was 37 °C for 20 min, followed by 80 °C for 10 min. The digested plasmid fragment was then subjected to a Gibson reaction with the sgRNA sequence. The Gibson reaction program was 50 °C for 5 min, followed by 55 °C for 10 s, for a total of 6 cycles. The Gibson reaction product was transformed into DH5α competent cells and cultured overnight at 37 °C. Positive strains were selected, and the plasmid was extracted to obtain the pUC19-ScCas12a-myc-sgRNA plasmid. The sgRNA sequence is as follows: AATTTCTACTGTCGTAGAT GCACACCTGAAGGAAGACCAACCT (Italicized text represents the crRNA sequence of ScCas12a, and underlined text represents the target sequence).

[0099] (2) Remove the husks from 9311 rice seeds and place them in a sterile triangular flask. Add 75% ethanol and shake well to sterilize for 1 min, then wash three times with sterile double-distilled water. Add an appropriate amount of 2% sodium hypochlorite, place in the dark, sterilize at 28 ℃ and 150 rpm for 30 min, wash five times with sterile double-distilled water, and sterilize again with an appropriate amount of 2% sodium hypochlorite. Use sterilized tweezers to place the seeds into 1 / 2 MS medium and culture in the dark for 13-15 days.

[0100] (3) Cut off the roots and leaves of the seedlings, leaving the leaf sheaths. Use a sharp blade to cut the leaf sheaths into small segments of about 0.5 mm and place them in W5 solution in the dark for 30 min. After removing the W5 solution, add an appropriate amount of EnzymeSolution solution and shake in the dark for 4 h at 28 ℃ and 50 rpm.

[0101] (4) Remove the enzyme solution, wash the leaf sheath tissue with W5 solution, filter the wash solution through 300-mesh and 400-mesh sieves, collect the wash solution in a 50 mL centrifuge tube, centrifuge at 150 g for 5 min, remove the supernatant, wash the precipitated cells with W5 solution, centrifuge at 150 g for 5 min, remove the supernatant, and resuspend the cells in 2 mL of W5 solution. Centrifuge at 200 g for 10 min, resuspend the cells in MMG, and adjust the concentration to 2×10⁻⁶. 6 / mL.

[0102] (5) Take 10 μg of plasmid into 200 μL of suspended cells and mix gently. Add 220 μL of PEG4000, mix well, and let stand in the dark at 25 °C for 20 min.

[0103] (6) Wash with 900 μL of W5 solution, centrifuge at 300 g for 10 min and remove the supernatant. Resuspend the cells in 1 mL of WI solution and incubate at 37 ℃ in the dark for 60 h.

[0104] (7) DNA was extracted from protoplasts cultured for 60 h, and a next-generation sequencing library was constructed using BADH2#-F and BADH2#-R primers, which were then sent to the company for sequencing. The sequences of BADH2#-F and BADH2#-R primers are as follows: BADH2#-F: CTCGGAGTGATCGCACGTTGTGGCATGTCCATGC; BADH2#-R: CTGAGAGGCTGGATGGCTGTACCTTGTCTACACCA.

[0105] The sequencing results were analyzed, and the results are as follows: Figure 7 As shown, ScCas12a can edit the rice genome, with an editing efficiency of 4.66% in rice protoplasts, and the main type of editing produced is fragment deletion.

[0106] Example 7: Editing efficiency of ScCas12a protein in rice This embodiment edits the ZH11 rice material, and the selected target point is... mir156f The gene contains GGTGGTGGCAGTTGACAGAAGAGA. The specific steps are as follows: The 1300 vector was double-digested using PstI (NEB) and AvrII (NEB) enzymes. The digestion system consisted of 6 μL of 1300 vector (100 ng / μL), 2 μL of CutSmart buffer (NEB), 0.5 μL of PstI (NEB) enzyme, 0.5 μL of AvrII (NEB) enzyme, 0.2 μL of FastAP (Thermo Fisher), and 10.8 μL of double-distilled water. The double digestion program was: incubation at 37 °C for 1 h followed by incubation at 85 °C for 10 min to obtain the digested 1300 vector fragment.

[0107] PCR was performed using artificially synthesized 1300-ScCas12a-F and 1300-ScCas12a-R primers to obtain ScCas12a fragments with NLS sequences. The PCR amplification system consisted of: 1 μL of rice codon-optimized ScCas12a (50 to 100 ng / μL), 2 μL each of 1300-ScCas12a-F (10 μmol / L) and 1300-ScCas12a-R (10 μmol / L), 25 μL of 2×PhantaMaxBuffer, 1 μL of dNTP, 1 μL of PhantaMax Super-Fidelity DNA Polymerase (1 U / μL), and 18 μL of double-distilled water. The PCR amplification program was as follows: denaturation at 94 ℃ for 3 min, denaturation at 94 ℃ for 15 s, annealing at 56 ℃ for 15 s, extension at 72 ℃ for 2 min 30 s, for a total of 30 cycles, followed by an extension at 72 ℃ for 5 min.

[0108] The ScCas12a fragment containing NLS was loaded into the 1300 restriction enzyme fragment using the Gibson reaction. The Gibson program was 50 °C for 5 min, 55 °C for 10 s, for a total of 6 cycles. The Gibson reaction product was transformed into TransDB3.1 competent cells and cultured overnight at 37 °C. Positive strains were picked and plasmids were extracted to obtain the 1300-ScCas12a-CCDB plasmid.

[0109] The sgRNA sequence was constructed into the 1300-ScCas12a-CCDB plasmid using a digest-and-ligation reaction. The digest-and-ligation system consisted of: 1.5 μL (100 ng / μL) of 1300-ScCas12a-CCDB plasmid, 1.5 μL (100 ng / μL) of sgRNA, 1.5 μL of CutSmart buffer (NEB), 1.5 μL of 10×ATP, 0.5 μL of BsaI-HF v2 (NEB), 0.2 μL of T4 DNA Ligase (TAKARA), and 8.3 μL of double-distilled water. The reaction program was: 37 ℃ for 15 min, 37 ℃ for 5 min, 10 ℃ for 3 min, 20 ℃ for 5 min, for a total of 17 cycles, followed by 37 ℃ for 10 min. After the reaction, the cells were transfected into DH5α competent cells and incubated at 37 ℃. After overnight culture at ℃, positive strains were selected and plasmids were extracted to obtain the 1300-ScCas12a-sgRNA plasmid. The 1300-ScCas12a-sgRNA plasmid was sent to the company for transformation, and the recipient material was ZH11.

[0110] The resulting transformed material was subjected to high-throughput sequencing to detect the editing of ScCas12a in ZH11. The results are as follows: Figure 8 As shown, a total of 27 transformed materials were obtained, of which 10 transformed materials... miR156f The gene was mutated, the editing efficiency was 37%, and the mutation results of the edited material were all deletions of fragments.

[0111] Example 8: Identification of PAM sequences recognized by ScCas12a-VV To broaden the PAM sequence recognized by ScCas12a and improve its editing efficiency in rice, this embodiment mutated the amino acid sequence of ScCas12a by replacing lysine (K) at positions 205 and 567 with valine (V), obtaining ScCas12a-VV. The PAM sequence recognized by the ScCas12a-VV protein was then identified using an in vivo PAM library subtraction experiment in *E. coli*. The specific steps are as follows: (1) Constructing the recombinant plasmid pET28a-ScCas12a-VV-sgRNA The sgRNA sequence used in this embodiment is: GGTCTCACTCGTAATACGACTCACTATAGGG AATTTCT ACTGTCGTAGAT AAGTGATTGAAGCATACACAAGAA AATTTCTACTGTCGTAGAT CTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCGGTTGAGACC (The bolded sequence is the T7 promoter sequence, the italicized sequence is the ScCas12a-VV crRNA sequence, and the underlined sequence is the target sequence).

[0112] Artificially synthesized sgRNA primer sequences are as follows: sgRNA-F: GGTCTCACTCGTAATACGACTCACTATAGGGAATTTCTACTGTCGTAGATAAGTGATTGAAGCATACACAAGAAAATTTCTACTGTCGT; sgRNA-R: GGTCTCAACCGCAAAAAACCCCTCAAGACCCGTTTAGAGGCCCCAAGGGGTTATGCTAGATCTACGACAGTAGAAATTTTCTTGTGTAT.

[0113] The complete sgRNA sequence was constructed by PCR using primers F and R. The PCR reaction system consisted of 2 μL each of sgRNA-F (10 μmol / L) and sgRNA-R (10 μmol / L), 25 μL of 2×PhantaMax Buffer, 1 μL of dNTP, 1 μL of PhantaMax Super-Fidelity DNA Polymerase (1 U / μL), and 19 μL of double-distilled water. The PCR amplification program was as follows: 30 cycles of denaturation at 94 °C for 3 min, denaturation at 94 °C for 15 s, annealing at 56 °C for 15 s, extension at 72 °C for 5 s, followed by a final extension at 72 °C for 5 min.

[0114] The sgRNA was ligated and digested simultaneously with the His-tag-removed pET28a-CCDB plasmid constructed in Example 2. The reaction mixture consisted of: 1.5 μL pET28a-CCDB plasmid (100 ng / μL), 1.5 μL sgRNA (100 ng / μL), 1.5 μL CutSmart buffer (NEB), 1.5 μL 10×ATP, 0.5 μL BsaI-HF v2 (NEB), 0.2 μL T4 DNA Ligase (TAKARA), and 8.3 μL double-distilled water. The reaction program was: 37 ℃ for 15 min, 37 ℃ for 5 min, 10 ℃ for 3 min, 20 ℃ for 5 min, for a total of 17 cycles, followed by 37 ℃ for 10 min. After the reaction, the cells were transfected into DH5α competent cells and cultured overnight at 37 ℃. Positive strains were then picked and the plasmid was extracted to obtain the pET28a-sgRNA plasmid.

[0115] The pET28a-sgRNA plasmid was double-digested using NcoI (Thermo Fisher) and BamHI (Thermo Fisher) enzymes. The double digestion system consisted of 6 μL (100 ng / μL) pET28a-sgRNA plasmid, 2 μL 10×FastDigest buffer, 0.5 μL NcoI (Thermo Fisher) enzyme, 0.5 μL BamHI (Thermo Fisher) enzyme, 0.2 μL FastAP (Thermo Fisher), and 10.8 μL double-distilled water. The reaction program was 37 ℃ for 45 min, followed by 80 ℃ for 15 min. After digestion, the ScCas12a-VV sequence was constructed into the pET28a-sgRNA plasmid using a Gibson reaction. The Gibson program was 50 ℃ for 5 min, 55 ℃ for 10 s, for a total of 6 cycles. The reaction product was then transformed into DH5α competent cells and incubated at 37 ℃. After overnight culture at ℃, positive strains were selected and plasmids were extracted to obtain pET28a-ScCas12a-VV-sgRNA plasmid.

[0116] (2) Preparation of competent cells The pET28a-ScCas12a-VV-sgRNA plasmid was transformed into BL21(DE3) competent cells. Positive strains were picked, diluted, and streaked in antibiotic-free solid medium, and incubated overnight at 37 °C. Single colonies were picked from the streaked plate and transferred to 3 mL of SOB liquid medium, and incubated at 37 °C for 4 h. 3 mL of the bacterial culture was then poured into 400 mL of SOB liquid medium and incubated at 37 °C until OD (October Expiratory Time) reached. 600 When the concentration reaches 0.6, remove the bacterial culture and shake it rapidly on ice for 10 min. After standing on ice for 10 min, transfer the bacterial culture to a centrifuge bottle and centrifuge at 1200 g for 10 min at 4 ℃. Wash with 10% glycerol, centrifuge at 1500 g for 15 min at 4 ℃, wash again with 10% glycerol, and aliquot into 1.5 mL centrifuge tubes. Store at -80 ℃ and name it BL21(DE3)-pET28a-ScCas12a-VV-sgRNA.

[0117] (4) PAM library reduction The PAM library constructed in Example 2 was transformed into BL21(DE3)-pET28a-ScCas12a-VV-sgRNA competent cells via electroporation. The cells were cultured overnight at 37 °C in a solid medium containing kanamycin, ampicillin, and IPTG. All bacterial cells were collected, and plasmids were extracted to obtain the subtracted PAM library. A subtracted PAM next-generation sequencing library was constructed using PCR. The unsubtracted PAM library served as the control group, and the subtracted PAM library served as the experimental group. Statistical analysis was performed on the sequencing data. The frequency of each PAM sequence was calculated in both the experimental and control groups, and normalization was performed. For each PAM sequence, log2 (normalized value of the control group / normalized value of the experimental group) was calculated. A result greater than 3.5 indicated that the PAM sequence was significantly consumed. The Weblogo3 (https: / / weblogo.berkeley.edu / logo.cgi) web tool was used to plot the significantly consumed PAM sequences. The results are shown below. Figure 9 As shown, the PAM sequence recognized by ScCas12a-VV is 5'-DTN-3' (D=A / T / G).

[0118] Example 8: Detection of ScCas12a-VV in vitro cleavage activity (1) Construction of recombinant plasmid pET28a-HisScCas12a-VV-sgRNA The pET28a-CCDB plasmid constructed in Example 2 was subjected to double digestion using BamHI (Thermo Fisher) enzyme and EcoRI (Thermo Fisher) enzyme. The double digestion system was as follows: 6 μL pET28a-CCDB plasmid (100 ng / μL), 2 μL 10×FastDigest buffer, 0.5 μL BamHI (Thermo Fisher) enzyme, 0.5 μL EcoRI (Thermo Fisher) enzyme, 0.2 μL FastAP (Thermo Fisher) and 10.8 μL double-distilled water. The reaction program was: 37 °C for 20 min, then 80 °C for 10 min. The enzyme-digested plasmid fragments were subjected to a Gibson reaction with the codon-optimized ScCas12a-VV sequence of E. coli (see SEQ ID NO.8). The Gibson program was 50 °C for 5 min, 55 °C for 10 s, for a total of 6 cycles. The Gibson reaction product was transformed into TransDB3.1 competent cells and cultured overnight at 37 °C. Positive strains were picked and plasmids were extracted to obtain the pET28a-HisScCas12a-VV-CCDB plasmid.

[0119] Artificially synthesized sgRNA primer sequences are as follows: sgRNA-F: GGTCTCACTCGTAATACGACTCACTATAGGGAATTTCTACTGTCGTAGATAAGTGATTGAAGCATACACAAGAAAATTTCTACTGTCGT; sgRNA-R: GGTCTCAACCGCAAAAAACCCCTCAAGACCCGTTTAGAGGCCCCAAGGGGTTATGCTAGATCTACGACAGTAGAAATTTTCTTGTGTAT.

[0120] The complete sgRNA sequence was constructed by PCR using primers F and R. The PCR reaction mixture consisted of 2 μL each of sgRNA-F (10 μmol / L) and sgRNA-R (10 μmol / L), 25 μL of 2×PhantaMax Buffer, 1 μL of dNTPs, 1 μL of PhantaMax Super-Fidelity DNA Polymerase (1 U / μL), and 19 μL of double-distilled water. The PCR amplification program was as follows: 94 °C denaturation for 3 min, 94 °C denaturation for 15 s, 56 °C annealing for 15 s, 72 °C extension for 5 s, for a total of 30 cycles, followed by a final extension at 72 °C for 5 min. The complete sgRNA sequence is as follows: GGTCTCACTCGTAATACGACTCACTATAGGG AATTTCTACTGTCGTAGAT AAGTGATTGAAGCATACA CAAGAA AATTTCTACTGTCGTAGAT CTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCGGTTGAGACC (The bolded sequence is the T7 promoter sequence, the italicized sequence is the ScCas12a-VV crRNA sequence, and the underlined sequence is the target sequence).

[0121] The sgRNA was ligated into the pET28a-HisScCas12a-VV-CCDB plasmid using a cleavage-ligation reaction. The reaction mixture consisted of: 1.5 μL (100 ng / μL) pET28a-HisScCas12a-VV-CCDB plasmid, 1.5 μL (100 ng / μL) sgRNA, 1.5 μL CutSmart buffer (NEB), 1.5 μL 10×ATP, 0.5 μL BsaI-HF v2 (NEB), 0.2 μL T4 DNA Ligase (TAKARA), and 8.3 μL double-distilled water. The reaction program was: 37 ℃ for 15 min, 37 ℃ for 5 min, 10 ℃ for 3 min, 20 ℃ for 5 min, for a total of 17 cycles, followed by 37 ℃ for 10 min. After the reaction, the cells were transfected into DH5α competent cells and incubated at 37 ℃. After overnight culture at ℃, positive strains were selected and plasmids were extracted to obtain pET28a-HisScCas12a-VV-sgRNA plasmid.

[0122] (2) Expression and purification of HisScCas12a-VV protein The pET28a-HisScCas12a-VV-sgRNA plasmid was transformed into BL21(DE3) competent cells. Positive single colonies were picked and transferred to 2 mL of LB broth. After culturing at 37 °C for 12 h, an appropriate amount of bacterial culture (1:100) was transferred to 100 mL of LB broth containing kanamycin resistance and cultured at 37 °C until the bacterial culture reached OD. 600 The concentration was 0.6, and IPTG was added to a final concentration of 0.5 mM. The mixture was then transferred to a 18 ℃ incubation chamber for 16 h. The cultured cells were collected by centrifugation at 8000 g for 5 min, and the cells were resuspended in protein wash buffer. The cells were then sonicated and centrifuged again at 8000 g for 5 min. The supernatant was transferred to an affinity chromatography column, and an appropriate amount of nickel beads (1:10) was added. The column was incubated at 4 ℃ for 3–4 h. After removing the protein wash buffer, protein elution buffer was added, and the column was incubated at 4 ℃ for 1 h. The eluent was collected in a 50 kDa ultrafiltration tube for protein concentration, and the buffer was replaced with protein preservation buffer. The concentrated protein was stored at -80 ℃. The expressed protein product was an RNP complex of sgRNA and HisScCas12a-VV.

[0123] (3) Construction of linear target fragments of ATN, CTN and GTN Artificial primers ATN-F, ATN-R, CTN-F, CTN-R, GTN-F, and GTN-R were synthesized. Equal amounts of primers F and R were added to a suitable amount of double-distilled water to achieve a concentration of 0.5 μM for both primers. The mixture was reacted at 95 °C for 5 min and then allowed to stand at room temperature to obtain target fragments containing ATN, CTN, and GTN. The primer sequences are as follows: ATN-F: TGTCGGGGCTGGCTTAAATN AAGTGATTGAAGCATACACAAGAA AGCTGTTTCCTGTGTGAAATTG (underlined part is the target sequence); ATN-R: CAATTTCACACAGGAAACAGCT TTCTTGTGTATGCTTCAATCACTT NATTTAAGCCAGCCCCGACA (underlined part is the target sequence); CTN-F: TGTCGGGGCTGGCTTAACTN AAGTGATTGAAGCATACACAAGAA AGCTGTTTCCTGTGTGAAATTG (underlined part is the target sequence); CTN-R: CAATTTCACACAGGAAACAGCT TTCTTGTGTATGCTTCAATCACTT NAGTTAAGCCAGCCCCGACA (underlined part is target sequence); GTN-F: TGTCGGGGCTGGCTTAAGTN AAGTGATTGAAGCATACACAAGAA AGCTGTTTCCTGTGTGAAATTG (underlined part is the target sequence); GTN-R: CAATTTCACACAGGAAACAGCT TTCTTGTGTATGCTTCAATCACTT NACTTAAGCCAGCCCCGACA (underlined is the target sequence).

[0124] The pUC19 vector was reverse-amplified. The PCR reaction system was as follows: 1 μL of pUC19 plasmid (50 to 100 ng / μL), 2 μL each of pUC19-F (10 μmol / L) and pUC19-R (10 μmol / L), 25 μL of 2×PhantaMax Buffer, 1 μL of dNTP, 1 μL of PhantaMax Super-Fidelity DNA Polymerase (1 U / μL), and 18 μL of double-distilled water. The PCR amplification program was: 94 °C denaturation for 3 min, 94 °C denaturation for 15 s, 56 °C annealing for 15 s, 72 °C extension for 1 min, for a total of 30 cycles, followed by a 5 min extension at 72 °C. The amplification primer sequences were: pUC19-F:AGCTGTTTCCTGTGTGAAAATTGTT; pUC19-R: TTAAGCCAGCCCCGACACCCGCCA.

[0125] The target fragment containing the PAM sequence was subjected to a Gibson reaction with the inversely amplified pUC19 fragment. The reaction program was: 50℃ for 5 min, 55℃ for 10 s, for a total of 6 cycles, to obtain a target library corresponding to the PAM sequence. The target library was then amplified by PCR using Vitro-F and Vitro-R primers to obtain a linearized target fragment of 1283 bp containing the corresponding PAM sequence. The primer sequences are as follows: Vitro-F: CTGTCATGCCATCCGTAAG; Vitro-R: AAGGCGGACAGGTATCCGGTAAG.

[0126] (4) Validation of HisScCas12a-VV protein in vitro cleavage activity The HisScCas12a-VV-sgRNA-bound RNP complex was used to cleave linearized target fragments containing ATN, CTN, GTN, and TTN from Example 4. The linearized target fragment was 1283 bp in size, and the target sequence was located at 720 bp of the linear target fragment. After cleavage, nucleic acid fragments of approximately 700 bp and 560 bp were expected to be obtained.

[0127] 500 ng of the linearized target fragment containing the PAM sequence, 4 μg of purified RNP complex, and 10 μL of protein reaction buffer were added. Double-distilled water was added to a final reaction volume of 20 μL. The mixture was incubated at 37 °C for 1 h, followed by the addition of 0.5 μL of proteinase K. The reaction was then incubated at 56 °C for 20 min to digest the protein in the system, and finally at 85 °C for 10 min to inactivate proteinase K, thus terminating the enzymatic reaction. The resulting product was electrophoresed on a 1.2% agarose gel to detect the in vitro protease activity of ScCas12a-VV. Results are as follows: Figure 10 As shown, after treatment with ScCas12a-VV protein, linearized target fragments containing ATN, TTN, and GTN can be cleaved, yielding nucleic acid fragments of approximately 700 bp and 560 bp, respectively. This indicates that ScCas12a-VV has nucleic acid cleavage activity in vitro, and that ScCas12a-VV can broaden the PAM recognition sequence of ScCas12a.

[0128] Example 9: Editing efficiency of ScCas12a-VV protein in rice protoplasts This embodiment edits rice protoplasts, and the selected target is rice. BADH2The gene contains GCACACCTGAAGGAAGACCAACCT. The specific steps are as follows: (1) Artificial primers ScCas12a-VV-F and ScCas12a-VV-R were synthesized to amplify the rice codon-optimized ScCas12a-VV sequence (see SEQ ID NO.9) by PCR, and NLS sequence was added. The PCR amplification system was as follows: 1 μL of rice codon-optimized ScCas12a-VV (50 to 100 ng / μL), 2 μL each of ScCas12a-VV-F (10 μmol / L) and ScCas12a-VV-R (10 μmol / L), 25 μL of 2×PhantaMax Buffer, 1 μL of dNTP, 1 μL of PhantaMax Super-Fidelity DNA Polymerase (1U / μL), and 18 μL of double-distilled water. The PCR amplification program was as follows: 94℃ denaturation for 3 min, 94℃ denaturation for 15 s, 56℃ annealing for 15 s, 72℃ extension for 2 min 30 s, for a total of 30 cycles, followed by a 72℃ extension for 5 min. The primer sequences for ScCas12a-VV-F and ScCas12a-VV-R are as follows: ScCas12a-VV-F: GATCAACGGTACACGCGTatggctcctaagaagaagcggaaggttggtattcacggggtgcctgcggctAAGGAGTTCACCAACCAGTAC (lowercase bases are NLS sequences). ScCas12a-VV-R: GAGCTCGAATTCACGCGTcttctttttcttagcctgtccggcctttttggtggcagcaggacgcttGAACTTCCTCTTCTGCAC (lowercase bases are NLS sequences).

[0129] The PCR amplification product was constructed into the pUC19-myc vector using the Gibson ligation method. The Gibson reaction program was 50 °C for 5 min, 55 °C for 10 s, for a total of 6 cycles. The Gibson reaction product was transformed into DH5α competent cells and cultured overnight at 37 °C. Positive strains were selected and plasmids were extracted to obtain the pUC19-ScCas12a-VV-myc plasmid.

[0130] The pUC19-ScCas12a-myc plasmid was digested with NcoI (Thermo Fisher enzyme) in the following reaction mixture: 6 μL pUC19-ScCas12a-VV-myc plasmid (100 ng / μL), 2 μL 10×FastDigest buffer, 0.5 μL NcoI (Thermo Fisher enzyme), 0.2 μL FastAP (Thermo Fisher), and 11.3 μL double-distilled water. The reaction program was 37 °C for 20 min followed by 80 °C for 10 min. The digested plasmid fragment was then subjected to a Gibson reaction with the sgRNA sequence. The Gibson reaction program was 50 °C for 5 min, 55 °C for 10 s, for a total of 6 cycles. The Gibson reaction product was transformed into DH5α competent cells and cultured overnight at 37 °C. Positive strains were picked, and the plasmid was extracted to obtain the pUC19-ScCas12a-VV-myc-sgRNA plasmid. The sgRNA sequence is as follows: AATTTCTACTGTCGTAGAT GCACACCTGAAGGAAGACCAACCT (Italicized text represents the crRNA sequence of ScCas12a-VV, and underlined text represents the target sequence).

[0131] (2) Remove the husks from 9311 rice seeds and place them in a sterile triangular flask. Add 75% ethanol and shake well to sterilize for 1 min, then wash three times with sterile double-distilled water. Add an appropriate amount of 2% sodium hypochlorite, place in the dark, sterilize at 28 ℃ and 150 rpm for 30 min, wash five times with sterile double-distilled water, and sterilize again with an appropriate amount of 2% sodium hypochlorite. Use sterilized tweezers to place the seeds into 1 / 2 MS medium and culture in the dark for 13-15 days.

[0132] (3) Cut off the roots and leaves of the seedlings, leaving the leaf sheaths. Use a sharp blade to cut the leaf sheaths into small segments of about 0.5 mm and place them in W5 solution in the dark for 30 min. After removing the W5 solution, add an appropriate amount of EnzymeSolution solution and shake in the dark for 4 h at 28 ℃ and 50 rpm.

[0133] (4) Remove the enzyme solution, wash the leaf sheath tissue with W5 solution, filter the wash solution through 300-mesh and 400-mesh sieves, collect the wash solution in a 50 mL centrifuge tube, centrifuge at 150 g for 5 min, remove the supernatant, wash the precipitated cells with W5 solution, centrifuge at 150 g for 5 min, remove the supernatant, and resuspend the cells in 2 mL of W5 solution. Centrifuge at 200 g for 10 min, resuspend the cells in MMG, and adjust the concentration to 2×10⁻⁶. 6 / mL.

[0134] (5) Take 10 μg of plasmid into 200 μL of suspended cells and mix gently. Add 220 μL of PEG4000, mix well, and let stand in the dark at 25 °C for 20 min.

[0135] (6) Wash with 900 μL of W5 solution, centrifuge at 300 g for 10 min and remove the supernatant. Resuspend the cells in 1 mL of WI solution and incubate at 37 ℃ in the dark for 60 h.

[0136] (7) DNA was extracted from protoplasts cultured for 60 h, and a next-generation sequencing library was constructed using BADH2#-F and BADH2#-R primers, which were then sent to the company for sequencing. The sequences of BADH2#-F and BADH2#-R primers are as follows: BADH2#-F: CTCGGAGTGATCGCACGTTGTGGCATGTCCATGC; BADH2#-R: CTGAGAGGCTGGATGGCTGTACCTTGTCTACACCA.

[0137] The sequencing results were analyzed, and the results are as follows: Figure 11 As shown, ScCas12a-VV can edit the rice genome, with an editing efficiency 1.7 times higher than ScCas12a, and the main type of editing produced is fragment deletion.

[0138] Example 10: Editing efficiency of ScCas12a-VV protein in rice This embodiment edits the ZH11 rice material, and the selected target point is... BADH2 The gene contains GTACAGCTGTACAGCCAGCAGCCA. The specific steps are as follows: The 1300 vector was double-digested using PstI (NEB) and AvrII (NEB) enzymes. The digestion system consisted of 6 μL of 1300 vector (100 ng / μL), 2 μL of CutSmart buffer (NEB), 0.5 μL of PstI (NEB) enzyme, 0.5 μL of AvrII (NEB) enzyme, 0.2 μL of FastAP (Thermo Fisher), and 10.8 μL of double-distilled water. The double digestion program was: incubation at 37 °C for 1 h followed by incubation at 85 °C for 10 min to obtain the digested 1300 vector fragment.

[0139] PCR was performed using artificially synthesized 1300-ScCas12a-VV-F and 1300-ScCas12a-VV-R primers to obtain the ScCas12a-VV fragment with the NLS sequence. The PCR amplification system was as follows: 1 μL of rice codon-optimized ScCas12a-VV (50 to 100 ng / μL), 2 μL each of 1300-ScCas12a-VV-F (10 μmol / L) and 1300-ScCas12a-VV-R (10 μmol / L), 25 μL of 2×PhantaMax Buffer, 1 μL of dNTP, 1 μL of PhantaMax Super-Fidelity DNAPolymerase (1U / μL), and 18 μL of double-distilled water. The PCR amplification program was as follows: denaturation at 94 ℃ for 3 min, denaturation at 94 ℃ for 15 s, annealing at 56 ℃ for 15 s, extension at 72 ℃ for 2 min 30 s, for a total of 30 cycles, followed by a final extension at 72 ℃ for 5 min.

[0140] The ScCas12a-VV fragment containing NLS was loaded into the 1300 restriction enzyme fragment using the Gibson reaction. The Gibson program was 50 °C for 5 min, 55 °C for 10 s, for a total of 6 cycles. The Gibson reaction product was transformed into TransDB3.1 competent cells and cultured overnight at 37 °C. Positive strains were picked, and plasmids were extracted to obtain the 1300-ScCas12a-VV-CCDB plasmid.

[0141] The sgRNA sequence was constructed into the 1300-ScCas12a-VV-CCDB plasmid using a digest-and-ligation reaction. The digest-and-ligation system consisted of: 1.5 μL (100 ng / μL) of 1300-ScCas12a-VV-CCDB plasmid, 1.5 μL (100 ng / μL) of sgRNA, 1.5 μL of CutSmart buffer (NEB), 1.5 μL of 10×ATP, 0.5 μL of BsaI-HF v2 (NEB), 0.2 μL of T4 DNA Ligase (TAKARA), and 8.3 μL of double-distilled water. The reaction program was: 37 ℃ for 15 min, 37 ℃ for 5 min, 10 ℃ for 3 min, 20 ℃ for 5 min, for a total of 17 cycles, followed by 37 ℃ for 10 min. After the reaction, the cells were transfected into DH5α competent cells and incubated at 37 ℃. After overnight culture at ℃, positive strains were selected and plasmids were extracted to obtain the 1300-ScCas12a-VV-sgRNA plasmid. The 1300-ScCas12a-VV-sgRNA plasmid was sent to the company for transformation, and the recipient material was ZH11.

[0142] The resulting transformed material was subjected to high-throughput sequencing to detect the editing of ScCas12a-VV in ZH11. The results are as follows: Figure 12 As shown, a total of 40 transformation materials were obtained, of which 26 transformation materials were... BADH2 The gene was mutated, the editing efficiency was 65%, and the mutation results of the edited material were all deletions of fragments.

[0143] Example 11: In vitro nucleic acid detection activity of ScCas12a-VV at different temperatures This embodiment identifies the in vitro nucleic acid detection activity of ScCas12a-VV. The selected double-stranded DNA fragment is the linearized target fragment with TTN, which was detected in vitro in Example 4 above. The target sequence is: AAGTGAGTTGAAGCATACACAAGAA. The specific steps are as follows: (1) Construction of recombinant plasmid pET28a-HisScCas12a-VV The pET28a plasmid was double-digested using BamHI (Thermo Fisher) enzyme and EcoRI (Thermo Fisher) enzyme. The double digestion system consisted of 6 μL pET28a plasmid (100 ng / μL), 2 μL 10×FastDigest buffer, 0.5 μL BamHI (Thermo Fisher) enzyme, 0.5 μL EcoRI (Thermo Fisher) enzyme, 0.2 μL FastAP (Thermo Fisher), and 10.8 μL double-distilled water. The reaction program was: 37 °C for 20 min, followed by 80 °C for 10 min. The enzyme-digested plasmid fragments were subjected to a Gibson reaction with the codon-optimized ScCas12a-VV sequence of E. coli. The Gibson program was 50 °C for 5 min, 55 °C for 10 s, for a total of 6 cycles. The Gibson reaction product was transformed into TransDB3.1 competent cells and cultured overnight at 37 °C. Positive strains were picked and plasmids were extracted to obtain the pET28a-HisScCas12a-VV plasmid.

[0144] (2) Expression and purification of HisScCas12a-VV protein The pET28a-HisScCas12a-VV plasmid was transformed into BL21(DE3) competent cells. Positive single colonies were picked and transferred to 2 mL of LB liquid medium. After culturing at 37 ℃ for 12 h, an appropriate amount of bacterial suspension (1:100) was transferred to 100 mL of LB liquid medium containing kanamycin resistance and cultured at 37 ℃ until the bacterial suspension reached its OD value. 600The concentration was 0.6, and IPTG was added to a final concentration of 0.5 mM. The mixture was then transferred to 18 °C and incubated for 16 h. The cultured cells were collected by centrifugation at 8000 g for 5 min. The cells were resuspended in protein wash buffer and then sonicated to disrupt the cell structure. The cells were then centrifuged at 8000 g for 5 min, and the supernatant was transferred to an affinity chromatography column. A suitable amount of nickel beads (1:10) was added, and the column was incubated at 4 °C for 3-4 h. After removing the protein wash buffer, protein elution buffer was added, and the column was incubated at 4 °C for 1 h. The eluent was collected in a 50 kDa ultrafiltration tube for protein concentration, and the buffer was replaced with protein preservation buffer. The concentrated protein was stored at -80 °C.

[0145] (3) In vitro transcription and purification of sgRNA Artificially synthesized sgRNA template DNA primers, with the primer sequences as follows: sgRNA-template-F:TAATACGACTCACTATAGGG AATTTCTACTGTCGTAGAT AAGTGATTGAAG CATACACAAGAA (The bolded sequence is the T7 promoter sequence, the italicized sequence is the ScCas12a-VV crRNA sequence, and the underlined sequence is the target sequence.) sgRNA-template-R: TTCTTGTGTATGCTTCAATCACTT ATCTACGACAGTAGAAATT CCCTATAGTGAGTCGTATTA (The bolded sequence is the T7 promoter sequence, the italicized sequence is the ScCas12a-VV crRNA sequence, and the underlined sequence is the target sequence).

[0146] Pipe 20 μL of sgRNA-template-F and 20 μL of sgRNA-template-R into a 1.5 mL centrifuge tube and mix thoroughly by pipetting. Incubate the mixture at 95°C for 5 min, then allow it to cool to room temperature to allow the sgRNA-template-F and sgRNA-template-R primers to form double strands. The complete sgRNA template DNA sequence is as follows: TAATACGACTCACTATAGGG AATTTCTACTGTCGTAGAT AAGTGATTGAAGCATACACAAGAA (The bolded sequence is the T7 promoter sequence, the italicized sequence is the ScCas12a-VV crRNA sequence, and the underlined sequence is the target sequence.)

[0147] The double-stranded DNA product of sgRNA was transcribed in vitro using NEB's T7 in vitro transcription kit to obtain ScCas12a-VV crRNA. The in vitro transcription system consisted of: 10 μL NTP Buffer Mix, 2 μL T7 RNA Polymerase Mix, 1 μg of the constructed sgRNA template DNA, and Nuclease-free Water to adjust the final volume to 30 μL. The mixed reaction system was incubated at 37°C for 16 h. The in vitro transcription product was then purified using NEB's RNA purification kit to obtain ScCas12a-VV crRNA.

[0148] (4) In vitro nucleic acid detection of ScCas12a-VV at different temperatures The artificially synthesized ssDNA reporter has the following sequence: FAM-TTATT-BHQ1; The in vitro detection system for ScCas12a-VV was prepared as follows: 2 μL NEBuffer™ 4 buffer, 1 μL crRNA (concentration of 1383 ng / μL), 0.8 μL ssDNA-reporter (concentration of 10 μM), 1 μL HisScCas12a-VV (concentration of 2400 ng / μL), 1 μL linearized target fragment with TTN (concentration of 210 ng / μL), and Nuclease-free Water was used to adjust the final volume of the system to 20 μL.

[0149] The negative control detection system consisted of: 2 μL NEBuffer™ 4 buffer, 1 μL crRNA (concentration 1383 ng / μL), 0.8 μL ssDNA-reporter (concentration 10 μM), 1 μL HisScCas12a-VV (concentration 2400 ng / μL), and Nuclease-free Water to adjust the final volume to 20 μL.

[0150] Eight temperature gradient reactions were set up in the PCR instrument at 35°C, 35.8°C, 37.5°C, 41°C, 44°C, 47.5°C, 49.2°C, and 50°C. For the in vitro detection of ScCas12a-VV, three replicates were prepared at each temperature. For the negative control, one replicate was prepared at each temperature. The prepared systems were added to PCR plates and incubated in the PCR instrument for 45 min. The resulting systems were then placed in a Jena qTower. 3 In a real-time quantitative PCR instrument, the fluorescence generated is measured in the FAM channel.

[0151] The results are as follows Figure 13 As shown, ScCas12a-VV exhibited in vitro nucleic acid detection activity at all eight temperatures, with the highest detection activity observed at 37.5°C.

[0152] Example 12: Comparison of in vitro nucleic acid detection activities of ScCas12a, ScCas12a-VV, LbCas12a, and FnCas12a. This embodiment identifies the in vitro nucleic acid detection activity of ScCas12a and ScCas12a-VV and compares it with the commonly used in vitro nucleic acid detection activity of LbCas12a and FnCas12a. The selected double-stranded DNA fragment is the linearized target fragment with TTN that was detected in vitro in Example 4 above. The target sequence is: AAGTGATTGAAGCATACACAAGAA.

[0153] The construction of recombinant plasmids for in vitro protein expression of ScCas12a, ScCas12a-VV, LbCas12a and FnCas12a, as well as the protein expression and purification, all used the methods in Example 11.

[0154] The crRNA for ScCas12a and ScCas12a-VV was obtained using the crRNA purified in Example 11.

[0155] Artificially synthesized sgRNA template DNA primers for LbCas12a and FnCas12a were used. The primer sequences are as follows: Lb-sgRNA-template-F:TAATACGACTCACTATAGGG TAATTTCTACTAAGTGTAGAT AAGTGAT TGAAGCATACACAAGAA (The bolded sequence is the T7 promoter sequence, the italicized sequence is the LbCas12a crRNA sequence, and the underlined sequence is the target sequence.) Lb-sgRNA-template-R: TTCTTGTGTATGCTTCAATCACTT ATCTACACTTAGTAGAAATTA CCCTATAGTGAGTCGTATTA (The bolded sequence is the T7 promoter sequence, the italicized sequence is the LbCas12a crRNA sequence, and the underlined sequence is the target sequence). Fn-sgRNA-template-F:TAATACGACTCACTATAGGG AATTTCTACTGTTGTAGAT AAGTGATTG AAGCATACACAAGAA (The bolded sequence is the T7 promoter sequence, the italicized sequence is the crRNA sequence of FnCas12a, and the underlined sequence is the target sequence.) Fn-sgRNA-template-R: TTCTTGTGTATGCTTCAATCACTTATCTACAACAGTAGAAATT CCCTATAGTGAGTCGTATTA (The bolded sequence is the T7 promoter sequence, the italicized sequence is the FnCas12a crRNA sequence, and the underlined sequence is the target sequence).

[0156] Using the in vitro nucleic acid detection system preparation method of ScCas12a-VV in Example 11, in vitro nucleic acid detection systems of LbCas12a, FnCas12a, ScCas12a and ScCas12a-VV were prepared respectively, with 5 replicates for each sample.

[0157] Using the method for preparing negative control systems for in vitro nucleic acid detection in Example 11, negative control systems for in vitro nucleic acid detection of LbCas12a, FnCas12a, ScCas12a and ScCas12a-VV were prepared, with three replicates for each sample.

[0158] The prepared in vitro nucleic acid detection systems for LbCas12a, FnCas12a, ScCas12a, and ScCas12a-VV, as well as the negative control systems for in vitro nucleic acid detection of LbCas12a, FnCas12a, ScCas12a, and ScCas12a-VV, were added to a 96-well real-time PCR plate and placed in a Jena qTower. 3 In a real-time quantitative PCR instrument, the fluorescence generated by the reaction was measured in the FAM channel. The reaction program was: 37°C for 45 min, with fluorescence values ​​read once per minute.

[0159] The results are as follows Figure 14 As shown, LbCas12a, FnCas12a, ScCas12a and ScCas12a-VV all have in vitro nucleic acid detection activity, while ScCas12a and ScCas12a-VV have higher in vitro nucleic acid detection activity than LbCas12a and FnCas12a, and ScCas12a-VV has the highest in vitro nucleic acid detection activity.

[0160] Example 13: The effect of target and crRNA mispairing on the in vitro nucleic acid detection activity of ScCas12a-VV This embodiment identifies the specificity of the in vitro nucleic acid detection activity of ScCas12a-VV. The selected double-stranded DNA fragment is the linearized target fragment with TTN in the in vitro cleavage activity detection of the above embodiment 4. The target sequence is: AAGTGATTGAAGCATACACAAGAA.

[0161] The specific steps for constructing 12 crRNAs with a single base mismatch with the linearized target fragment are as follows: Twelve primers for mismatched crRNAs were artificially synthesized, with the following sequences: sgRNA-template-mm1-F:TAATACGACTCACTATAGGG AATTTCTACTGTCGTAGAT TAGTGATT GAAGCATACACAAGAA (The bolded sequence is the T7 promoter sequence, the italicized sequence is the ScCas12a-VV crRNA sequence, and the underlined sequence is the target sequence.) sgRNA-template-mm1-R: TTCTTGTGTATGCTTCAATCACTA ATCTACGACAGTAGAAATT CCCTATAGTGAGTCGTATTA (The bolded sequence is the T7 promoter sequence, the italicized sequence is the ScCas12a-VV crRNA sequence, and the underlined sequence is the target sequence). sgRNA-template-mm2-F:TAATACGACTCACTATAGGG AATTTCTACTGTCGTAGAT AACTGATT GAAGCATACACAAGAA (The bolded sequence is the T7 promoter sequence, the italicized sequence is the ScCas12a-VV crRNA sequence, and the underlined sequence is the target sequence.) sgRNA-template-mm2-R: TTCTTGTGTATGCTTCAATCAGTT ATCTACGACAGTAGAAATT CCCTATAGTGAGTCGTATTA (The bolded sequence is the T7 promoter sequence, the italicized sequence is the ScCas12a-VV crRNA sequence, and the underlined sequence is the target sequence). sgRNA-template-mm3-F:TAATACGACTCACTATAGGG AATTTCTACTGTCGTAGAT AAGTCATT GAAGCATACACAAGAA (The bolded sequence is the T7 promoter sequence, the italicized sequence is the ScCas12a-VV crRNA sequence, and the underlined sequence is the target sequence.) sgRNA-template-mm3-R: TTCTTGTGTATGCTTCAATGACTT ATCTACGACAGTAGAAATT CCCTATAGTGAGTCGTATTA (The bolded sequence is the T7 promoter sequence, the italicized sequence is the ScCas12a-VV crRNA sequence, and the underlined sequence is the target sequence). sgRNA-template-mm4-F:TAATACGACTCACTATAGGG AATTTCTACTGTCGTAGAT AAGTGAAT GAAGCATACACAAGAA (The bolded sequence is the T7 promoter sequence, the italicized sequence is the ScCas12a-VV crRNA sequence, and the underlined sequence is the target sequence.) sgRNA-template-mm4-R:TTCTTGTGTATGCTTCATTCACTT ATCTACGACAGTAGAAATT CCCTATAGTGAGTCGTATTA (The bolded sequence is the T7 promoter sequence, the italicized sequence is the ScCas12a-VV crRNA sequence, and the underlined sequence is the target sequence). sgRNA-template-mm5-F:TAATACGACTCACTATAGGG AATTTCTACTGTCGTAGAT AAGTGATT CAAGCATACACAAGAA (The bolded sequence is the T7 promoter sequence, the italicized sequence is the ScCas12a-VV crRNA sequence, and the underlined sequence is the target sequence.) sgRNA-template-mm5-R: TTCTTGTGTATGCTTGAATCACTT ATCTACGACAGTAGAAATT CCCTATAGTGAGTCGTATTA (The bolded sequence is the T7 promoter sequence, the italicized sequence is the ScCas12a-VV crRNA sequence, and the underlined sequence is the target sequence). sgRNA-template-mm6-F:TAATACGACTCACTATAGGG AATTTCTACTGTCGTAGAT AAGTGATT GATGCATACACAAGAA (The bolded sequence is the T7 promoter sequence, the italicized sequence is the ScCas12a-VV crRNA sequence, and the underlined sequence is the target sequence.) sgRNA-template-mm6-R: TTCTTGTGTATGCATCAATCACTT ATCTACGACAGTAGAAATT CCCTATAGTGAGTCGTATTA (The bolded sequence is the T7 promoter sequence, the italicized sequence is the ScCas12a-VV crRNA sequence, and the underlined sequence is the target sequence). sgRNA-template-mm7-F:TAATACGACTCACTATAGGG AATTTCTACTGTCGTAGAT AAGTGATT GAAGGATACACAAGAA (The bolded sequence is the T7 promoter sequence, the italicized sequence is the ScCas12a-VV crRNA sequence, and the underlined sequence is the target sequence.) sgRNA-template-mm7-R: TTCTTGTGTATCCTTCAATCACTT ATCTACGACAGTAGAAATT CCCTATAGTGAGTCGTATTA (The bolded sequence is the T7 promoter sequence, the italicized sequence is the ScCas12a-VV crRNA sequence, and the underlined sequence is the target sequence). sgRNA-template-mm8-F:TAATACGACTCACTATAGGG AATTTCTACTGTCGTAGAT AAGTGATT GAAGCAAACACAAGAA(The bolded sequence is the T7 promoter sequence, the italicized sequence is the ScCas12a-VV crRNA sequence, and the underlined sequence is the target sequence.) sgRNA-template-mm8-R: TTCTTGTGTTTGCTTCAATCACTT ATCTACGACAGTAGAAATT CCCTATAGTGAGTCGTATTA (The bolded sequence is the T7 promoter sequence, the italicized sequence is the ScCas12a-VV crRNA sequence, and the underlined sequence is the target sequence). sgRNA-template-mm9-F:TAATACGACTCACTATAGGG AATTTCTACTGTCGTAGAT AAGTGATT GAAGCATAGACAAGAA (The bolded sequence is the T7 promoter sequence, the italicized sequence is the ScCas12a-VV crRNA sequence, and the underlined sequence is the target sequence.) sgRNA-template-mm9-R: TTCTTGTCTATGCTTCAATCACTT ATCTACGACAGTAGAAATT CCCTATAGTGAGTCGTATTA (The bolded sequence is the T7 promoter sequence, the italicized sequence is the ScCas12a-VV crRNA sequence, and the underlined sequence is the target sequence). sgRNA-template-mm10-F:TAATACGACTCACTATAGGG AATTTCTACTGTCGTAGAT AAGTGAT TGAAGCATACAGAAGAA (The bolded sequence is the T7 promoter sequence, the italicized sequence is the ScCas12a-VV crRNA sequence, and the underlined sequence is the target sequence.) sgRNA-template-mm10-R: TTCTTCTGTATGCTTCAATCACTT ATCTACGACAGTAGAAATT CCCTATAGTGAGTCGTATTA (The bolded sequence is the T7 promoter sequence, the italicized sequence is the ScCas12a-VV crRNA sequence, and the underlined sequence is the target sequence). sgRNA-template-mm11-F:TAATACGACTCACTATAGGG AATTTCTACTGTCGTAGAT AAGTGAT TGAAGCATACACATGAA (The bolded sequence is the T7 promoter sequence, the italicized sequence is the ScCas12a-VV crRNA sequence, and the underlined sequence is the target sequence.) sgRNA-template-mm11-R: TTCATGTGTATGCTTCAATCACTT ATCTACGACAGTAGAAATT CCCTATAGTGAGTCGTATTA (The bolded sequence is the T7 promoter sequence, the italicized sequence is the ScCas12a-VV crRNA sequence, and the underlined sequence is the target sequence). sgRNA-template-mm12-F:TAATACGACTCACTATAGGG AATTTCTACTGTCGTAGAT AAGTGAT TGAAGCATACACAAGTA (The bolded sequence is the T7 promoter sequence, the italicized sequence is the ScCas12a-VV crRNA sequence, and the underlined sequence is the target sequence.) sgRNA-template-mm12-R: TACTTGTGTATGCTTCAATCACTT ATCTACGACAGTAGAAATT CCCTATAGTGAGTCGTATTA (The bolded sequence is the T7 promoter sequence, the italicized sequence is the ScCas12a-VV crRNA sequence, and the underlined sequence is the target sequence).

[0162] Twelve mismatched crRNAs were constructed according to the crRNA template construction, in vitro transcription, and purification steps in Example 11.

[0163] According to the preparation method of the ScCas12a-VV in vitro nucleic acid detection system in Example 11, in vitro nucleic acid activity detection systems with no mismatch crRNA and 12 mismatch crRNA were prepared, and three replicates of each were prepared.

[0164] Add the prepared mixture to a 96-well real-time PCR plate and place it in a Jena qTower. 3 In a real-time quantitative PCR instrument, the fluorescence generated by the reaction was measured in the FAM channel. The reaction program was: 37°C for 45 min, with fluorescence values ​​read once per minute. The results are as follows: Figure 15 As shown, ScCas12a-VV exhibits high in vitro nucleic acid detection activity in the absence of mismatched crRNA, while its in vitro nucleic acid detection activity significantly decreases when there is a mismatch between the crRNA and the target site, indicating that ScCas12a-VV has high specificity for in vitro nucleic acid detection.

[0165] Example 14: Detection of PAM recognition range in in vitro nucleic acid detection of LbCas12a, FnCas12a, ScCas12a and ScCas12a-VV In the in vitro cleavage of ScCas12a-VV in Example 8, ScCas12a effectively cleaved target fragments containing ATN, TTN, and GTN. To determine whether ScCas12a-VV could also detect ATN, TTN, and GTN target fragments in in vitro nucleic acid detection, this example identified the in vitro nucleic acid detection activity of the linear target fragments containing ATN, TTN, CTN, and GTN constructed in Examples 4 and 8, and compared them with LbCas12a and FnCas12a. The specific implementation steps are as follows: Using the method for preparing the ScCas12a-VV in vitro nucleic acid detection system in Example 11, in vitro nucleic acid detection systems for LbCas12a, FnCas12a, ScCas12a, and ScCas12a-VV at linear target fragments of ATN, TTN, CTN, and GTN were prepared, for a total of 16 systems, with 3 replicates for each system.

[0166] Using the method for preparing the negative control system in Example 11, negative control systems for in vitro nucleic acid detection of LbCas12a, FnCas12a, ScCas12a and ScCas12a-VV were prepared, with three replicates for each system.

[0167] Add the prepared mixture to a 96-well real-time PCR plate and place it in a Jena qTower. 3 In a real-time quantitative PCR instrument, the fluorescence generated by the reaction was measured in the FAM channel. The reaction program was: 37°C for 45 min, with fluorescence values ​​read once per minute. The results are as follows: Figure 16 As shown, ScCas12a and ScCas12a-VV all exhibit certain nucleic acid detection activities under ATN, TTN, CTN, and GTN conditions, while LbCas12a shows high activity under TTN and CTN conditions. FnCas12a only shows high nucleic acid detection activity when the PAM sequence is TTN. Among LbCas12a, FnCas12a, ScCas12a, and ScCas12a-VV, ScCas12a-VV has the highest nucleic acid detection activity and the widest range of PAM recognition.

[0168] Example 15: In vitro nucleic acid detection signal amplifier based on ScCas12a-VV This embodiment utilizes the principle that splitting crRNA can detect RNA samples and that amplification-free one-pot method can amplify fluorescence signals. Based on ScCas12a-VV, a highly sensitive nucleic acid detection system capable of detecting RNA samples without reverse transcription is constructed. This system comprises five components: ScCas12a-VV protein, scaffold RNA, TCC amplifier, TCC-crRNA, and ssDNAReporter. The specific steps are as follows: (1) Prokaryotic expression and purification of ScCas12a-VV protein were obtained according to the method of prokaryotic expression and purification of ScCas12a-VV protein in Example 11.

[0169] (2) Scaffold RNA preparation: The artificially synthesized scaffold RNA template DNA primer sequence is as follows: scaffold RNA-F: TAATACGACTCACTATAGGG AATTTCTACTGTCGTAGAT (The bolded sequence is the T7 promoter sequence, and the italicized sequence is the crRNA sequence of ScCas12a-VV). scaffold RNA-R: ATCTACGACAGTAGAAATT CCCTATAGTGAGTCGTATTA (The bolded sequence is the T7 promoter sequence, and the italicized sequence is the ScCas12a-VV crRNA sequence).

[0170] Following the crRNA template construction, in vitro transcription, and purification steps in Example 11, scaffold RNA double-stranded DNA templates were constructed from scaffold RNA-F and scaffold RNA-R, and scaffold RNA was transcribed. The prepared scaffold RNA was stored at -80°C for later use.

[0171] (3) Preparation of TCC amplifier.

[0172] The artificially synthesized TCC amplifier heterocyclic oligonucleotide has the following sequence: GGTCTCCTCAAACAAAGCATCCTTTTTTTTGGATTGTTTTTTTTCTTTGTTTGAGGAGACC; Prepare the annealing reaction system in a 1.5 mL sterile centrifuge tube. Take 5 μL of single-stranded TNH heterocyclic oligonucleotides (100 μM stock solution), add 5 μL of 10× annealing buffer, and finally bring the volume to 50 μL with RNase-free water. After mixing, incubate the reaction system at 95°C for 5 min to allow the oligonucleotides to completely denature into single-stranded DNA. Then, place the reaction system at room temperature for 30 min to allow the system to cool to room temperature. The oligonucleotides will fold to form a double stem-loop structure, i.e., a TCC amplifier. Store the prepared TCC amplifier at 4°C for later use.

[0173] (4) TCC-crRNA preparation.

[0174] Artificially synthesized primers were used to construct a TCC amplifier that recognizes linearization. The specific primer sequences are as follows: TCC-crRNA-template-F:TAATACGACTCACTATAGGG AATTTCTACTGTCGTAGAT GGATGCTT TGTTTGAGGAGACC (The bolded sequence is the T7 promoter sequence, the italicized sequence is the ScCas12a-VV crRNA sequence, and the underlined sequence is the target sequence.)

[0175] TCC-crRNA-template-R: GGTCTCCTCAAACAAAGCATCC ATCTACGACAGTAGAAATT CCCTATAGTGAGTCGTATTA (The bolded sequence is the T7 promoter sequence, the italicized sequence is the ScCas12a-VV crRNA sequence, and the underlined sequence is the target sequence).

[0176] Following the crRNA template construction, in vitro transcription, and purification steps in Example 11, a TCC-crRNA-template was constructed, and TCC-crRNA was prepared using the TCC-crRNA-template as a double-stranded DNA template and stored at -80°C for later use.

[0177] (5) The ssDNA Reporter uses the ssDNA-reporter from Example 11, with the following specific sequence: FAM-TTATT-BHQ1.

[0178] (6) Preparation of long RNA samples Artificially synthesized primers were used to construct double-stranded DNA templates for long RNA samples, and the RMSV target sequence was added at position 556 of the fragment. The specific primer sequences are as follows: RNA-F1: TAATACGACTCACTATAGGGG; RNA-R1: GCTGTCTTGACTTCTGTCACGTCG GAACTGCCCATTCTTTGGTA (underlined part is target sequence); RNA-F2: GACAGAAGTCAAGACAGCATGGGTGAGAACAACGAAC; RNA-R2: AAAGCTGATTGCAGTGGC; PCR amplification was performed using pET28a-HisScCas12a-sgRNA plasmid constructed in Example 3 as templates, with RNA-F1 / RNA-R1 and RNA-F2 / RNA-R2 as templates, respectively. The PCR reaction system consisted of 2 μL each of RNA-F1 / RNA-F2 (10 μmol / L) and RNA-R1 / RNA-R2 (10 μmol / L), 1 μL of pET28a-HisScCas12a-sgRNA, 25 μL of 2×PhantaMax Buffer, 1 μL of dNTP, 1 μL of PhantaMax Super-Fidelity DNA Polymerase (1 U / μL), and 18 μL of double-distilled water. The PCR amplification program was as follows: denaturation at 94 °C for 3 min, denaturation at 94 °C for 15 s, annealing at 56 °C for 15 s, extension at 72 °C for 20 s, for a total of 30 cycles, followed by a final extension at 72 °C for 5 min. The fragment amplified by RNA-F1 / RNA-R1 is designated as RNA-1, and the fragment amplified by RNA-F2 / RNA-R2 is designated as RNA-2. Their specific sequences are as follows: RNA-template1: TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGGGCAGCAGCCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCG GCAGCCATATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGCGGATCCATGAAAGAATTCACCAACCAGTATTCTCTGACTAAGACCTTGCGTTTCGAGCTTCGTCCAGTAGGTGAAACTGCGGAGAAGATCGAAGAC TTCAAATCCGGTGGCCTCAAACAAACTGTAGAGAAAGACCGCGAACGTACCGAAGCTTACAAACAGCTCAAAGAGGTAATCGACTCTTATCATCGCGACTTCATCGAGCAGGCCTTCGCACGCCAACAGACCTTGAGCGA AGAAGACTTCAAACAGACCTACCAGCTGTACAAAGAAGCACAGAAAGAAAAAGACGGCGAAACGCTGACCAAACAGTACGAGCACCTTCGCAAGAAGATCGCTGCTATGTTCTCCAAAGCTACCAAAGAATGGGCAGTTC CGACGTGACAGAAGTCAAGACAGC (The bolded sequence is the T7 promoter sequence, and the underlined sequence is the target sequence).

[0179] RNA-template2: GACAGAAGTCAAGACAGC ATGGGTGAGAACAACGAACTGATCGGCAAGAATAAAGAGAGCAAGCTGTATCAGTGGCTCGAGAAGAACTACCGTGCGGCCGTATTGAGAAAGAAGAATTCGACCATAACGCCGGTCTGATCGAGTACTTTGAGAAGTTCTCTACCTATTTCGTTGGCTTCGATAAGAACCGTGCTAACATGTACAGCAAAGAGGCAAAGGCCACTGCAATCAGCTTT (Underlined part is the target sequence).

[0180] Subsequently, RNA-template1 and RNA-template2 samples were gel-cleaved and recovered. PCR amplification was then performed using primers RNA-F1 and RNA-R2 to splice the RNA1 and RNA2 samples into a complete long-chain double-stranded DNA template, RNA-template. The PCR reaction system consisted of 2 μL each of RNA-F1 (10 μmol / L) and RNA-R2 (10 μmol / L), 1 μL each of RNA-template1 and RNA-template2, 25 μL of 2×PhantaMax Buffer, 1 μL of dNTPs, 1 μL of PhantaMax Super-Fidelity DNA Polymerase (1 U / μL), and 1 μL of double-distilled water. The PCR amplification program was: 94 °C denaturation for 3 min, 94 °C denaturation for 15 s, 56 °C annealing for 15 s, 72 °C extension for 25 s, for a total of 30 cycles, followed by a 5 min extension at 72 °C. The specific sequences of the amplified RNA-template are as follows: RNA-template: TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGGGCAGCAGCCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCG GCAGCCATATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGCGGATCCATGAAAGAATTCACCAACCAGTATTCTCTGACTAAGACCTTGCGTTTCGAGCTTCGTCCAGTAGGTGAAACTGCGGAGAAGATCGAAGAC TTCAAATCCGGTGGCCTCAAACAAACTGTAGAGAAAGACCGCGAACGTACCGAAGCTTACAAACAGCTCAAAGAGGTAATCGACTCTTATCATCGCGACTTCATCGAGCAGGCCTTCGCACGCCAACAGACCTTGAGCGA AGAAGACTTCAAACAGACCTACCAGCTGTACAAAGAAGCACAGAAAGAAAAAGACGGCGAAACGCTGACCAAACAGTACGAGCACCTTCGCAAGAAGATCGCTGCTATGTTCTCCAAAGCTACCAAAGAATGGGCAGTTC CGACGTGACAGAAGTCAAGACAGC ATGGGTGAGAACAACGAACTGATCGGCAAGAATAAAGAGAGCAAGCTGTATCAGTGGCTCGAGAAGAACTACCGTGCGGCCGTATTGAGAAAGAAGAATTCGACCATAACGCCGGTCTGATCGAGTACTTTGAGAAGTTCTCTACCTATTTCGTTGGCTTCGATAAGAACCGTGCTAACATGTACAGCAAAGAGGCAAAGGCCACTGCAATCAGCTTT (The bold sequence is the T7 promoter sequence, and the underlined sequence is the target sequence).

[0181] Using the in vitro transcription and purification steps in Example 11, long RNA samples were transcribed using the RNA-template as a template and stored at -80°C for later use.

[0182] Artificially synthesized primers are complementary to the target sequence in the long RNA fragment. The specific sequences are as follows: ssDNA: CGACGTGACAGAAGTCAAGACAGC.

[0183] (7) Detection of long RNA samples based on ScCas12a-VV The in vitro detection system containing TNH was prepared as follows: 5 μL of NEBuffer™ 4 buffer, 150 nM scaffold RNA, 100 nM TCC-crRNA, 200 nM HisScCas12a-VV protein, 100 nM TCC amplifier, 250 nM ssDNA reporter, 400 ng of long RNA sample, 150 nM ssDNA, and Nuclease-free water was used to adjust the final volume of the system to 50 μL (TNH+).

[0184] The negative control system containing TNH was prepared as follows: 5 μL NEBuffer™ 4 buffer, scaffold RNA with a final concentration of 150 nM, TCC-crRNA with a final concentration of 100 nM, HisScCas12a-VV protein with a final concentration of 200 nM, TCC amplifier with a final concentration of 100 nM, ssDNAReporter with a final concentration of 250 nM, ssDNA with a final concentration of 150 nM, and Nuclease-free Water was used to adjust the final volume of the system to 50 μL (NC-TNH+).

[0185] Prepare an in vitro detection system free of TNH as follows: 5 μL NEBuffer™ 4 buffer, scaffold RNA with a final concentration of 150 nM, TCC-crRNA with a final concentration of 100 nM, HisScCas12a-VV protein with a final concentration of 200 nM, ssDNA Reporter with a final concentration of 250 nM, 400 ng of long RNA sample, ssDNA with a final concentration of 150 nM, and adjust the final volume of the system to 50 μL (TNH-) with Nuclease-free Water.

[0186] Prepare an in vitro negative control system that does not contain TNH, specifically: 5 μL NEBuffer™ 4 buffer, 150 nM scaffold RNA, 100 nM TCC-crRNA, 200 nM HisScCas12a-VV protein, 250 nM ssDNA Reporter, 150 nM ssDNA, and adjust the final volume of the system to 50 μL (NC-TNH-) with Nuclease-free Water.

[0187] The four reaction systems were wrapped in aluminum foil and placed at room temperature for 1 hour. After the reaction, the results were visualized using a handheld UV lamp and laser goggles. Figure 17 As shown, no obvious green fluorescence was observed in the NC-TNH-, TNH-, and NC-TNH+ reaction systems, while TNH+ showed obvious green fluorescence under ultraviolet light irradiation, indicating that the TNH system amplifies the fluorescence signal.

[0188] Example 16: In vitro detection of cDNA based on ScCas12a-VV This embodiment uses the long RNA sample and TNH system constructed in Example 15 to build a high-sensitivity reverse transcription detection platform based on ScCas12a-VV. The specific steps are as follows: (1) Construction of cDNA.

[0189] The long RNA sample from Example 15 was reverse transcribed using the ABScript Neo RT Master Mix for qPCR with gDNA Remover (ABclonal) kit to obtain a cDNA sample.

[0190] (2) The stock solution for the in vitro cDNA detection reaction system was constructed based on the TNH system constructed in Example 15, specifically: 5 μL of NEBuffer™ 4 buffer, 150 nM crRNA, 100 nM TCC-crRNA, 200 nM HisScCas12a-VV protein, 100 nM TCC amplifier, and 250 nM ssDNA Reporter.

[0191] Based on the stock solution of the in vitro cDNA detection reaction system containing the TNH system, reaction systems with final cDNA concentrations of 100 pM, 10 pM, 1 pM and 100 aM were constructed, and Nuclease-free Water was added to adjust the final volume of the system to 50 μL.

[0192] The stock solution for the in vitro cDNA detection reaction system without the TNH system was prepared as follows: 5 μL of NEBuffer™ 4 buffer, 150 nM crRNA, 100 nM TCC-crRNA, 200 nM HisScCas12a-VV protein, and 250 nM ssDNA Reporter.

[0193] Based on the stock solution of the in vitro cDNA detection reaction system without TNH, reaction systems with final cDNA concentrations of 100 pM, 10 pM, 1 pM and 100 aM were constructed, and Nuclease-free Water was added to adjust the final volume of the system to 50 μL.

[0194] All the above reaction systems were wrapped in aluminum foil and placed at room temperature for 1 hour. After the reaction, the results were visualized using a handheld UV lamp and laser goggles. Figure 18 As shown, the reaction system with the addition of the TNH system exhibits more pronounced green fluorescence, indicating that the TNH system can effectively improve the sensitivity of cDNA detection.

[0195] Simultaneously, based on the stock solution of the in vitro cDNA detection reaction system containing the TNH system, reaction systems with final cDNA concentrations of 100 pM, 10 pM, 1 pM, and 100 aM were constructed, and Nuclease-free Water was added to adjust the final volume of the system to 50 μL. Three replicates were prepared for each concentration system.

[0196] Based on the stock solution of the in vitro cDNA detection reaction system containing the TNH system, a negative control system was prepared, and Nuclease-free Water was added to adjust the final volume of the system to 50 μL.

[0197] Based on the stock solution of the in vitro cDNA detection reaction system without TNH, reaction systems with final cDNA concentrations of 100 pM, 10 pM, 1 pM, and 100 aM were constructed, and nuclease-free water was added to adjust the final volume of the system to 50 μL. Triple replicates were prepared for each concentration system.

[0198] Based on the stock solution of the in vitro cDNA detection reaction system without TNH, a negative control system was prepared, and Nuclease-free Water was added to adjust the final volume of the system to 50 μL.

[0199] Add the prepared mixture to a 96-well real-time PCR plate and place it in a Jena qTower. 3In a real-time quantitative PCR instrument, the fluorescence generated by the reaction was measured in the FAM channel. The reaction program was: 37°C for 45 min, with fluorescence values ​​read once per minute. The results are as follows: Figure 19 As shown, the TNH amplifier significantly amplified the intensity of the fluorescence signal and improved the sensitivity of the in vitro detection system in the ScCas12a-VV in vitro detection system.

[0200] In summary, the ScCas12a protein of this invention can recognize the 5'-TTN-3' PAM sequence, exhibits typical Cas12a nucleic acid cleavage activity (cis and trans cleavage), and achieves gene editing in the rice genome. The obtained ScCas12a-VV mutant protein broadens the PAM recognition sequence from 5'-TTN-3' to 5'-DTN-3' (D=A / T / G), expanding the PAM coverage from 3 types (TTA / TTC / TTG) to 9 types (ATA / ATC / ATG, TTA / TTC / TTG, GTA / GTC / GTG), significantly improving target site coverage. The editing efficiency of ScCas12a-VV in rice protoplasts is 1.7 times that of ScCas12a, and the editing efficiency in stable transgenic rice reaches 65%, significantly superior to ScCas12a and several previously reported Cas12a homologs. ScCas12a-VV maintains trans-cleavage activity over a wide temperature range of 35 °C to 50 °C, with the highest activity at 37.5 °C. Its in vitro nucleic acid detection activity is higher than that of LbCas12a and FnCas12a. The ScCas12a-VV mutant protein exhibits good resolution for single base mismatches. ScCas12a-VV shows nucleic acid detection activity under four PAMs: ATN, TTN, CTN, and GTN, with a significantly better PAM recognition range than LbCas12a (TTN and CTN only) and FnCas12a (TTN only). Finally, the nucleic acid detection system constructed in this invention, with its TNH amplifier, significantly amplifies the fluorescence signal intensity in the ScCas12a-VV in vitro detection system, improving the sensitivity of the in vitro detection system.

[0201] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. A ScCas12a protein, characterized in that, The ScCas12a protein has one of the following amino acid sequences: (a) The amino acid sequence as shown in SEQ ID NO.1; (b) An amino acid sequence that has at least 95% sequence homology with SEQ ID NO.1 and has nucleic acid cleavage function; (c) An amino acid sequence that retains the same nucleic acid cleavage function as SEQ ID NO.1 after one or more amino acid substitutions, deletions or additions based on the amino acid sequence shown in SEQ ID NO.

1.

2. The ScCas12a protein according to claim 1, characterized in that, The ScCas12a protein is a ScCas12a-VV mutant protein, and its amino acid sequence is shown in SEQ ID NO.

7.

3. The ScCas12a protein according to claim 1 or 2, characterized in that, The ScCas12a protein is linked to a His tag.

4. An isolated nucleic acid molecule, characterized in that, The nucleic acid molecule comprises a nucleotide sequence encoding the ScCas12a protein according to any one of claims 1-3, and the nucleic acid molecule has a nucleotide sequence having one of the following sequences: (a) A nucleotide sequence as shown in SEQ ID NO.2 or SEQ ID NO.5; (b) Codon-optimized nucleotide sequences as shown in SEQ ID NO.3 or SEQ ID NO.4; (c) A nucleotide sequence having one or more base substitutions, deletions or additions compared to SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO.5; or a nucleotide sequence having at least 70% sequence identity compared to SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO.5 and encoding a protein with nucleic acid cleavage function.

5. A recombinant vector, characterized in that, The recombinant vector comprises the nucleic acid molecule of claim 4.

6. A recombinant cell, characterized in that, The recombinant cell comprises the recombinant vector of claim 5, or has the nucleic acid molecule of claim 4 integrated into its genome.

7. The use of the ScCas12a protein as described in any one of claims 1-3 or the nucleic acid molecule as described in claim 4 in the preparation of gene editing tools or nucleic acid detection tools.

8. A gene editing method, characterized in that, The method includes introducing the ScCas12a protein or its coding sequence according to any one of claims 1-3 into a target cell, so that the ScCas12a protein recognizes and cleaves the in situ adjacent motif of the target nucleic acid sequence under the guidance of guide RNA.

9. The gene editing method according to claim 8, characterized in that: When the ScCas12a protein is the ScCas12a protein shown in SEQ ID NO.1, its recognized in-situ neighboring motif is 5'-TTN-3', where N is A / T / C / G; When the ScCas12a protein is the ScCas12a-VV mutant protein shown in SEQ ID NO.7, its recognized in situ neighboring motif is 5'-DTN-3', where D stands for A / T / G.

10. A nucleic acid detection system based on CRISPR / Cas12a, characterized in that, The system includes: The ScCas12a-VV mutant protein or its coding sequence as described in claim 2; Guide RNA, the guide RNA comprising a guide sequence capable of hybridizing with a target nucleic acid sequence; A signal reporter molecule, wherein the signal reporter molecule is a single-stranded DNA with a fluorescent group and a quenching group; scaffold RNA; TCC-crRNA, wherein the TCC amplifier is a DNA oligonucleotide with a double stem-loop structure; The system utilizes the trans-cleavage activity activated by the ScCas12a-VV mutant protein after recognizing the target nucleic acid under the guidance of guide RNA, and the cleavage signal reporter molecule generates a detectable signal.

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