A gene editing system NovaCas-2 with multiple PAM selection and its applications

Abstract

The present invention discloses a high-efficiency gene editing system NovaCas 2 with multiple PAM selections and its application, which belongs to the field of gene editing technology. The gene editing system NovaCas 2 is a complex of NovaCas 2 protein and sgRNA, which can accurately locate the target DNA sequence and produce cutting, causing double-strand break damage to the DNA, wherein the amino acid sequence of NovaCas 2 protein is shown in SEQ ID NO: 1, and the nucleotide sequence of sgRNA is shown in SEQ ID NO: 7 or SEQ ID NO: 9. The benefits of the present invention are: (1) NovaCas 2 protein can recognize up to 36 PAM sequences, which greatly broadens the scope of targeted gene editing of existing gene editing systems; (2) It has been experimentally confirmed that the gene editing system NovaCas 2 has higher gene editing efficiency than existing gene editing systems.

Description

Technical Field

[0001] The present invention relates to a gene editing system and applications thereof, and in particular to a high-efficiency gene editing system NovaCas-2 with multiple PAM selections and applications thereof, belonging to the technical field of gene editing. Background Art

[0002] CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) / Cas (CRISPR-associated protein) is an adaptive immune system evolved by bacteria and archaea to defend against invading viruses or plasmids. Currently, 33 Cas enzyme subtypes have been discovered, divided into two major classes and six distinct types. CRISPR / Cas12 belongs to the second class of V-type RNA-guided endonucleases. The most studied protein in this family is Cas12a. The most commonly used Cas12a proteins are AsCas12a, from the genus Acidaminococcus Rogosa, and LbCas12a, from the family Lachnospiraceae. Cas12a proteins have been widely used in a variety of species, including bacteria, yeast, plants, and human cells.

[0003] In the CRISPR / Cas12a gene editing system, the Cas12a protein forms a complex with a single guide RNA (sgRNA) and recognizes the PAM (Protospacer Adjacent Motif) sequence at the target site. After recognizing the PAM sequence, the CRISPR-derived RNA (crRNA) forms a complementary structure with the target DNA sequence, and the Cas12a protein cleaves the DNA, causing double-strand breaks. In cells, two main DNA repair mechanisms are responsible for repairing DNA damage: non-homologous end-joining (NHEJ) and homologous recombination (HR). NHEJ repair results in base deletions or insertions, enabling gene knockout. HR repair, when provided with a homologous template, allows for site-specific insertions and precise base substitutions.

[0004] The existing CRISPR / Cas12a gene editing system has the following main problems in actual use:

[0005] (1) Cas12a proteins are mainly AsCas12a and LbCas12a, and the PAM sequences they can recognize are mainly TTTA, TTTC, and TTTG. The choice of PAM sequences is relatively small, which limits the targeted gene editing range of the CRISPR / Cas12a gene editing system;

[0006] (2) Due to the differences in target site sequences and the different locations of related genes in the genome, the existing CRISPR / Cas12a gene editing system has DNA sequences that are difficult to edit, which limits the use of the CRISPR / Cas12a gene editing system at related sites or genes. Summary of the Invention

[0007] To address the deficiencies of the prior art, the present invention aims to provide a highly efficient gene editing system NovaCas-2 with multiple PAM selections and its applications.

[0008] In order to achieve the above objectives, the present invention adopts the following technical solutions:

[0009] A highly efficient gene editing system NovaCas-2 with multiple PAM selections, comprising a NovaCas-2 protein and sgRNA complex, which can precisely locate a targeted DNA sequence and produce cuts, causing double-strand breaks in the DNA. The amino acid sequence of the NovaCas-2 protein is shown in SEQ ID NO: 1, and the nucleotide sequence of the sgRNA is shown in SEQ ID NO: 7 or SEQ ID NO: 9.

[0010] Preferably, the precise positioning of the targeting DNA sequence includes a PAM sequence on the targeting DNA sequence recognized by the NovaCas-2 protein and sgRNA complex, and the PAM sequence includes ACTA, ATTA, ATTC, ATTG, CCTA, CCTC, CCTG, CTAC, CTCC, CTGC, CTTA, CTTC, CTTG, CTTT, GTTA, GTTC, GTTG, TATA, TCCA, TCCC, TCCG, TCGC, TCTA, TCTC, TCTG, TTAC, TTCA, TTCC, TTCG, TTC T, TTGA, TTGC, TTTA, TTTC, TTTG and TTTT; preferably, the PAM sequence includes ATTA, ATTG, CTTA, CTTC, CTTG, GTTA, GTTG, TCTA, TCTC, TCTG, TTAC, TTCA, TTCC, TTCG, TTCT, TTGC, TTTA, TTTC, TTTG and TTTT; more preferably, the PAM sequence includes ATTA, CTTA, CTTG, GTTA, TCTA, TTCA, TTCC, TTTA, TTTG, TTTC and TTTT.

[0011] Preferably, the NovaCas-2 protein is obtained by codon-optimizing the nucleotide sequence of the original NovaCas-2 protein and then transcribing and translating it, wherein the nucleotide sequence of the original NovaCas-2 protein is shown in SEQ ID NO: 2, and the nucleotide sequence of the optimized NovaCas-2 protein is shown in SEQ ID NO: 3.

[0012] Preferably, the NovaCas-2 protein includes NovaCas-2 protein variants having no cleavage activity, single-stranded cleavage activity, or double-stranded cleavage activity.

[0013] Preferably, the sgRNA includes sgRNA modified by phosphorylation, sulfation, methylation or hydroxylation.

[0014] Preferably, the NovaCas-2 protein and sgRNA complex can accurately locate the targeted DNA sequence, which means that the NovaCas-2 protein and sgRNA complex can accurately recognize and target the DNA sequence, or refers to bringing other proteins fused to the NovaCas-2 protein or proteins that specifically recognize sgRNA to the targeted DNA position, and the other proteins fused to the NovaCas-2 protein or proteins that specifically recognize sgRNA can modify or regulate the targeted DNA sequence, and the modification or regulation includes regulation of the gene transcription level and single base conversion, and the single base conversion includes conversion between adenine to guanine, cytosine to thymine or other bases.

[0015] The application of the aforementioned multi-PAM selected efficient gene editing system NovaCas-2 in gene editing, wherein the gene editing includes gene editing in cells and gene editing in vitro, wherein the cells include eukaryotic cells and prokaryotic cells, and the eukaryotic cells include mammalian cells and plant cells, and the application is for non-disease diagnosis and treatment purposes.

[0016] The aforementioned multi-PAM selected efficient gene editing system NovaCas-2 is used to quickly detect whether there is a targeted DNA sequence in double-stranded DNA. The single-stranded DNA fluorescent reporter probe is contacted with the NovaCas-2 protein and sgRNA complex, and the presence of a targeted DNA sequence in the double-stranded DNA is judged based on the change in the fluorescence signal. Specifically: when there is a targeted DNA sequence in the double-stranded DNA, the single-stranded DNA cleavage activity of the NovaCas-2 protein is activated, the single-stranded DNA fluorescent reporter probe is degraded to release the fluorescent group, and the fluorescence signal is enhanced; when there is no targeted DNA sequence in the double-stranded DNA, the single-stranded DNA cleavage activity of the NovaCas-2 protein is not activated, and the fluorescence signal remains unchanged.

[0017] The present invention is beneficial in that:

[0018] (1) The nuclease selected in the present invention is the NovaCas-2 protein. In addition to recognizing TTTA, TTTC, and TTTG, the NovaCas-2 protein can also recognize ATTA, CTTA, CTTG, GTTA, TCTA, TTCA, TTCC, and TTTT, etc., and can recognize up to 36 PAM sequences, which greatly broadens the scope of targeted gene editing of the existing CRISPR / Cas12a gene editing system;

[0019] (2) Experiments have confirmed that the gene editing system NovaCas-2 provided by the present invention has higher gene editing efficiency than the existing CRISPR / Cas12a gene editing system for the Escherichia coli targeting DNA sequence shown in SEQ ID NO: 11 and the HEK293 cell targeting DNA sequence shown in SEQ ID NO: 12. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a schematic diagram of the gene editing system NovaCas-2 provided by the present invention cutting targeted DNA;

[0021] Figure 2 This is a graph showing the PAM preference detection results of the gene editing system NovaCas-2 (NovaCas-2 protein + sgRNA1) provided by the present invention;

[0022] Figure 3This is a comparison chart of the gene editing efficiency of the gene editing system NovaCas-2 (NovaCas-2 protein + sgRNA1) provided by the present invention and the existing CRISPR / Cas12a gene editing system (AsCas12a protein + sgRNA2);

[0023] Figure 4 This is the electrophoresis result diagram of the experimental group (NovaCas-2 protein + sgRNA1) and the control group (AsCas12a protein + sgRNA2) outside the cell;

[0024] Figure 5 This is a comparison chart of the gene editing efficiency of the experimental group (NovaCas-2 protein + sgRNA1) and the control group (AsCas12a protein + sgRNA2) outside the cell;

[0025] Figure 6 This is the electrophoresis result of the experimental group (NovaCas-2 protein + sgRNA3) and the control group (AsCas12a protein + sgRNA4) in HEK293 cells;

[0026] Figure 7 This is a comparison of the gene editing efficiency of the experimental group (NovaCas-2 protein + sgRNA3) and the control group (AsCas12a protein + sgRNA4) in HEK293 cells;

[0027] Figure 8 This is the fluorescence intensity detection result of the experimental group (NovaCas-2 protein + sgRNA1) and the control group (AsCas12a protein + sgRNA2) outside the cell. DETAILED DESCRIPTION

[0028] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0029] 1. Composition of the NovaCas-2 gene editing system

[0030] like Figure 1 As shown, the gene editing system NovaCas-2 provided by the present invention is a complex of NovaCas-2 protein and sgRNA, which can accurately locate the targeted DNA sequence and produce cutting, causing double-strand break damage to the DNA.

[0031] The ability of the NovaCas-2 protein and sgRNA complex to precisely locate the targeted DNA sequence means that the NovaCas-2 protein and sgRNA complex can precisely identify and target the DNA sequence, or refers to bringing other proteins fused to the NovaCas-2 protein or proteins that specifically recognize the sgRNA to the targeted DNA position, wherein other proteins fused to the NovaCas-2 protein or proteins that specifically recognize the sgRNA can modify or regulate the targeted DNA sequence, and the modification or regulation includes regulation of the gene transcription level and single base conversion, and the single base conversion includes conversion from adenine to guanine, cytosine to thymine or other bases.

[0032] The NovaCas-2 protein and sgRNA are introduced below.

[0033] 1. NovaCas-2 protein

[0034] The NovaCas-2 protein is derived from bacteria of the Lachnospiraceae family, with the NCBI accession number MBQ9120454. The amino acid sequence is shown in SEQ ID NO: 1, and the nucleotide sequence is shown in SEQ ID NO: 2. Studies have shown that the protein belongs to Class 2 V-type RNA-guided nucleases. The present invention names the protein NovaCas-2 protein.

[0035] In order to enable efficient expression of the NovaCas-2 protein in Escherichia coli, the original nucleotide sequence (SEQ ID NO: 2) was codon-optimized. The nucleotide sequence after codon optimization is shown in SEQ ID NO: 3.

[0036] Of course, the NovaCas-2 protein can also include NovaCas-2 protein variants with no cleavage activity, single-stranded cleavage activity, or double-stranded cleavage activity.

[0037] 2. sgRNA

[0038] CRISPRCasdb was used to mine nucleotide sequences capable of transcribing crRNAs from the 5000 bp downstream nucleotide sequence of the NovaCas-2 protein-encoding gene in the metagenomics of Lachnospiraceae bacteria. The resulting crRNA nucleotide sequence is shown in SEQ ID NO: 5 (denoted as crRNA1). Based on this crRNA, two sgRNAs (denoted as sgRNA1 and sgRNA3) were designed, targeting a DNA sequence targeting Escherichia coli (SEQ ID NO: 11) and a DNA sequence targeting HEK293 cells (SEQ ID NO: 12), respectively. The nucleotide sequences of these two sgRNAs are shown in SEQ ID NO: 7 (sgRNA1) and SEQ ID NO: 9 (sgRNA3), respectively. Both sgRNAs recognize the PAM sequence (TTTA, TTTC, TTTG) of the target site, and the 20 bp or 21 bp nucleotide sequence at the 3' end can form base complementary pairing structures with the DNA sequence of the target site.

[0039] Of course, sgRNA also includes sgRNA modified by phosphorylation, sulfation, methylation or hydroxylation.

[0040] 2. Experimental Preparation

[0041] 1. Prepare PAM library plasmid

[0042] The method for obtaining the PAM library plasmid is as follows:

[0043] (1) Enzyme digestion of plasmid

[0044] Linearize the pUC19 plasmid using HindIII and KpnI restriction enzymes. Digestion system: 1 μg pUC19 plasmid, 5 μL buffer, 1 μL HindIII enzyme, 1 μL KpnI enzyme, and make up to 50 μL with sterile water. Digestion reaction conditions: 37°C for 1 hour.

[0045] (2) Gel electrophoresis

[0046] The products obtained by enzyme digestion were electrophoresed on 1% agarose gel at 120V for 30min.

[0047] (3) Recovering plasmids

[0048] The 2643 bp DNA fragment on the gel was excised, and the plasmid was recovered using a gel recovery kit, and finally eluted with ultrapure water to obtain the pUC19 plasmid linearized DNA fragment.

[0049] (4) Determination of DNA concentration

[0050] The recovered pUC19 plasmid linearized DNA fragment was measured for DNA concentration using NanoDrop and stored in a -20°C refrigerator.

[0051] (5) Design and synthesize primers

[0052] Design and synthesize long primers containing NNNN (two single-stranded oligonucleotide DNAs, both with sticky end sequences corresponding to the linearized DNA fragment of the pUC19 plasmid). The nucleotide sequence of the long primer is as follows:

[0053] Oligo-F1:AGCTCgtcgacTACCCTACTGATTAGATCCGTTCANNNNCGGTCATCGCGTGGTGCACATCCGTTAAGTCATAGCGAgacgtcAGTAC (SEQ ID NO: 19);

[0054] Oligo-R1:GTACTgacgtcTCGCTATGACTTAACGGATGTGCACCACGCGATGACCGNNNNTGAACGGATCTAATCAGTAGGGTAgtcgacGAGCT (SEQ ID NO: 20).

[0055] (6) Annealing

[0056] Two oligonucleotide single-stranded DNAs were annealed into double-stranded DNA. The annealing reaction system was as follows: 1 μL 100 μM Oligo-F1, 1 μL 100 μM Oligo-R1, and 28 μL sterile water. After thorough mixing, the mixture was placed in a PCR instrument and the annealing program was run (cooling rate 0.3°C / s): 95°C for 5 min, 85°C for 1 min, 75°C for 1 min, 65°C for 1 min, 55°C for 1 min, 45°C for 1 min, 35°C for 1 min, and 25°C for 1 min. The mixture was then stored at 4°C.

[0057] (7) Connection

[0058] Ligate the annealed product to the linearized pUC19 plasmid DNA fragment using DNA ligase. Ligation system: 2 μL buffer, 50 ng of the linearized pUC19 plasmid DNA fragment, 20 ng of the annealed product, 1 μL of T4 DNA ligase, and make up to 20 μL with sterile water. Ligation reaction conditions: 16°C for 2 hours.

[0059] (8) Verify library abundance

[0060] Take 5 μL of ligation product for chemical competence transformation, add it to 900 μL of SOB liquid medium, and resuscitate at 37°C for 1 hour. Then take 10 μL of bacterial liquid and spread it on SOB solid medium to verify the library abundance (library abundance = number of colonies × 100, the library abundance is required to be no less than 10,000) and perform Sanger sequencing verification on the grown bacteria.

[0061] (9) Obtaining PAM library plasmid

[0062] The remaining bacterial liquid was inoculated into 20 mL of LB liquid medium and cultured at 37°C for 12 h. The plasmid was then extracted to obtain the PAM library plasmid for later use.

[0063] 2. Prepare NovaCas-2 protein and AsCas12a protein

[0064] The nucleotide sequence shown in SEQ ID NO: 3 was synthesized and constructed on the pET28a plasmid using conventional methods. Then, conventional methods were used to synthesize the protein expression plasmid, express the protein, and purify the protein to finally obtain the NovaCas-2 protein.

[0065] AsCas12a protein was purchased directly from the market as a control.

[0066] 3. Prepare sgRNA

[0067] Targeting the NovaCas-2 protein, the sgRNA (sgRNA1) shown in SEQ ID NO: 7 and the sgRNA (sgRNA3) shown in SEQ ID NO: 9 were synthesized using conventional methods.

[0068] The nucleotide sequence of the reported crRNA for the AsCas12a protein is shown in SEQ ID NO: 6 (denoted as crRNA2). Based on the crRNA, two sgRNAs (denoted as sgRNA2 and sgRNA4, respectively) were designed for the Escherichia coli targeting DNA sequence (SEQ ID NO: 11) and the HEK293 cell targeting DNA sequence (SEQ ID NO: 12), respectively. The nucleotide sequences of the two sgRNAs are shown in SEQ ID NO: 8 (sgRNA2) and SEQ ID NO10 (sgRNA4), respectively. The sgRNA (sgRNA2) shown in SEQ ID NO: 8 and the sgRNA (sgRNA4) shown in SEQ ID NO: 10 were synthesized by conventional methods.

[0069] 3. Detecting the PAM preference of the NovaCas-2 gene editing system

[0070] The principle of PAM preference detection is to detect the abundance of PAM libraries without gene editing (negative control group) and the abundance of PAM libraries with gene editing (experimental group), respectively. The changes in PAM library abundance before and after gene editing are analyzed by bioinformatics methods to finally obtain the PAM preference of the gene editing system NovaCas-2.

[0071] The specific method for detecting PAM preference is as follows:

[0072] 1. Enzyme digestion of plasmid

[0073] Digest the previously obtained PAM library plasmid with HaeII restriction enzyme to obtain a linearized plasmid. Digestion system: 1 μg PAM library plasmid, 5 μL buffer, 1 μL HaeII enzyme, and make up to 50 μL with sterile water. Digestion reaction conditions: 37°C for 1 hour.

[0074] 2. Gel electrophoresis

[0075] The products obtained by enzyme digestion were electrophoresed on 1% agarose gel at 120V for 30min.

[0076] 3. Recover plasmid

[0077] The 2676 bp DNA fragment on the gel was excised and the plasmid was recovered using a gel recovery kit and finally eluted with ultrapure water to obtain the linearized DNA fragment of the PAM library plasmid.

[0078] 4. Determine DNA concentration

[0079] The recovered PAM library plasmid linearized DNA fragment was measured for DNA concentration using NanoDrop, and the DNA was diluted to a concentration of 30.8 ng / μL for later use (stored in a -20°C refrigerator).

[0080] 5. Co-incubation of NovaCas-2 protein and sgRNA1

[0081] 20 μL DEPC water, 3 μL buffer solution, 1 μL 1 μM NovaCas-2 protein and 3 μL 300 nM sgRNA1 were shaken and mixed, and placed in a PCR instrument and incubated at 25°C for 10 min.

[0082] 6. Gene Editing

[0083] Experimental group: Add 3 μL of PAM library plasmid linearized DNA fragment to the incubation product, shake and mix, place in a PCR instrument and run the gene editing program: 37°C for 30 min.

[0084] Negative control group: Add 3 μL of PAM library plasmid linearized DNA fragment to 27 μL of DEPC water, shake and mix, place in a PCR instrument and run the gene editing program: 37°C for 30 min.

[0085] 7. Protease inactivation

[0086] 1 μL of proteinase K was added to the experimental group and negative control group respectively, mixed thoroughly, incubated at room temperature for 10 min, and then placed in a PCR instrument to run the protease inactivation program: 70°C for 30 min to obtain the cleavage product.

[0087] 8. PCR amplification

[0088] The cleavage products of the experimental group and the negative control group were amplified by PCR using primers containing different characteristic sequences (barcodes), where:

[0089] For the cleavage products of the experimental group, the nucleotide sequences of the PCR primers are shown in SEQ ID NO: 13 and SEQ ID NO: 14;

[0090] For the cleavage products of the negative control group, the nucleotide sequences of the PCR primers are shown in SEQ ID NO: 15 and SEQ ID NO: 16.

[0091] The PCR reaction system was as follows: 25 μL Q5 High-Fidelity 2× Master Mix, 1.25 μL 10 μM forward primer, 1.25 μL 10 μM reverse primer, 2 μL cleavage product, and sterile water was added to 50 μL.

[0092] After vortexing and mixing, place the plate in a PCR instrument and run the amplification program: 98°C for 30 seconds, followed by 30 cycles of 98°C for 10 seconds, 65°C for 30 seconds, and 72°C for 10 seconds. After the above cycles, incubate at 72°C for 2 minutes and store at 4°C.

[0093] 9. Gel electrophoresis

[0094] The PCR amplification products were placed on a 2% agarose gel for electrophoresis at 120V for 30min.

[0095] 10. Recover sample DNA

[0096] The 201 bp DNA fragment on the gel was excised, and the sample DNA was recovered using a gel recovery kit, and finally eluted with ultrapure water to obtain different sample DNAs.

[0097] 11. Sequencing

[0098] 100 ng of DNA from different samples were mixed and then subjected to Illumina second-generation sequencing. The second-generation sequencing results were analyzed by bioinformatics, and the following results were obtained: Figure 2 The results of PAM preference detection of the gene editing system NovaCas-2 (NovaCas-2 protein and sgRNA1 complex) are shown.

[0099] Depend on Figure 2 It can be seen that the gene editing system NovaCas-2 (NovaCas-2 protein and sgRNA1 complex) provided by the present invention can recognize TTTA, TTTC and TTTG that can be recognized by the existing CRISPR / Cas12a gene editing system, as well as ACTA, ATTA, ATTC, ATTG, CCTA, CCTC, CCTG, CTAC, CTCC, CTGC, CTTA, CTTC, CTTG, CTTT, GTTA, GTTC, GTTG, TATA, TCCA, TCCC, TCCG, TCGC, TCTA, TCTC, TCTG, TTAC, TTCA, TTCC, TTCG, TTCT, TTGA, TTG C and TTTT, and can recognize as many as 36 PAM sequences. Among them, it has low recognition activity for 16 PAM sequences including ATTC, CCTA, TCCC, GTTC, CCTC, TCCA, CTTT, ACTA, CCTG, CTCC, TATA, TCGC, TCCG, TTGA, CTAC and CTGC, has high recognition activity for 9 PAM sequences including ATTG, TTGC, GTTG, TCTG, TTAC, TTCG, TTCT, TCTC and CTTC, and has very high recognition activity for 11 PAM sequences including TTTG, TTTA, TTTT, TCTA, CTTA, CTTG, ATTA, TTCC, TTCA, GTTA and TTTC.

[0100] Since sgRNA3 also contains the sequence of crRNA1, it can be determined that the NovaCas-2 protein and sgRNA3 complex can also recognize the above 47 PAM sequences.

[0101] In summary, the gene editing system NovaCas-2 (NovaCas-2 protein and sgRNA1 or sgRNA3 complex) provided by the present invention greatly broadens the targeted gene editing range of the existing CRISPR / Cas12a gene editing system.

[0102] 4. Detecting the gene editing efficiency of the NovaCas-2 gene editing system

[0103] The gene editing system NovaCas-2 provided by the present invention can perform gene editing in vitro and in cells, wherein the cells include eukaryotic cells and prokaryotic cells, eukaryotic cells include mammalian cells and plant cells, and prokaryotic cells include Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Pseudomonas aeruginosa, Pseudomonas putida, actinomycetes and cyanobacteria.

[0104] The following uses Escherichia coli to test the gene editing efficiency of the gene editing system NovaCas-2 provided by the present invention. Specifically, Escherichia coli is used. galK The red and white spot screening system detects the gene editing efficiency of the gene editing system NovaCas-2 provided by the present invention. The detection principle is:

[0105] When Escherichia coli galK Once the functional sequence on the gene is recognized, the NovaCas-2 protein will galK The gene is cut to create a double-strand break, and then homologous recombination occurs. galk Gene inactivation results in Escherichia coli being unable to degrade galactose and produce acid, and white colonies grow on MacConkey medium containing galactose. However, Escherichia coli that has not undergone gene editing still has the ability to degrade galactose and produce acid, and red colonies grow on MacConkey medium containing galactose.

[0106] AsCas12a protein and sgRNA2 complex were used as controls. The gene editing efficiency was tested as follows:

[0107] 1. Construction of Cas protein plasmid

[0108] The nucleotide sequence shown in SEQ ID NO: 3 (the codon-optimized NovaCas-2 protein encoding gene) was synthesized and constructed into the pSC101 plasmid to obtain the pNovaCas-2 protein plasmid (concentration 100 ng / μL).

[0109] In order to enable efficient expression of the AsCas12a protein in Escherichia coli, the original nucleotide sequence of the AsCas12a protein encoding gene was codon optimized, and the nucleotide sequence after codon optimization is shown in SEQ ID NO: 4. The nucleotide sequence shown in SEQ ID NO: 4 was synthesized and constructed into the pSC101 plasmid to obtain the pAsCas12a protein plasmid (concentration 100 ng / μL).

[0110] 2. Construction of sgRNA plasmid

[0111] To construct a plasmid containing sgRNAs (sgRNA1, sgRNA2) and homology repair templates, different primers were designed for sgRNA1 and sgRNA2, respectively.

[0112] For sgRNA1, the nucleotide sequences of the designed primers (Oligo-F2, Oligo-R2) are shown in SEQ ID NO: 21 and SEQ ID NO: 22;

[0113] The nucleotide sequences of the designed primers (Oligo-F3, Oligo-R3) for sgRNA2 are shown in SEQ ID NO: 23 and SEQ ID NO: 24.

[0114] Primers Oligo-F2 and Oligo-R2 were synthesized according to the nucleotide sequences shown in SEQ ID NO: 21 and SEQ ID NO: 22, and primers Oligo-F3 and Oligo-R3 were synthesized according to the nucleotide sequences shown in SEQ ID NO: 23 and SEQ ID NO: 24.

[0115] Two pairs of primers (Oligo-F2 and Oligo-R2, Oligo-F3 and Oligo-R3) were annealed separately to obtain two double-stranded DNAs with sticky ends at both ends. The annealing reaction system was as follows: 1 μL 100 μM Oligo-F2 (or Oligo-F3), 1 μL 100 μM Oligo-R2 (or Oligo-R3), 28 μL sterile water. After thorough mixing, the tubes were placed in a PCR instrument and the annealing program was run (cooling rate 0.3°C / s): 95°C for 5 min, 85°C for 1 min, 75°C for 1 min, 65°C for 1 min, 55°C for 1 min, 45°C for 1 min, 35°C for 1 min, 25°C for 1 min, and stored at 4°C.

[0116] Ligate the annealed products to the previously obtained linearized pUC19 plasmid DNA fragment using DNA ligase. Ligation system: 2 μL buffer, 50 ng of the linearized pUC19 plasmid DNA fragment, 20 ng of the annealed product, 1 μL of T4 DNA ligase, and make up to 20 μL with sterile water. Ligation reaction conditions: 16°C for 2 hours.

[0117] Take 1 μL of the ligation product for chemical competent transformation, and verify the grown bacteria by Sanger sequencing.

[0118] Plasmids were extracted from bacteria verified to have been correctly connected by sequencing to obtain psgRNA1 plasmid (concentration 100 ng / μL) and psgRNA2 plasmid (concentration 100 ng / μL).

[0119] 3. E. coli transfection with Cas protein plasmid

[0120] The pNovaCas-2 protein plasmid and pAsCas12a protein plasmid obtained above were transformed into MG1655 competent cells (high-efficiency competent cells made from Escherichia coli MG1655) respectively. The specific operations are as follows:

[0121] (1) Take 50 μL of MG1655 competent cells and place them in a centrifuge tube and place them in an ice bath. Add 1 μL of pNovaCas-2 protein particles or 1 μL of pAsCas12a protein particles to the cell suspension, flick to mix, and let it stand in an ice bath for 30 minutes.

[0122] (2) Heat shock the centrifuge tube in a 42°C water bath for 90 seconds, then quickly transfer it to an ice bath and cool it for 3 minutes.

[0123] (3) Add 950 μL of sterile LB liquid culture medium without antibiotics to the centrifuge tube, mix well, and place in a shaker at 37°C and 150 rpm for 45 minutes to allow E. coli to recover.

[0124] (4) Mix the bacterial solution in the centrifuge tube and pipette 100 μL of the bacterial solution onto LB solid agar medium containing 34 μg / mL chloramphenicol. Use a sterile spreading stick to spread the solution evenly. After the bacterial solution is absorbed by the medium, invert the plate and culture it at 30°C for 16 h.

[0125] (5) Use a sterilized white pipette tip to pick a single colony from the plate and place it in 10 mL of LB liquid culture medium. Place it in a shaker at 30°C and 200 rpm for 16 hours to amplify the E. coli and obtain E. coli transfected with the Cas protein vector. Among them, the E. coli transfected with the pNovaCas-2 protein vector is recorded as E. coli Nova2, and the E. coli transfected with the pAsCas12a protein vector is recorded as E. coli As.

[0126] 4. Preparation of E. coli competent cells transfected with Cas protein plasmids

[0127] The method for preparing Escherichia coli competent cells transfected with Cas protein plasmids is as follows:

[0128] (1) E. coli Nova2 (bacterial solution) and E. coli As (bacterial solution) were inoculated into 50 mL LB liquid medium at a 1% inoculum volume, and cultured in a shaking incubator at 30°C and 200 rpm until the OD 600 =0.55, and then immediately placed in an ice bath for 15 min.

[0129] (2) Centrifuge the bacterial solution at 6000 rpm for 4 min at 4°C and collect the precipitate into a 50 mL centrifuge tube.

[0130] (3) Add 25 mL of pre-cooled 10% glycerol to the centrifuge tube and place it in an ice bath to resuspend the precipitate.

[0131] (4) Centrifuge the bacterial solution at 6000 rpm for 3 min at 4°C and collect the precipitate into a 50 mL centrifuge tube.

[0132] (5) Add 25 mL of pre-cooled 10% glycerol to the centrifuge tube and place it in an ice bath to resuspend the precipitate.

[0133] (6) Centrifuge the bacterial solution again at 6000 rpm for 3 min at 4°C and collect the precipitate into a 50 mL centrifuge tube.

[0134] (7) Add 200 μL of pre-cooled 10% glycerol to the centrifuge tube, and then adjust the volume to 500 μL with 10% glycerol to obtain Escherichia coli Nova2 competent cells (bacterial solution) and Escherichia coli As competent cells (bacterial solution), respectively.

[0135] 5. E. coli transfection with sgRNA plasmid

[0136] The specific method for transfecting E. coli with sgRNA plasmid is as follows:

[0137] (1) Pre-cool the 1 mm electroporation cuvette on ice for 30 min, then add 0.1 μg of psgRNA1 plasmid and psgRNA2 plasmid respectively.

[0138] (2) Add 50 μL of E. coli Nova2 competent cells to the electroporation cup containing the psgRNA1 plasmid, and add 50 μL of E. coli As competent cells to the electroporation cup containing the psgRNA2 plasmid, flick to mix, and then electroporate at 1800 V. Immediately after electroporation, add 950 μL of sterile LB liquid culture medium without antibiotics, pipet to mix, and transfer to a centrifuge tube. Place it in a shaker at 30°C and shake at 150 rpm for 2 hours to allow the E. coli to recover.

[0139] (3) Mix the bacterial solution in the centrifuge tube and pipette 50 μL of the bacterial solution onto MacConkey solid agar medium containing 34 μg / mL chloramphenicol, 100 μg / mL ampicillin and 0.2% galactose. Use a sterile spreading stick to spread the solution evenly. After the bacterial solution is absorbed by the culture medium, invert the plate and culture it at 30°C for 16 h.

[0140] 6. Calculating gene editing efficiency

[0141] When observed with the naked eye, if the colony on the MacConkey solid agar medium is red, it indicates that the Escherichia coli in the colony has not undergone gene editing (negative); if the colony is white, it indicates that the Escherichia coli in the colony has undergone gene editing (positive).

[0142] Count the number of red and white colonies on MacConkey solid agar medium and calculate the gene editing efficiency according to the following formula:

[0143] Gene editing efficiency = number of white colonies / (number of red colonies + number of white colonies) × 100%

[0144] Calculations show that in E. coli, the gene editing efficiency of the gene editing system NovaCas-2 (NovaCas-2 protein + sgRNA1) provided by the present invention is 74.8%, while the gene editing efficiency of the existing CRISPR / Cas12a gene editing system (AsCas12a protein + sgRNA2) is 48.1%. See the comparison chart of the gene editing efficiency of the two gene editing systems for details. Figure 3 .

[0145] In summary, in Escherichia coli, compared with the existing CRISPR / Cas12a gene editing system (AsCas12a protein + sgRNA2), the gene editing system NovaCas-2 (NovaCas-2 protein + sgRNA1) provided by the present invention exhibits higher gene editing efficiency.

[0146] 5. Verifying the gene editing activity of the NovaCas-2 gene editing system outside cells

[0147] Before verifying gene editing activity, prepare a linear double-stranded DNA substrate. The method for preparing a linear double-stranded DNA substrate is as follows:

[0148] (1) Obtaining the E. coli genome: E. coli MG1655 was inoculated with 1% of the inoculum into 3 mL of LB liquid culture medium, and the culture was shaken at 200 rpm at 37°C for 12 h to amplify the E. coli. The genome was then extracted using a bacterial genome extraction kit and eluted with ultrapure water to obtain the E. coli MG1655 genome. The E. coli genome was finally diluted to 500 ng / μL with ultrapure water and stored in a -20°C refrigerator.

[0149] (2) PCR to obtain in vitro cleavage double-stranded DNA substrate: PCR amplification of the E. coli MG1655 genome was performed using the forward primer and reverse primer shown in SEQ ID NO: 25 and SEQ ID NO: 26. The PCR reaction system is as follows: 25 μL Q5 High-Fidelity 2× Master Mix, 1.25 μL 10 μM forward primer, 1.25 μL 10 μM reverse primer, 1 μL E. coli MG1655 genome, and sterile water to 50 μL. After shaking and mixing, place in a PCR instrument and run the amplification program: 98°C for 30 seconds, the following reaction is carried out for 30 cycles: 98°C for 10 seconds, 65°C for 30 seconds, and 72°C for 30 seconds. After the above cycles are completed, incubate at 72°C for 2 minutes and store at 4°C.

[0150] (3) Gel electrophoresis: Place the PCR product on a 1% agarose gel for electrophoresis at 120V for 30 minutes.

[0151] (4) Recover the linear double-stranded DNA substrate: excise the 3124 bp DNA fragment on the gel, use a gel recovery kit to recover the sample DNA, and finally elute with ultrapure water to obtain a linear double-stranded DNA substrate (template), dilute to a concentration of 225 ng / μL, and set aside (store in a -20°C refrigerator).

[0152] After the linear double-stranded DNA substrate is prepared, the gene editing activity of the gene editing system NovaCas-2 is verified outside the cell. The method for verifying the gene editing activity of the gene editing system NovaCas-2 outside the cell is as follows:

[0153] (1) Experimental group: 20 μL DEPC water, 3 μL buffer solution, 1 μL 1 μM NovaCas-2 protein and 3 μL 300 nM sgRNA1 were shaken and mixed, and then placed in a PCR instrument and incubated at 25°C for 10 min.

[0154] (2) Control group: 20 μL DEPC water, 3 μL buffer solution, 1 μL 1 μM AsCas12a protein and 3 μL 300 nM sgRNA2 were shaken and mixed, and then placed in a PCR instrument and incubated at 25°C for 10 min.

[0155] (3) Add 3 μL of linear double-stranded DNA substrate to the incubation product, add 3 μL of linear double-stranded DNA substrate to 27 μL of DEPC water (as a negative control group), shake to mix, and then place in a PCR instrument and run the editing program: 37°C for 30 min.

[0156] (4) Add 1 μL of proteinase K to the system, shake and mix, incubate at room temperature for 10 min, and then place in a PCR instrument to run the protease inactivation program: 70°C for 30 min.

[0157] (5) Continue to add 6 μL of 6× loading buffer solution to the system, pipette to mix well, and then perform electrophoresis on 1% agarose gel at 120V for 30 minutes.

[0158] (6) Use a gel imager to observe the electrophoresis results and preliminarily determine the gene editing activity of the gene editing system. Specifically:

[0159] If there is only one band with a size of 3124 bp, it indicates that the linear double-stranded DNA substrate has not been cut and the corresponding gene editing system is inactive;

[0160] If there are two bands with sizes of 2531 bp and 593 bp respectively, it indicates that the linear double-stranded DNA substrate has been cut and the cutting is very thorough, and the corresponding gene editing system has high gene editing activity;

[0161] If there are three bands with sizes of 3124bp, 2531bp and 593bp respectively, it indicates that the linear double-stranded DNA substrate is cut but not thoroughly, and the corresponding gene editing system has gene editing activity but it is not high.

[0162] The electrophoresis results of the experimental group (NovaCas-2 protein + sgRNA1), the control group (AsCas12a protein + sgRNA2) and the negative control group are shown in Figure 4 .Depend on Figure 4 A preliminary conclusion can be drawn: the gene editing system in the experimental group has high gene editing activity, and the gene editing system in the control group also has gene editing activity but the gene editing activity is not high.

[0163] (7) The grayscale of the template band on the gel can reflect the shearing amount of the linear double-stranded DNA substrate, and then the gene editing efficiency of the gene editing system can be calculated. Specifically, the gene editing efficiency of the experimental group and the control group can be calculated according to the following formula:

[0164] Gene editing efficiency = (grayscale value of the template band in the negative control group - grayscale value of the template band in the experimental group or control group) / grayscale value of the template band in the negative control group × 100%

[0165] After calculation, the gene editing efficiency of the gene editing system (NovaCas-2 protein + sgRNA1) in the experimental group was 69.1%, and the gene editing efficiency of the gene editing system (AsCas12a protein + sgRNA2) in the control group was 47.2%. The gene editing efficiency comparison chart of the two gene editing systems is shown in Figure 5 .

[0166] In summary, outside the cell, compared with the existing CRISPR / Cas12a gene editing system (AsCas12a protein + sgRNA2), the gene editing system NovaCas-2 (NovaCas-2 protein + sgRNA1) provided by the present invention exhibits higher gene editing efficiency.

[0167] 6. Verifying the gene editing activity of the NovaCas-2 gene editing system in cells

[0168] HEK293 cells were used to verify the gene editing activity of the NovaCas-2 gene editing system in cells. Before verifying the gene editing activity, HEK293 cells were transfected with Cas protein and sgRNA, and the AsCas12a protein and sgRNA4 complex was used as a control. The specific method for HEK293 cell transfection with Cas protein and sgRNA is as follows:

[0169] (1) HEK293 cells were plated on 6-well plates and cultured until the cell density reached 30%.

[0170] (2) Experimental group: 20 μg of the previously prepared NovaCas-2 protein and 2 μg of the previously prepared sgRNA3 were added to 450 μL of Nucleofector culture medium, gently pipetted to mix, and incubated the transfection solution at room temperature for 10 min.

[0171] (3) Control group: Take 20 μg of the previously prepared AsCas12a protein and 2 μg of the previously prepared sgRNA4 and add them to 450 μL Nucleofector culture medium, gently pipette to mix, and incubate the transfection solution at room temperature for 10 minutes.

[0172] (4) Negative control group: Take 500 μL of Nucleofector culture medium and incubate the transfection solution at room temperature for 10 minutes.

[0173] (5) Pipette the HEK293 cell line into the electroporation plate and add the mixed transfection solution (transfection solution of the experimental group, transfection solution of the control group, and transfection solution of the negative control group) respectively.

[0174] (6) Place the electroporation plate in a cell electroporator for electroporation. After electroporation, aspirate all the cells and add them to 500 μL of 37°C preheated Nucleofector culture medium. Continue to culture in a 37°C, 5% CO2 incubator for 24 hours.

[0175] After HEK293 cells were transfected with Cas proteins (NovaCas-2 protein, AsCas12a protein) and sgRNAs (sgRNA3, sgRNA4), the endogenous target gene editing activity was verified. The specific method for verifying the endogenous target gene editing activity is as follows:

[0176] (1) HEK293 cells (experimental group, control group, and negative control group) were collected 3 days after gene editing, and genomic DNA (100 ng / μL) was extracted using a DNA kit.

[0177] (2) PCR amplification was performed using the forward primer and reverse primer shown in SEQ ID NO: 17 and SEQ ID NO: 18. The PCR reaction system was as follows: 25 μL Q5 High-Fidelity 2× Master Mix, 1.25 μL 10 μM forward primer, 1.25 μL 10 μM reverse primer, 2 μL genomic DNA, and sterile water was added to make up to 50 μL. After vortexing and mixing, the mixture was placed in a PCR instrument and the amplification program was run: 98°C for 30 seconds, and the following reaction was performed for 30 cycles: 98°C for 10 seconds, 65°C for 30 seconds, and 72°C for 10 seconds. After the above cycles were completed, the reaction was incubated at 72°C for 2 minutes and stored at 4°C.

[0178] (3) Purify a 776 bp DNA fragment from the PCR product using a PCR product purification kit. Determine the DNA concentration using NanoDrop and dilute to 200 ng / μL. Denature and anneal the purified product. The reaction system is as follows: 1 μL purified product, 2 μL buffer solution, and make up to 20 μL with sterile water. After vortexing and mixing, place the fragment in a PCR instrument and run the program (cooling rate 0.3°C / s): 95°C for 5 min, 85°C for 1 min, 75°C for 1 min, 65°C for 1 min, 55°C for 1 min, 45°C for 1 min, 35°C for 1 min, 25°C for 1 min, and store at 4°C (template).

[0179] (4) Digest the previously prepared template with T7 EI enzyme, and electrophorese the digested product on a 2% agarose gel at 120 V for 30 min.

[0180] (5) Use a gel imager to observe the electrophoresis results and preliminarily determine the gene editing activity of the gene editing system. Specifically:

[0181] If there is only one band with a size of 776 bp, it indicates that the template has not been cut and the corresponding gene editing system is inactive;

[0182] If there are two bands with sizes of 490 bp and 286 bp respectively, it indicates that the template has been cut and the cutting is very thorough, and the corresponding gene editing system has high gene editing activity;

[0183] If there are three bands with sizes of 776bp, 490bp and 286bp respectively, it means that the template has been cut but not thoroughly, and the corresponding gene editing system has gene editing activity but it is not high.

[0184] The electrophoresis results of the experimental group (NovaCas-2 protein + sgRNA3), the control group (AsCas12a protein + sgRNA4) and the negative control group are shown in Figure 6 .Depend on Figure 6 It can be preliminarily determined that the gene editing systems of both the experimental and control groups have gene editing activity, and the gene editing activity of the experimental group is slightly higher than that of the control group.

[0185] (6) The grayscale of the template bands on the gel can reflect the amount of template shearing. The gene editing efficiency is calculated based on the grayscale value of each group of template bands using the same method as above.

[0186] After calculation, the gene editing efficiency of the gene editing system (NovaCas-2 protein + sgRNA3) in the experimental group was 11.5%, and the gene editing efficiency of the gene editing system (AsCas12a protein + sgRNA4) in the control group was 6.12%. The gene editing efficiency comparison chart of the two gene editing systems is shown in Figure 7 .

[0187] In summary, in HEK293 cells, compared with the existing CRISPR / Cas12a gene editing system (AsCas12a protein + sgRNA4), the gene editing system NovaCas-2 (NovaCas-2 protein + sgRNA3) provided by the present invention exhibited higher gene editing efficiency.

[0188] 7. Other Applications of the NovaCas-2 Gene Editing System

[0189] In addition to being used for gene editing, the gene editing system NovaCas-2 provided by the present invention can also be used to detect whether there is a targeted DNA sequence in double-stranded DNA.

[0190] When used to detect whether there is a targeted DNA sequence in double-stranded DNA, a single-stranded DNA fluorescent reporter probe is required. The detection method is as follows:

[0191] The single-stranded DNA fluorescent reporter probe is exposed to the NovaCas-2 protein and sgRNA complex. When there is a targeted DNA sequence in the double-stranded DNA, the NovaCas-2 protein and gRNA complex accurately locates the targeted DNA sequence and produces cutting to cause double-strand break damage to the DNA. After completing the double-stranded DNA gene editing, the conformation of the NovaCas-2 protein changes, activating the single-stranded DNA cutting activity ("trans-cutting" activity). After the single-stranded DNA cutting activity is activated, the NovaCas-2 protein will cut (degrade) the single-stranded DNA fluorescent reporter probe, thereby releasing the fluorescent group. A strong fluorescence value can be detected using a microplate reader. When there is no targeted DNA sequence in the double-stranded DNA, the conformation of the NovaCas-2 protein will not change, the single-stranded DNA cutting activity will not be activated, the single-stranded DNA fluorescent reporter probe will not be cut (degraded), and the fluorescence signal detected by the microplate reader will remain unchanged (the fluorescence value is still the base value).

[0192] The method for verifying that the gene editing system NovaCas-2 provided by the present invention can detect whether there is a targeted DNA sequence in double-stranded DNA is as follows:

[0193] (1) Experimental group: 19 μL DEPC water, 3 μL buffer solution, 1 μL 1 μM NovaCas-2 protein, 3 μL 300 nM sgRNA1, and 1 μL single-stranded DNA fluorescent reporter probe (Guangzhou Meige Biotechnology Co., Ltd., product number C009S, concentration 2 μM) were shaken and mixed, and then transferred to a 96-well black ELISA plate;

[0194] (2) Control group: 19 μL DEPC water, 3 μL buffer solution, 1 μL 1 μM AsCas12a protein, 3 μL 300 nM sgRNA2 and 1 μL single-stranded DNA fluorescent reporter probe were shaken and mixed, and transferred to a 96-well black ELISA plate;

[0195] (3) Add 3 μL of the linear double-stranded DNA substrate prepared above to the above system, shake and mix, and then place it in a microplate reader for fluorescence detection. The reaction procedure is: 37°C, 25 min, and collect fluorescence every 0.5 min.

[0196] (4) Determine whether there is a targeted DNA sequence in the double-stranded DNA based on the change in fluorescence signal. Specifically:

[0197] If the fluorescence intensity does not change over time (remains at the base value) or does not change significantly, it indicates that the single-stranded DNA fluorescent reporter probe has not been cut, indicating that there is no targeted DNA sequence in the double-stranded DNA;

[0198] If the fluorescence intensity increases over time, it indicates that the single-stranded DNA fluorescent reporter probe is cleaved, indicating that there is a targeted DNA sequence in the double-stranded DNA.

[0199] The curves of fluorescence intensity changes with cycle number (time) in the experimental group (NovaCas-2 protein + sgRNA1) and the control group (AsCas12a protein + sgRNA2) are shown in Figure 8 .Depend on Figure 8 It can be determined that there is a targeted DNA sequence in the double-stranded DNA. The gene editing system of the experimental group can make a judgment that there is a targeted DNA sequence in the double-stranded DNA within 16 cycles (8 minutes), while the gene editing system of the control group can make a judgment that there is a targeted DNA sequence in the double-stranded DNA after 20 cycles (10 minutes). Compared with the gene editing system of the control group, the gene editing system of the experimental group has a shorter detection time and a faster detection speed.

[0200] It should be noted that the above embodiments are merely examples for the purpose of clearly illustrating the present invention and are not intended to limit the embodiments of the present invention. A person skilled in the art would be able to make other variations or modifications based on the above description. It is not possible to enumerate all embodiments here. Any obvious variations or modifications arising from the technical solution of the present invention remain within the scope of protection of the present invention.

Claims

1. A gene editing system NovaCas-2 with multiple PAM selection, characterized by: The gene editing system NovaCas-2 is a complex of NovaCas-2 protein and sgRNA, which can accurately locate the targeted DNA sequence and produce cutting, causing double-strand break damage to the DNA, wherein the amino acid sequence of the NovaCas-2 protein is shown in SEQ ID NO: 1, and the nucleotide sequence of the sgRNA is shown in SEQ ID NO: 7 or SEQ ID NO:

9.

2. The multi-PAM selection gene editing system NovaCas-2 according to claim 1, characterized in that The precise positioning of the targeting DNA sequence includes that the NovaCas-2 protein and sgRNA complex recognizes the PAM sequence on the targeting DNA sequence, and the PAM sequence includes ACTA, ATTA, ATTC, ATTG, CCTA, CCTC, CCTG, CTAC, CTCC, CTGC, CTTA, CTTC, CTTG, CTTT, GTTA, GTTC, GTTG, TATA, TCCA, TCCC, TCCG, TCGC, TCTA, TCTC, TCTG, TTAC, TTCA, TTCC, TTCG, TTCT, TTGA, TTGC, TTTA, TTTC, TTTG or TTTT.

3. The multi-PAM selection gene editing system NovaCas-2 according to claim 2, characterized in that The PAM sequence includes ATTA, ATTG, CTTA, CTTC, CTTG, GTTA, GTTG, TCTA, TCTC, TCTG, TTAC, TTCA, TTCC, TTCG, TTCT, TTGC, TTTA, TTTC, TTTG or TTTT.

4. The multi-PAM selection gene editing system NovaCas-2 according to claim 3, characterized in that The PAM sequence includes ATTA, CTTA, CTTG, GTTA, TCTA, TTCA, TTCC, TTTA, TTTG, TTTC or TTTT.

5. The multi-PAM selection gene editing system NovaCas-2 according to claim 1, characterized in that The NovaCas-2 protein is obtained by codon-optimizing the nucleotide sequence of the original NovaCas-2 protein and then transcribing and translating it, wherein the nucleotide sequence of the original NovaCas-2 protein is shown in SEQ ID NO: 2, and the nucleotide sequence of the optimized NovaCas-2 protein is shown in SEQ ID NO:

3.

6. The multi-PAM selection gene editing system NovaCas-2 according to claim 1, characterized in that The NovaCas-2 protein and sgRNA complex can accurately locate the targeted DNA sequence, which means that the NovaCas-2 protein and sgRNA complex can accurately identify and target the DNA sequence.

7. The multi-PAM selection gene editing system NovaCas-2 according to claim 6, characterized in that The other proteins fused to the NovaCas-2 protein or the protein that specifically recognizes the sgRNA can modify or regulate the targeted DNA sequence, and the modification or regulation includes regulation of gene transcription level and single base conversion.

8. The multi-PAM selection gene editing system NovaCas-2 according to claim 7, characterized in that The single base conversion includes conversion from adenine to guanine, from cytosine to thymine or between other bases.

9. The use of the multi-PAM selection gene editing system NovaCas-2 according to claim 1 in gene editing, characterized in that: The gene editing includes gene editing in cells and gene editing in vitro, the cells include eukaryotic cells and prokaryotic cells, the eukaryotic cells include mammalian cells and plant cells, and the application is not for the purpose of disease diagnosis and treatment.

10. Use of the multi-PAM selection gene editing system NovaCas-2 according to claim 1 in rapid detection of the presence of a targeted DNA sequence in double-stranded DNA, characterized in that: The single-stranded DNA fluorescent reporter probe is exposed to the NovaCas-2 protein and sgRNA complex, and the presence of the targeted DNA sequence in the double-stranded DNA is determined based on the change in the fluorescent signal. Specifically: When there is a targeted DNA sequence in double-stranded DNA, the single-stranded DNA cleavage activity of the NovaCas-2 protein is activated, the single-stranded DNA fluorescent reporter probe is degraded to release the fluorescent group, and the fluorescence signal is enhanced; When there is no targeted DNA sequence in the double-stranded DNA, the single-stranded DNA cleavage activity of the NovaCas-2 protein is not activated and the fluorescence signal remains unchanged.

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