Multi-PAM-selection efficient gene editing system NovaCas-3 and application thereof

Abstract

The invention discloses a multi-PAM selection efficient gene editing system NovaCas-3 and application thereof, and belongs to the technical field of gene editing. The gene editing system NovaCas-3 is a complex of NovaCas-3 protein and sgRNA, and can accurately locate a targeted DNA sequence and generate cleavage to enable DNA to generate double-strand breakage damage, the amino acid sequence of the NovaCas-3 protein is as shown in SEQ ID NO: 1, and the nucleotide sequence of the sgRNA is as shown in SEQ ID NO: 7 or SEQ ID NO: 9. The NovaCas-3 protein has the beneficial effects that (1) as many as 38 PAM sequences can be recognized by the NovaCas-3 protein, so that the target gene editing range of the existing gene editing system is greatly widened; and (2) experiments prove that the gene editing system NovaCas-3 has higher gene editing efficiency than the existing gene editing system.

Description

Technical Field

[0001] The present invention relates to a gene editing system and applications thereof, and in particular to a multi-PAM selection efficient gene editing system NovaCas-3 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 acquired immune system evolved by bacteria and archaea to resist invasion by exogenous viruses or plasmids. At present, a total of 33 Cas enzyme subtypes have been discovered, which are divided into 2 major categories and 6 different types. CRISPR / Cas12 belongs to 2 types of V-type RNA-guided nuclease. The Cas12a protein is the most studied in this protein family. The most commonly used Cas12a proteins are AsCas12a and LbCas12a. The former comes from Acidaminococcus Rogosa and the latter comes from Lachnospiraceae. Cas12a protein has been widely used in many species including bacteria, yeast, plants and human cells.

[0003] In the CRISPR / Cas12a gene editing system, after the Cas12a protein forms a complex with sgRNA (single guide RNA), it recognizes the PAM (Protospacer Adjacent Motif) sequence of the target site. After recognizing the PAM sequence, crRNA (CRISPR-derived RNA) will form a complementary structure with the targeted DNA sequence, and the Cas12a protein will perform the function of cutting DNA, causing double-strand break damage to the DNA. In cells, there are two main DNA damage repair mechanisms responsible for repairing DNA damage: non-homologous end joining (NHEJ) and homologous recombination (HR). The result of NHEJ repair will cause the deletion or insertion of bases, and gene knockout can be performed; in the case of providing a homologous template, HR repair can be used to perform site-specific insertion of genes and precise replacement of bases.

[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 that 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 the 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-3 with multiple PAM selections and its applications.

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

[0009] A highly efficient gene editing system NovaCas-3 with multiple PAM selections, wherein the gene editing system NovaCas-3 is a complex of NovaCas-3 protein and sgRNA, which can accurately locate a targeted DNA sequence and produce cutting to cause double-strand break damage to the DNA, wherein the amino acid sequence of the NovaCas-3 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-3 protein and the sgRNA complex, and the PAM sequence includes ACTA, ACTG, CCTA, CCTC, CCTG, GCTA, GCTC, GCTG, TCCA, TCCC, TCCG, TCTA, TCTC, TCTG, ATTA, ATTC, ATTG, CTAC, CTCA, CTCC, CTGC, CTTA, CTTC, CTTG, CTTT, GTTA, GTTC, GTTG, TTAC, TTCA, TTCC, TTC G, TTCT, TTGC, TTTA, TTTC, TTTG and TTTT; preferably, the PAM sequence includes CCTA, TCTA, TCTC, TCTG, ATTA, ATTG, CTTA, CTTC, CTTG, GTTA, GTTG, TTCA, TTCC, TTCG, TTCT, TTTA, TTTC, TTTG and TTTT; more preferably, the PAM sequence includes TCTA, TCTG, ATTA, ATTG, CTTA, CTTG, GTTA, GTTG, TTCA, TTCC, TTTA, TTTC, TTTG and TTTT.

[0011] Preferably, the NovaCas-3 protein is obtained by codon optimization and transcription and translation of the nucleotide sequence of the original NovaCas-3 protein, wherein the nucleotide sequence of the original NovaCas-3 protein is shown in SEQ ID NO: 2, and the nucleotide sequence of the optimized NovaCas-3 protein is shown in SEQ ID NO: 3.

[0012] Preferably, the NovaCas-3 protein includes NovaCas-3 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-3 protein and sgRNA complex can accurately locate the targeted DNA sequence, which means that the NovaCas-3 protein and sgRNA complex can accurately recognize and target the DNA sequence, or refers to bringing other proteins fused to the NovaCas-3 protein or proteins that specifically recognize sgRNA to the targeted DNA position, and the other proteins fused to the NovaCas-3 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, 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-3 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, wherein 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-3 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-3 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-3 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-3 protein is not activated, and the fluorescence signal remains unchanged.

[0017] The present invention is beneficial in that:

[0018] (1) The nuclease selected by the present invention is NovaCas-3 protein. In addition to recognizing TTTA, TTTC and TTTG, NovaCas-3 protein can also recognize CTTA, TTCA, ATTA, TTTT, TCTA, TTCC, CTTG, GTTA, TCTG, GTTG and ATTG, etc. There are as many as 39 recognizable 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 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, the gene editing system NovaCas-3 provided by the present invention has higher gene editing efficiency than the existing CRISPR / Cas12a gene editing system. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a schematic diagram of the gene editing system NovaCas-3 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-3 (NovaCas-3 protein+sgRNA1) provided by the present invention;

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

[0023] Figure 4 It is the electrophoresis result diagram of the experimental group (NovaCas-3 protein + sgRNA1) and the control group (AsCas12a protein + sgRNA2) outside the cell;

[0024] Figure 5 This is a comparison chart of gene editing efficiency between the experimental group (NovaCas-3 protein + sgRNA1) and the control group (AsCas12a protein + sgRNA2) outside the cell;

[0025] Figure 6 It is the electrophoresis result diagram of the experimental group (NovaCas-3 protein + sgRNA3) and the control group (AsCas12a protein + sgRNA4) in HEK293 cells;

[0026] Figure 7 This is a comparison chart of the gene editing efficiency of the experimental group (NovaCas-3 protein + sgRNA3) and the control group (AsCas12a protein + sgRNA4) in HEK293 cells;

[0027] Figure 8 This is a graph of the fluorescence intensity detection results of the experimental group (NovaCas-3 protein + sgRNA1) and the control group (AsCas12a protein + sgRNA2) outside the cell. DETAILED DESCRIPTION

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

[0029] 1. Composition of the gene editing system NovaCas-3

[0030] like Figure 1 As shown, the gene editing system NovaCas-3 provided by the present invention is a complex of NovaCas-3 protein and sgRNA, which can accurately locate the targeted DNA sequence and produce cutting, causing double-strand break damage to the DNA.

[0031] The NovaCas-3 protein and sgRNA complex can accurately locate the targeted DNA sequence, which means that the NovaCas-3 protein and sgRNA complex can accurately identify and target the DNA sequence, or refers to bringing other proteins fused with the NovaCas-3 protein or proteins that specifically recognize the sgRNA to the targeted DNA position, wherein other proteins fused with the NovaCas-3 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-3 protein and sgRNA are introduced below.

[0033] 1. NovaCas-3 protein

[0034] The NovaCas-3 protein comes from the genus Acidaminococcaceae, with the NCBI retrieval number MBR1591151, the amino acid sequence as shown in SEQ ID NO: 1, and the nucleotide sequence as shown in SEQ ID NO: 2. Studies have shown that the protein belongs to Class 2 V-type RNA-guided nucleases, and the present invention names the protein NovaCas-3 protein.

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

[0036] Of course, the NovaCas-3 protein may also include NovaCas-3 protein variants having no cleavage activity, single-stranded cleavage activity, or double-stranded cleavage activity.

[0037] 2. sgRNA

[0038] CRISPRCasdb was used to mine the nucleotide sequence that can transcribe crRNA from the 5000bp nucleotide sequence downstream of the NovaCas-3 protein encoding gene in the metagenomic genome of Aminoacillus, and the nucleotide sequence of the crRNA finally obtained was shown in SEQ ID NO: 5 (denoted as crRNA1). Based on the crRNA, two sgRNAs (denoted as sgRNA1 and sgRNA3, 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: 7 (sgRNA1) and SEQ ID NO: 9 (sgRNA3), respectively. Both sgRNAs can recognize the PAM sequence (TTTA, TTTC, TTTG) of the targeting site and the 20bp or 21bp nucleotide sequence at the 3' end can form a base complementary pairing structure with the DNA sequence of the targeting 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] Use HindIII and KpnI restriction enzymes to digest the pUC19 plasmid to obtain a linearized plasmid. Enzyme digestion system: 1 μg pUC19 plasmid, 5 μL buffer, 1 μL HindIII endonuclease, 1 μL KpnI endonuclease, and sterile water to 50 μL. Enzyme digestion reaction conditions: 37℃ for 1h.

[0045] (2) Gel electrophoresis

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

[0047] (3) Recovering plasmid

[0048] The DNA fragment of 2643 bp on the gel was cut out, 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 a long primer containing NNNN (two oligonucleotide single-stranded DNAs, both with sticky end sequences corresponding to the two sides of the linearized DNA fragment of the pUC19 plasmid), the nucleotide sequence of the long primer is specifically:

[0053] Oligo-F1:AGTCCgtcgacTACCCTACTGATTAGATCCGTTCANN NNCGGTCATCGCGTGGTGCACATCCGTTAAGTCATAGCGAgacgtc AGTAC (SEQ ID NO: 19);

[0054] Oligo-R1:GTACTgacgtcTCGCTATGACTTAACGGATGTGCACCA CGCGATGACCGNNNNTGAACGGATCTAATCAGTAGGGTAgtcgac GAGCT (SEQ ID NO: 20).

[0055] (6) Annealing

[0056] Two oligonucleotide single-stranded DNAs were annealed to become 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, 25 °C for 1 min, and stored at 4 °C.

[0057] (7) Connection

[0058] The annealed product and the pUC19 plasmid linearized DNA fragment were connected under the action of DNA ligase. The connection system: 2μL buffer, 50ng pUC19 plasmid linearized DNA fragment, 20ng annealed product, 1μL T4 DNA ligase, and sterile water to make up to 20μL. Connection reaction conditions: 16℃ reaction for 2h.

[0059] (8) Verify library abundance

[0060] Take 5 μL of the ligation product for chemical transformation, add it to 900 μL of SOB liquid medium, resuscitate at 37 °C for 1 h, then take 10 μL of the bacterial solution 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 not 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 LB liquid culture medium and cultured at 37°C for 12 h, and then the plasmid was extracted to obtain the PAM library plasmid for later use.

[0063] 2. Prepare NovaCas-3 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, and then protein expression plasmid synthesis, protein expression and protein purification were performed using conventional methods to finally obtain the NovaCas-3 protein.

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

[0066] 3. Prepare sgRNA

[0067] For the NovaCas-3 protein, the sgRNA (sgRNA1) shown in SEQ ID NO: 7 and the sgRNA (sgRNA3) shown in SEQ ID NO: 9 were synthesized by conventional methods.

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

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

[0070] The detection principle of PAM preference: The abundance of the PAM library without gene editing (negative control group) and the abundance of the PAM library with gene editing (experimental group) were detected respectively, and the changes in the abundance of the PAM library before and after gene editing were analyzed by bioinformatics methods, and finally the PAM preference of the gene editing system NovaCas-3 was obtained.

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

[0072] 1. Enzyme digestion of plasmid

[0073] Use HaeII restriction endonuclease to digest the PAM library plasmid obtained above to obtain a linearized plasmid. Enzyme digestion system: 1μg PAM library plasmid, 5μL buffer, 1μL HaeII endonuclease, and sterile water to 50μL. Enzyme digestion reaction conditions: 37℃ for 1h.

[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 DNA fragment of 2676 bp on the gel was cut out, 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. Determination of 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-3 protein and sgRNA1

[0081] 20 μL DEPC water, 3 μL buffer solution, 1 μL 1 μM NovaCas-3 protein and 3 μL 300 nM sgRNA1 were shaken and mixed, and placed in a PCR instrument to run the incubation program: 25°C for 10 min.

[0082] 6. Gene Editing

[0083] Experimental group: Add 3 μL 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 PCR instrument and run gene editing program: 37°C for 30 min.

[0085] 7. Protease inactivation

[0086] Add 1 μL of proteinase K to the experimental group and negative control group respectively, mix well, 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 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), wherein:

[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 product 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×MasterMix, 1.25 μL 10 μM forward primer, 1.25 μL 10 μM reverse primer, 2 μL cleavage product, and filled to 50 μL with sterile water.

[0092] After shaking and mixing, place it in a PCR instrument and run the amplification program: 98℃30s, the following reaction is carried out for 30 cycles, 98℃10s, 65℃30s, 72℃10s. After the above cycles are completed, 72℃2min and stored at 4℃.

[0093] 9. Gel electrophoresis

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

[0095] 10. Recover sample DNA

[0096] The 201 bp DNA fragment on the gel was cut out, 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 PAM preference detection results of the gene editing system NovaCas-3 (NovaCas-3 protein and sgRNA1 complex) are shown.

[0099] Depend on Figure 2 It can be seen that the gene editing system NovaCas-3 (NovaCas-3 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, and can also recognize ACTA, ACTG, CCTA, CCTC, CCTG, GCTA, GCTC, GCTG, TCCA, TCCC, TCCG, TCTA, TCTC, TCTG, ATTA, ATTC, ATTG, CTAC, CTCA, CTCC, CTGC, CTTA, CTTC, CTTG, CTTT, GTTA, GTTC, GTTG, TTAC, TTCA, TTCC, TTCG, TTCT, TTG C and TTTT, and can recognize as many as 38 PAM sequences. Among them, it has low recognition activity for 19 PAM sequences including ACTA, ACTG, CCTC, CCTG, GCTA, GCTC, GCTG, TCCA, TCCC, TCCG, ATTC, CTAC, CTCA, CTCC, CTGC, CTTT, GTTC, TTAC and TTGC, has high recognition activity for 5 PAM sequences including CCTA, TCTC, CTTC, TTCG and TTCT, and has very high recognition activity for 14 PAM sequences including TCTA, TCTG, ATTA, ATTG, CTTA, CTTG, GTTA, GTTG, TTCA, TTCC, TTTA, TTTC, TTTG and TTTT.

[0100] Since sgRNA3 also contains the sequence of crRNA1, it can be determined that the NovaCas-3 protein and sgRNA3 complex can also recognize the above 38 PAM sequences.

[0101] In summary, the gene editing system NovaCas-3 (NovaCas-3 protein and sgRNA1 or sgRNA3 complex) provided by the present invention greatly broadens the scope of targeted gene editing of the existing CRISPR / Cas12a gene editing system.

[0102] 4. Detecting the gene editing efficiency of the gene editing system NovaCas-3

[0103] The gene editing system NovaCas-3 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] Escherichia coli is used to detect the gene editing efficiency of the gene editing system NovaCas-3 provided by the present invention. Specifically, the gene editing efficiency of the gene editing system NovaCas-3 provided by the present invention is detected using the Escherichia coli galK red and white spot screening system. The detection principle is:

[0105] When the functional sequence on the galK gene of Escherichia coli is recognized, the NovaCas-3 protein will cut the galK gene to cause a double-strand break, and then homologous recombination will occur. The inactivation of the galk gene causes Escherichia coli to lose the ability to degrade galactose and produce acid, and white colonies will grow on the 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 will grow on the MacConkey medium containing galactose.

[0106] AsCas12a protein and sgRNA2 complex were used as controls. The specific method for detecting gene editing efficiency is as follows:

[0107] 1. Construction of Cas protein plasmid

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

[0109] In order to enable the AsCas12a protein to be efficiently expressed in Escherichia coli, the original nucleotide sequence of the AsCas12a protein encoding gene was codon optimized, and the nucleotide sequence after codon optimization was 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 sgRNA (sgRNA1, sgRNA2) and a homologous repair template, different primers were designed for sgRNA1 and sgRNA2, respectively, where:

[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: 1μL 100μM Oligo-F2 (or Oligo-F3), 1μL 100μM Oligo-R2 (or Oligo-R3), 28μL sterile water. After thorough mixing, place in a PCR instrument and run the annealing program (cooling rate 0.3℃ / s): 95℃5min, 85℃1min, 75℃1min, 65℃1min, 55℃1min, 45℃1min, 35℃1min, 25℃1min, and store at 4℃.

[0116] The annealed products were connected to the previously obtained pUC19 plasmid linearized DNA fragments under the action of DNA ligase. Ligation system: 2μL buffer, 50ng pUC19 plasmid linearized DNA fragments, 20ng annealed products, 1μLT4 DNA ligase, and sterile water to make up to 20μL. Ligation reaction conditions: 16℃ reaction for 2h.

[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 whose connection was verified to be correct 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-3 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-3 protein particle or 1 μL of pAsCas12a protein particle to the cell suspension, flick to mix, and let stand in an ice bath for 30 min.

[0122] (2) Heat shock the centrifuge tube in a 42°C water bath for 90 seconds, then quickly transfer to an ice bath and cool 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 min to allow the 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 LB liquid culture medium, place it in a 30°C shaker at 200 rpm for 16 h to amplify the E. coli and obtain E. coli transfected with the Cas protein plasmid. Among them, the E. coli transfected with the pNovaCas-3 protein plasmid is recorded as E. coli Nova3, and the E. coli transfected with the pAsCas12a protein plasmid 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 Nova3 (bacterial liquid) and E. coli As (bacterial liquid) were inoculated with 1% of each other into 50 mL of LB liquid medium 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 Nova3 competent cells (bacterial solution) and Escherichia coli As competent cells (bacterial solution), respectively.

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

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

[0137] (1) Precool a 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 Nova3 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. Gently flick to mix, and then electroporate at 1800 V. Immediately after electroporate, add 950 μL of sterile LB liquid culture medium without antibiotics, pipette to mix, and transfer to a centrifuge tube. Place in a 30°C shaker at 150 rpm for 2 h 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 medium, invert the plate and culture it at 30°C for 16 h.

[0140] 6. Calculate gene editing efficiency

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

[0142] The number of red colonies and white colonies on MacConkey solid agar medium was counted, and the gene editing efficiency was calculated according to the following formula:

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

[0144] It has been calculated that in Escherichia coli, the gene editing efficiency of the gene editing system NovaCas-3 (NovaCas-3 protein + sgRNA1) provided by the present invention is 90.82%, and the gene editing efficiency of the existing CRISPR / Cas12a gene editing system (AsCas12a protein + sgRNA2) is 48.13%. The gene editing efficiency comparison chart of the two gene editing systems is shown in 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-3 (NovaCas-3 protein + sgRNA1) provided by the present invention exhibits higher gene editing efficiency.

[0146] 5. Verification of the gene editing activity of NovaCas-3 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 culture medium into 3 mL of LB liquid culture medium, and the culture was placed in a shaker at 37°C and 200 rpm for 12 h to amplify the E. coli. The genome was then extracted using a bacterial genome extraction kit, and then eluted with ultrapure water to obtain the E. coli MG1655 genome. Finally, the E. coli genome was diluted to 500 ng / μL with ultrapure water for later use (stored in a -20°C refrigerator).

[0149] (2) PCR to obtain in vitro sheared double-stranded DNA substrate: PCR amplification of the Escherichia 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 Escherichia coli MG1655 genome, and sterile water is added to 50 μL. After shaking and mixing, place it in a PCR instrument and run the amplification program: 98°C 30s, the following reaction is carried out for 30 cycles, 98°C 10s, 65°C 30s, 72°C 30s, after the above cycles are completed, 72°C 2min, and stored at 4°C.

[0150] (3) Gel electrophoresis: The PCR products were electrophoresed on a 1% agarose gel at 120 V for 30 min.

[0151] (4) Recovering 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 keep for later use (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-3 outside the cell is verified. The method for verifying the gene editing activity of the gene editing system NovaCas-3 outside the cell is as follows:

[0153] (1) Experimental group: 20 μL DEPC water, 3 μL buffer solution, 1 μL 1 μM NovaCas-3 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 to 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, mix by pipetting, and then perform electrophoresis on 1% agarose gel at 120 V for 30 min.

[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 and the size is 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 and their sizes are 3124bp, 2531bp and 593bp respectively, it indicates that the linear double-stranded DNA substrate is cut but not thoroughly enough, and the corresponding gene editing system has gene editing activity but it is not high.

[0162] The electrophoresis results of the experimental group (NovaCas-3 protein + sgRNA1), the control group (AsCas12a protein + sgRNA2) and the negative control group are shown in Figure 4 .Depend on Figure 4 It can be preliminarily judged that the gene editing system of the experimental group has high gene editing activity, and the gene editing system of 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. The gene editing efficiency of the experimental group and the control group can be calculated according to the following formula:

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

[0165] After calculation, the gene editing efficiency of the gene editing system (NovaCas-3 protein + sgRNA1) in the experimental group was 98.7%, 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-3 (NovaCas-3 protein + sgRNA1) provided by the present invention exhibits higher gene editing efficiency.

[0167] 6. Verify the gene editing activity of NovaCas-3 gene editing system in cells

[0168] HEK293 cells were used to verify the gene editing activity of the gene editing system NovaCas-3 in cells. Before verifying the gene editing activity, HEK293 cells were transfected with Cas protein and sgRNA, and AsCas12a protein and sgRNA4 complex were used as controls. The specific method for transfecting HEK293 cells 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 NovaCas-3 protein prepared previously and 2 μg of the sgRNA3 prepared previously 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 min.

[0173] (5) The HEK293 cell line was added to the electroporation plate, and the mixed transfection solution (transfection solution of the experimental group, transfection solution of the control group, and transfection solution of the negative control group) was added 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 Nucleofector medium preheated at 37°C. Continue culturing in a 37°C, 5% CO2 incubator for 24 h.

[0175] After HEK293 cells were transfected with Cas proteins (NovaCas-3 protein, AsCas12a protein) and sgRNAs (sgRNA3, sgRNA4), the endogenous target gene editing activity was verified. The 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 the mixture was made up to 50 μL with sterile water. After vortexing and mixing, the mixture was placed in a PCR instrument and the amplification program was run: 98°C for 30 s, and the following reaction was performed for 30 cycles: 98°C for 10 s, 65°C for 30 s, and 72°C for 10 s. After the above cycles were completed, the mixture was incubated at 72°C for 2 min and stored at 4°C.

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

[0179] (4) The template prepared above was digested with T7 EI enzyme, and the digested product was electrophoresed on 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 and the size is 776 bp, it means 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 and their sizes are 776bp, 490bp and 286bp respectively, it means that the template is cut but not thoroughly enough, and the corresponding gene editing system has gene editing activity but it is not high.

[0184] The electrophoresis results of the experimental group (NovaCas-3 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 judged that the gene editing systems of both the experimental group and the control group 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 before.

[0186] After calculation, the gene editing efficiency of the gene editing system (NovaCas-3 protein + sgRNA3) in the experimental group was 67.2%, and the gene editing efficiency of the gene editing system (AsCas12a protein + sgRNA4) in the control group was 31.7%. The gene editing efficiency comparison 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-3 (NovaCas-3 protein + sgRNA3) provided by the present invention exhibits higher gene editing efficiency.

[0188] 7. Other applications of the NovaCas-3 gene editing system

[0189] In addition to being used for gene editing, the gene editing system NovaCas-3 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 contacted with the NovaCas-3 protein and sgRNA complex. When there is a targeted DNA sequence in the double-stranded DNA, the NovaCas-3 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-3 protein changes, activating the single-stranded DNA cutting activity ("trans-cutting" activity). After the single-stranded DNA cutting activity is activated, the NovaCas-3 protein will cut (degrade) the single-stranded DNA fluorescent reporter probe, thereby releasing the fluorescent group. A strong fluorescence value can be detected using an enzyme reader. When there is no targeted DNA sequence in the double-stranded DNA, the conformation of the NovaCas-3 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 enzyme reader will remain unchanged (the fluorescence value is still the base value).

[0192] The method for verifying that the gene editing system NovaCas-3 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-3 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 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 in a microplate reader for fluorescence detection. The reaction procedure is: 37°C, 25 min, and collect fluorescence every 0.5 min.

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

[0197] If the fluorescence intensity does not change over time (still at the base value) or does not change significantly, it indicates that the single-stranded DNA fluorescent reporter probe has not been cleaved, 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 the fluorescence intensity of the experimental group (NovaCas-3 protein + sgRNA1) and the control group (AsCas12a protein + sgRNA2) versus the number of cycles (time) 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 5 cycles (2.5 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 clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the embodiments here. Any obvious changes or modifications derived from the technical solution of the present invention are still within the scope of protection of the present invention.

Claims

1. A highly efficient gene editing system NovaCas-3 with multiple PAM selections, characterized in that: The gene editing system NovaCas-3 is a complex of NovaCas-3 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-3 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 efficient gene editing system NovaCas-3 with multiple PAM selections according to claim 1, characterized in that: The precise positioning of the targeting DNA sequence includes the NovaCas-3 protein and the sgRNA complex recognizing the PAM sequence on the targeting DNA sequence, and the PAM sequence includes ACTA, ACTG, CCTA, CCTC, CCTG, GCTA, GCTC, GCTG, TCCA, TCCC, TCCG, TCTA, TCTC, TCTG, ATTA, ATTC, ATTG, CTAC, CTCA, CTCC, CTGC, CTTA, CTTC, CTTG, CTTT, GTTA, GTTC, GTTG, TTAC, TTCA, TTCC, TTCG, TTCT, TTGC, TTTA, TTTC, TTTG and TTTT; preferably, the PAM sequence includes CCTA, TCTA, TCTC, TCTG, ATTA, ATTG, CTTA, CTTC, CTTG, GTTA, GTTG, TTCA, TTCC, TTCG, TTCT, TTTA, TTTC, TTTG and TTTT; more preferably, the PAM sequence includes TCTA, TCTG, ATTA, ATTG, CTTA, CTTG, GTTA, GTTG, TTCA, TTCC, TTTA, TTTC, TTTG and TTTT.

3. The efficient gene editing system NovaCas-3 with multiple PAM selections according to claim 1, characterized in that: The NovaCas-3 protein is obtained by codon-optimizing the nucleotide sequence of the original NovaCas-3 protein and then transcribing and translating it, wherein the nucleotide sequence of the original NovaCas-3 protein is shown in SEQ ID NO: 2, and the nucleotide sequence of the optimized NovaCas-3 protein is shown in SEQ ID NO:

3.

4. The efficient gene editing system NovaCas-3 with multiple PAM selections according to claim 1, characterized in that: The NovaCas-3 protein includes a NovaCas-3 protein variant having no cleavage activity, having single-stranded cleavage activity or having double-stranded cleavage activity.

5. The efficient gene editing system NovaCas-3 with multiple PAM selections according to claim 1, characterized in that: The sgRNA includes sgRNA modified by phosphorylation, sulfation, methylation or hydroxylation.

6. The efficient gene editing system NovaCas-3 with multiple PAM selections according to claim 1, characterized in that: The NovaCas-3 protein and sgRNA complex can accurately locate the targeted DNA sequence, which means that the NovaCas-3 protein and sgRNA complex can accurately recognize and target the DNA sequence, or refers to bringing other proteins fused with the NovaCas-3 protein or proteins that specifically recognize the sgRNA to the targeted DNA position.

7. The efficient gene editing system NovaCas-3 with multiple PAM selections according to claim 6, characterized in that: The other proteins fused with the NovaCas-3 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 efficient gene editing system NovaCas-3 with multiple PAM selections 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 NovaCas-3, a highly efficient gene editing system with multiple PAM selections 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 for non-disease diagnosis and treatment purposes.

10. The use of NovaCas-3, a high-efficiency gene editing system with multiple PAM selections according to claim 1, in rapid detection of the presence or absence of a targeted DNA sequence in double-stranded DNA, characterized in that: The single-stranded DNA fluorescent reporter probe is exposed to the NovaCas-3 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-3 protein is activated, the single-stranded DNA fluorescent reporter probe is degraded to release the fluorescent group, and the fluorescent signal is enhanced; When there is no targeted DNA sequence in the double-stranded DNA, the single-stranded DNA cleavage activity of the NovaCas-3 protein is not activated and the fluorescence signal remains unchanged.

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