A multi-pam selection gene editing system NovaCas-1 and application thereof

Abstract

The application discloses a kind of multi-PAM selection efficient gene editing system NovaCas-1 and application thereof, belong to gene editing technical field.The gene editing system NovaCas-1 is NovaCas-1 protein and sgRNA complex, can accurately locate targeted DNA sequence, and produce cutting, make DNA double-strand break damage, wherein, the amino acid sequence of NovaCas-1 protein is as shown in SEQ ID NO:1, the nucleotide sequence of sgRNA is as shown in SEQ ID NO:7 or SEQ ID NO:9.The application has the advantages that (1) NovaCas-1 protein can recognize up to 47 PAM sequences, greatly widening the existing gene editing system targeted gene editing range;(2) experiment proves that gene editing system NovaCas-1 has higher gene editing efficiency than existing gene editing system.

Description

TECHNICAL FIELD

[0001] The present application relates to a gene editing system and its application, in particular to a multi-PAM selected gene editing system NovaCas-1 and its application, belonging to the technical field of gene editing. BACKGROUND

[0002] CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) / Cas (CRISPR-associated protein) is an acquired immune system evolved by bacteria and archaea to resist the invasion of foreign viruses or plasmids. At present, a total of 33 Cas enzyme subtypes have been found, which are divided into two large categories and six different types. CRISPR / Cas12 belongs to type 2 V RNA-guided endonuclease. The most studied protein in this protein family is Cas12a protein, and the most commonly used Cas12a proteins are AsCas12a and LbCas12a, the former is from Acidaminococcus Rogosa, and the latter is 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 the sgRNA (single guide RNA), it recognizes the PAM (Protospacer Adjacent Motif) sequence of the target site. After recognizing the PAM sequence, the crRNA (CRISPR-derived RNA) forms a complementary structure with the target DNA sequence, and the Cas12a protein performs the function of cutting DNA, causing double-strand breakage damage to DNA. In cells, there are mainly two 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 deletion or insertion of bases, which can be used for gene knockout; under the condition of providing a homologous template, HR repair can be used for gene site insertion and precise base replacement.

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

[0005] (1) The Cas12a protein is mainly AsCas12a and LbCas12a, and the PAM sequences that can be recognized by the two are mainly TTTA, TTTC and TTTG, and the selection of PAM sequences is less, which limits the targeted gene editing range of the CRISPR / Cas12a gene editing system;

[0006] (2) Due to the difference of the target site sequence and the different positions of the related genes in the genome, the existing CRISPR / Cas12a gene editing system has difficult-to-edit DNA sequences, which limits the use of the CRISPR / Cas12a gene editing system in related sites or genes. SUMMARY

[0007] In order to solve the problems of the prior art, the purpose of the present application is to provide a multi-PAM selection gene editing system NovaCas-1 and its application.

[0008] In order to achieve the above-mentioned target, the technical scheme adopted by the present application is as follows:

[0009] A multi-PAM selection gene editing system NovaCas-1, the gene editing system NovaCas-1 is a NovaCas-1 protein and sgRNA complex, which can accurately locate the target DNA sequence and produce cutting to cause double-strand breakage damage of DNA, wherein the amino acid sequence of the NovaCas-1 protein is shown as SEQ ID NO: 1, and the nucleotide sequence of the sgRNA is shown as SEQ ID NO: 7 or SEQ ID NO: 9.

[0010] Preferably, the precisely targeted DNA sequence comprises a PAM sequence recognized by the NovaCas-1 protein and sgRNA complex on the targeted DNA sequence, the PAM sequence comprising ACTA, ACTC, ACTG, CCCA, CCC, CCCG, CCTA, CCTC, CCTG, GCTA, GCTC, GCTG, TCAC, TCCA, TCCC, TCCG, TCGC, TCTA, TCTC, TCTG, TCTT, ATTA, ATTC, ATTG, CTAC, CTCA, CTCC, CTCG, CTGC, CTTA, CTTC, CTTG, CTTT, GTCA, GTTA, GTTC, GTTG, GTTT, TTAC, TTCA, TTCC, TTCG, TTCT, TTGC, TTT, TTTC, TTTC, and TTTT; preferably, the PAM sequence comprises ACTA, CCTA, CCTG, TCCA, TCCC, TCTA, TCTC, TCTG, ATTA, ATTG, CTCA, CTCC, CTTA, CTTC, CTTG, GTTA, GTTG, TTCA, TTCC, TTCG, TTCT, TTGC, TTT, TTTC, TTTC, and TTTT; more preferably, the PAM sequence comprises CCTA, TCTA, TCTG, ATTA, ATTG, CTTA, CTTG, GTTA, GTTG, TTCA, TTCC, TTCT, TTT, TTTC, TTTC, and TTTT.

[0011] Preferably, the NovaCas-1 protein is obtained by transcription and translation of a nucleotide sequence of an original NovaCas-1 protein after codon optimization, wherein the nucleotide sequence of the original NovaCas-1 protein is shown as SEQ ID NO: 2, and the nucleotide sequence of the NovaCas-1 protein after optimization is shown as SEQ ID NO: 3.

[0012] Preferably, the NovaCas-1 protein comprises a NovaCas-1 protein variant without cleavage activity, with single-strand cleavage activity, or with double-strand cleavage activity.

[0013] Preferably, the sgRNA comprises an sgRNA modified by phosphorylation, sulfuration, methylation, or hydroxylation.

[0014] The NovaCas-1 protein and sgRNA complex can preferably accurately locate the target DNA sequence, that is, the NovaCas-1 protein and sgRNA complex can accurately recognize and target the DNA sequence, or other proteins fused with the NovaCas-1 protein or proteins specifically recognizing the sgRNA are brought to the target DNA position, and the other proteins fused with the NovaCas-1 protein or the proteins specifically recognizing the sgRNA can modify or regulate the target DNA sequence, and the modification or regulation includes regulation at a 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 foregoing multi-PAM selected gene editing system NovaCas-1 is applied to gene editing, including gene editing in cells and gene editing in vitro, and the cells include eukaryotic cells and prokaryotic cells, and the eukaryotic cells include mammalian cells and plant cells, and the application is a non-disease diagnosis and treatment purpose application.

[0016] The foregoing multi-PAM selected gene editing system NovaCas-1 is applied to rapid detection of whether there is a target DNA sequence in double-stranded DNA, and a single-stranded DNA fluorescent reporter probe is contacted with the NovaCas-1 protein and sgRNA complex, and whether there is a target DNA sequence in the double-stranded DNA is judged according to a fluorescent signal change, and specifically: when there is a target DNA sequence in the double-stranded DNA, the NovaCas-1 protein single-stranded DNA cleavage activity is activated, the single-stranded DNA fluorescent reporter probe is degraded to release a fluorescent group, and the fluorescent signal is enhanced; when there is no target DNA sequence in the double-stranded DNA, the NovaCas-1 protein single-stranded DNA cleavage activity is not activated, and the fluorescent signal is unchanged.

[0017] The present application has the advantages that:

[0018] (1) The nuclease selected in the present application is the NovaCas-1 protein, and the NovaCas-1 protein can recognize CCTA, TCTA, TCTG, ATTA, ATTG, CTTA, CTTG, GTTA, GTTG, TTCA, TTCC, TTCT and TTTT in addition to TTTA, TTTC and TTTG, and can recognize up to 47 PAM sequences, greatly widening the range of targeted gene editing of the existing CRISPR / Cas12a gene editing system;

[0019] (2) Experiments have confirmed that the gene editing system NovaCas-1 provided by the application has higher gene editing efficiency than the existing CRISPR / Cas12a gene editing system for the E. 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 DRAWINGS

[0020] Figure 1 is a schematic diagram of the target DNA cleaved by the gene editing system NovaCas-1 provided by the application;

[0021] Figure 2 is a PAM preference detection result diagram of the gene editing system NovaCas-1 (NovaCas-1 protein + sgRNA1) provided by the application;

[0022] Figure 3 is a gene editing efficiency comparison diagram of the gene editing system NovaCas-1 (NovaCas-1 protein + sgRNA1) provided by the application and the existing CRISPR / Cas12a gene editing system (AsCas12a protein + sgRNA2);

[0023] Figure 4 is an electrophoresis result diagram of the experimental group (NovaCas-1 protein + sgRNA1) and the control group (AsCas12a protein + sgRNA2) outside cells;

[0024] Figure 5 is a gene editing efficiency comparison diagram of the experimental group (NovaCas-1 protein + sgRNA1) and the control group (AsCas12a protein + sgRNA2) outside cells;

[0025] Figure 6 is an electrophoresis result diagram of the experimental group (NovaCas-1 protein + sgRNA3) and the control group (AsCas12a protein + sgRNA4) in HEK293 cells;

[0026] Figure 7 is a gene editing efficiency comparison diagram of the experimental group (NovaCas-1 protein + sgRNA3) and the control group (AsCas12a protein + sgRNA4) in HEK293 cells;

[0027] Figure 8 is a fluorescence intensity detection result diagram of the experimental group (NovaCas-1 protein + sgRNA1) and the control group (AsCas12a protein + sgRNA2) outside cells. DETAILED DESCRIPTION

[0028] The application will be specifically introduced below in combination with the drawings and specific embodiments.

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

[0030] As shown in Figure 1 The gene editing system NovaCas-1 provided by the application is a NovaCas-1 protein and sgRNA complex, which can accurately locate and target a DNA sequence and produce a cut to cause a double-stranded break damage to the DNA.

[0031] The NovaCas-1 protein and sgRNA complex can accurately locate and target a DNA sequence, which means that the NovaCas-1 protein and sgRNA complex can accurately recognize and target a DNA sequence, or means that other proteins fused with the NovaCas-1 protein or proteins specifically recognizing sgRNA are brought to the target DNA position, wherein the other proteins fused with the NovaCas-1 protein or the proteins specifically recognizing sgRNA can modify or regulate the target DNA sequence, and the modification or regulation includes regulation at the transcription level of a gene, single base conversion, and the single base conversion includes conversion between adenine to guanine, cytosine to thymine or other bases.

[0032] The NovaCas-1 protein and sgRNA are introduced below respectively.

[0033] 1. NovaCas-1 protein

[0034] The NovaCas-1 protein is from a bacterium named Jingyaoa shaoxingensis, and the NCBI search number is WP_249309341.1. The amino acid sequence is shown as SEQ ID NO: 1, and the nucleotide sequence is shown as SEQ ID NO: 2. It is found through research that the protein belongs to class 2 V-type RNA-guided endonuclease, and the protein is named NovaCas-1 protein in the application.

[0035] In order to enable the NovaCas-1 protein to be efficiently expressed in Escherichia coli, the original nucleotide sequence (SEQ ID NO: 2) is subjected to codon optimization treatment, and the nucleotide sequence after codon optimization treatment is shown as SEQ ID NO: 3.

[0036] Of course, the NovaCas-1 protein can also include NovaCas-1 protein variants without cutting activity, with single-strand cutting activity or with double-strand cutting activity.

[0037] 2. sgRNA

[0038] The nucleotide sequence of the crRNA is shown in SEQ ID NO: 5 (denoted as crRNA1) by using CRISPRCasdb to mine the nucleotide sequence capable of transcribing crRNA from the 5000bp nucleotide sequence downstream of the Jingyaoa shaoxingensis macrogenome NovaCas-1 protein coding gene, and two sgRNAs (denoted as sgRNA1 and sgRNA3, respectively) targeting the DNA sequence of E. coli (SEQ ID NO: 11) and the DNA sequence of HEK293 cells (SEQ ID NO: 12), respectively, are designed according to the crRNA, and the nucleotide sequences of the two sgRNAs are shown in SEQ ID NO: 7 (sgRNA1) and SEQ ID NO: 9 (sgRNA3), respectively. Both of the two sgRNAs can recognize the PAM sequence (TTTA, TTTC, TTTC) of the target site and the 3' end 20bp or 21bp nucleotide sequence can form a base complementary pairing structure with the DNA sequence of the target site.

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

[0040] II. Experimental preparation

[0041] 1. Preparation of PAM library plasmid

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

[0043] (1) Enzymatic digestion of plasmid

[0044] The pUC19 plasmid was digested with HindIII and KpnI restriction enzymes to obtain linearized plasmid. The enzyme digestion system: 1 μg pUC19 plasmid, 5 μL buffer, 1 μL HindIII endonuclease, 1 μL KpnI endonuclease, and sterile water to 50 μL. The enzyme digestion reaction conditions: 37℃ reaction for 1h.

[0045] (2) Gel electrophoresis

[0046] The product obtained by enzyme digestion was subjected to electrophoresis on a 1% agarose gel at 120V for 30min.

[0047] (3) Recovery of plasmid

[0048] The 2643bp DNA fragment was removed from the gel, 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 was stored in a refrigerator at -20°C.

[0051] (5) Design and synthesis of primers

[0052] A long primer containing NNN was designed and synthesized (two oligonucleotide single-stranded DNAs, both with the corresponding sticky end sequence on both sides of the pUC19 plasmid linearized DNA fragment), and the nucleotide sequence of the long primer was specifically as follows:

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

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

[0055] (6) Annealing

[0056] The two oligonucleotide single-stranded DNAs were annealed into double-stranded DNA, and the annealing reaction system was as follows: 1 μL of 100 μM Oligo-F1, 1 μL of 100 μM Oligo-R1, and 28 μL of sterile water. After being mixed thoroughly, the mixture was placed in a PCR instrument to run the annealing 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 4°C for storage.

[0057] (7) Ligation

[0058] The annealing product was ligated with the pUC19 plasmid linearized DNA fragment under the action of DNA ligase. The ligation system was as follows: 2 μL of buffer, 50 ng of pUC19 plasmid linearized DNA fragment, 20 ng of annealing product, 1 μL of T4 DNA ligase, and sterile water to make up to 20 μL. The ligation reaction condition was 16°C for 2 h.

[0059] (8) Verification of library abundance

[0060] Take 5 μL of the ligation product for chemical competent transformation, add to 900 μL of SOB liquid medium, recover at 37°C for 1 h, then take 10 μL of bacterial liquid to spread on SOB solid medium, verify the library abundance (library abundance = number of colonies x 100, and the library abundance should not be less than 10,000), and perform Sanger sequencing verification on the grown bacteria.

[0061] (9) Obtain PAM library plasmid

[0062] Inoculate the remaining bacterial liquid into 20 mL of LB liquid medium and culture at 37°C for 12 h, then extract the plasmid to obtain the PAM library plasmid, which is ready for use.

[0063] 2. Prepare NovaCas-1 protein and AsCas12a protein

[0064] Synthesize the nucleotide sequence shown in SEQ ID NO: 3 and construct it on the pET28a plasmid using conventional methods, then perform protein expression plasmid synthesis, protein expression and protein purification using conventional methods, and finally obtain the NovaCas-1 protein.

[0065] Directly purchase AsCas12a protein from the market as a control.

[0066] 3. Prepare sgRNA

[0067] For NovaCas-1 protein, synthesize sgRNA (sgRNA1) shown in SEQ ID NO: 7 and sgRNA (sgRNA3) shown in SEQ ID NO: 9 using 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 targeting E. coli target DNA sequence (SEQ ID NO: 11) and HEK293 cell target DNA sequence (SEQ ID NO: 12) are designed according to the crRNA (denoted as sgRNA2 and sgRNA4, respectively), and the nucleotide sequences of the two sgRNAs are shown in SEQ ID NO: 8 (sgRNA2) and SEQ ID NO: 10 (sgRNA4), respectively. Synthesize sgRNA (sgRNA2) shown in SEQ ID NO: 8 and sgRNA (sgRNA4) shown in SEQ ID NO: 10 using conventional methods.

[0069] III. Detection of PAM preference of gene editing system NovaCas-1

[0070] The principle of detecting PAM preference is as follows: the abundance of PAM library without gene editing (negative control group) and the abundance of PAM library with gene editing (experimental group) are detected respectively, the change of PAM library abundance before and after gene editing is analyzed by bioinformatics method, and finally the PAM preference of gene editing system NovaCas-1 is obtained.

[0071] The detection method of PAM preference is as follows:

[0072] 1. Enzymatic digestion of plasmid

[0073] The PAM library plasmid obtained in the foregoing is subjected to enzyme digestion using HaeII restriction endonuclease to obtain linearized plasmid. The enzyme digestion system is as follows: 1 μg of PAM library plasmid, 5 μL of buffer, 1 μL of HaeII endonuclease, and sterile water is added to 50 μL. The enzyme digestion reaction conditions are as follows: 37°C for 1 h.

[0074] 2. Gel electrophoresis

[0075] The product obtained by enzyme digestion is subjected to electrophoresis on a 1% agarose gel at 120 V for 30 min.

[0076] 3. Recovery of plasmid

[0077] The 2676 bp DNA fragment on the gel is removed, and the plasmid is recovered using a gel recovery kit. Finally, the plasmid is eluted with ultrapure water to obtain the PAM library plasmid linearized DNA fragment.

[0078] 4. Determination of DNA concentration

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

[0080] 5. Co-incubation of NovaCas-1 protein and sgRNA1

[0081] 20 μL of DEPC water, 3 μL of buffer solution, 1 μL of 1 μM NovaCas-1 protein and 3 μL of 300 nM sgRNA1 are 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: 3 μL of PAM library plasmid linearized DNA fragment is added to the incubation product, shaken and mixed, and placed in a PCR instrument to run the gene editing program: 37°C for 30 min.

[0084] Negative control group: 3 μL PAM library plasmid linearized DNA fragments were added to 27 μL DEPC water, shaken and mixed, and placed in a PCR instrument to run the gene editing program: 37°C for 30 min.

[0085] 7. Proteinase inactivation

[0086] 1 μL proteinase K was added to the system of the experimental group and the negative control group respectively, mixed thoroughly, incubated at room temperature for 10 min, and then placed in a PCR instrument to run the proteinase 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 subjected to PCR amplification using primers containing different characteristic sequences (barcodes), wherein:

[0089] For the cleavage product of the experimental group, the nucleotide sequence of the PCR primer is shown in SEQ ID NO: 13 and SEQ ID NO: 14;

[0090] For the cleavage product of the negative control group, the nucleotide sequence of the PCR primer is shown in SEQ ID NO: 15 and SEQ ID NO: 16.

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

[0092] After shaking and mixing, it was placed in a PCR instrument to run the amplification program: 98°C for 30 s, 30 cycles of 98°C for 10 s, 65°C for 30 s, and 72°C for 10 s, followed by 72°C for 2 min and 4°C storage.

[0093] 9. Gel electrophoresis

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

[0095] 10. Recovering sample DNA

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

[0097] 11. Sequencing

[0098] 100 ng of DNA from different samples was mixed and then subjected to Illumina next-generation sequencing. The sequencing results were then analyzed using bioinformatics to obtain the final results. Figure 2 The results of PAM preference testing for the gene editing system NovaCas-1 (NovaCas-1 protein and sgRNA1 complex) are shown.

[0099] Depend on Figure 2 It is understood that the gene editing system NovaCas-1 (NovaCas-1 protein and sgRNA1 complex) provided by this invention can recognize not only TTTA, TTTC, and TTTG, which are recognized by existing CRISPR / Cas12a gene editing systems, but also ACTA, ACTC, ACTG, CCCA, CCCC, CCCG, CCTA, CCTC, CCTG, GCTA, GCTC, GCTG, TCAC, TCCA, TCCC, TCCG, TCGC, TCTA, TCTC, TCTG, TCTT, ATTA, ATTC, ATTG, CTAC, CTCA, CTCC, CTCG, CTGC, CTTA, CTTC, CTTG, CTTT, GTCA, GTTA, GTTC, GTTG, GTTT, TTAC, TTCA, TTCC, TTCG, TTCT, and TTGC. The algorithm can identify up to 47 PAM sequences, including GCTA, ATTC, CTTT, TTAC, GTTC, CCTC, CCCA, ACTG, TCCG, CCCC, CTCG, CTAC, GCTG, ACTC, TCGC, CTGC, GTTT, TCAC, GCTC, TCTT, CCCG, and GTCA. It exhibits low recognition activity for 21 PAM sequences, TTCG, TCCA, TTGC, CTCA, ACTA, CCTG, TCTC, CTTC, TCCC, and CTCC. It also exhibits high recognition activity for 10 PAM sequences, including TTTG, TTTA, TTTT, CTTA, TTCC, TTCA, CTTG, GTTA, TCTA, ATTA, ATTG, GTTG, TTCT, TCTG, TTTC, and CCTA.

[0100] Since sgRNA3 also contains the sequence of crRNA1, it can be determined that the NovaCas-1 protein and sgRNA3 complex can also recognize the above 47 PAM sequences.

[0101] In summary, the gene editing system NovaCas-1 (NovaCas-1 protein and sgRNA1 or sgRNA3 complex) provided by this invention greatly expands the target gene editing range of existing CRISPR / Cas12a gene editing systems.

[0102] IV. Testing the gene editing efficiency of the NovaCas-1 gene editing system

[0103] The gene editing system NovaCas-1 provided by this invention can perform gene editing in vitro and in cells. The cells include eukaryotic cells and prokaryotic cells. Eukaryotic cells include mammalian cells and plant cells. Prokaryotic cells include Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Pseudomonas aeruginosa, Pseudomonas putida, actinomycetes, and cyanobacteria.

[0104] The gene editing efficiency of the NovaCas-1 gene editing system provided in this invention was then tested using E. coli. Specifically, E. coli was used. galK The red-white screening system detects the gene editing efficiency of the NovaCas-1 gene editing system provided by this invention. The detection principle is as follows:

[0105] When E. coli galK Once the functional sequence on the gene is identified, the NovaCas-1 protein will... galK Genes are cut, causing double-strand breaks, which then lead to homologous recombination. galk Gene inactivation prevents E. coli from degrading galactose and producing acid, resulting in white colonies growing on MacConkey medium containing galactose. In contrast, E. coli that has not undergone gene editing still has the ability to degrade galactose and produce acid, resulting in red colonies growing on MacConkey medium containing galactose.

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

[0107] 1. Constructing Cas protein particles

[0108] The nucleotide sequence shown in SEQ ID NO:3 (the NovaCas-1 protein-coding gene after codon optimization) was synthesized and constructed into the pSC101 plasmid to obtain the pNovaCas-1 protein granule (concentration 100 ng / μL).

[0109] In order to enable the AsCas12a protein to be expressed efficiently in E. coli, the original nucleotide sequence of the AsCas12a protein coding gene is subjected to codon optimization treatment, and the nucleotide sequence after codon optimization treatment is shown in SEQ ID NO: 4. The nucleotide sequence shown in SEQ ID NO: 4 is synthesized and constructed into a pSC101 plasmid to obtain a pAsCas12a protein plasmid (concentration 100 ng / μL).

[0110] 2. Construction of sgRNA plasmid

[0111] In order to construct a plasmid containing sgRNA (sgRNA1, sgRNA2) and a homologous repair template, different primers are designed for sgRNA1 and sgRNA2, wherein:

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

[0114] The primers Oligo-F2 and Oligo-R2 are synthesized according to the nucleotide sequences shown in SEQ ID NO: 21 and SEQ ID NO: 22, and the primers Oligo-F3 and Oligo-R3 are 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) are annealed to obtain two double-stranded DNAs with sticky ends at both ends, and the annealing reaction system is as follows: 1 μL 100 μM Oligo-F2 (or Oligo-F3), 1 μL 100 μM Oligo-R2 (or Oligo-R3), 28 μL sterile water, fully mixed, and then placed in a PCR instrument to 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, 4℃ storage.

[0116] The annealed product and the previously obtained pUC19 plasmid linearized DNA fragment were connected under the action of DNA ligase. The connection system: 2 μL buffer, 50 ng pUC19 plasmid linearized DNA fragment, 20 ng annealed product, 1 μL T4 DNA ligase, and sterile water to 20 μL. The connection reaction condition: 16°C for 2h.

[0117] 1 μL of the connection product was subjected to chemical competent transformation, and the growing bacteria were subjected to Sanger sequencing verification.

[0118] The plasmid was extracted from the bacteria verified by sequencing to be correctly connected, obtaining the psgRNA1 plasmid (concentration 100 ng / μL) and the psgRNA2 plasmid (concentration 100 ng / μL).

[0119] 3. Escherichia coli transfected Cas protein plasmid

[0120] The previously obtained pNovaCas-1 protein plasmid and pAsCas12a protein plasmid were transformed into MG1655 competent cells (high-efficiency competent cells made of Escherichia coli MG1655) respectively, and the specific operation was as follows:

[0121] (1) 50 μL of MG1655 competent cells were taken into a centrifuge tube and placed in an ice bath, 1 μL of pNovaCas-1 protein plasmid or 1 μL of pAsCas12a protein plasmid was added to the cell suspension, and it was mixed by flicking and placed in an ice bath for 30 min.

[0122] (2) The centrifuge tube was placed in a 42°C water bath for 90 s, and then quickly transferred to an ice bath for 3 min.

[0123] (3) 950 μL of sterile LB liquid medium without antibiotics was added to the centrifuge tube, mixed, and then placed in a 37°C shaker at 150 rpm for 45 min to recover the Escherichia coli.

[0124] (4) The bacterial solution in the centrifuge tube was mixed, 100 μL of bacterial solution was taken to LB solid agar medium containing 34 μg / mL chloramphenicol, and a sterile spreader was used to spread it evenly. After the bacterial solution was absorbed by the medium, the plate was incubated at 30°C for 16 h.

[0125] (5) A sterile white gun head was used to pick a single colony from the plate and place it in 10 mL of LB liquid medium, which was incubated at 30°C on a shaker at 200 rpm for 16 h to amplify the Escherichia coli. The Escherichia coli transfected with the pNovaCas-1 protein plasmid was recorded as Escherichia coli Nova1, and the Escherichia coli transfected with the pAsCas12a protein plasmid was recorded as Escherichia coli As.

[0126] 4. E. coli competent cells for transfecting Cas protein plasmid

[0127] The method for preparing E. coli competent cells for transfecting Cas protein plasmid is as follows:

[0128] (1) E. coli Nova1 (bacterial liquid) and E. coli As (bacterial liquid) were respectively inoculated into 50 mL LB liquid medium at a inoculation amount of 1%, and cultured at 30°C on a shaker at 200 rpm until OD 600 = 0.55, and then immediately placed in an ice bath for 15 min.

[0129] (2) The bacterial liquid was centrifuged at 6000 rpm for 4 min at 4°C, and the precipitate was collected into a 50 mL centrifuge tube.

[0130] (3) 25 mL of pre-cooled 10% glycerol was added to the centrifuge tube, and the precipitate was resuspended in an ice bath.

[0131] (4) The bacterial liquid was centrifuged at 6000 rpm for 3 min at 4°C, and the precipitate was collected into a 50 mL centrifuge tube.

[0132] (5) 25 mL of pre-cooled 10% glycerol was added to the centrifuge tube again, and the precipitate was resuspended in an ice bath.

[0133] (6) The bacterial liquid was centrifuged at 6000 rpm for 3 min at 4°C, and the precipitate was collected into a 50 mL centrifuge tube.

[0134] (7) 200 μL of pre-cooled 10% glycerol was added to the centrifuge tube, and then 10% glycerol was added to a volume of 500 μL, respectively, to obtain E. coli Nova1 competent cells (bacterial liquid) and E. coli As competent cells (bacterial liquid).

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

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

[0137] (1) 1 mm electroporation cup was pre-cooled on ice for 30 min, and then 0.1 μg of psgRNA1 plasmid and psgRNA2 plasmid were added, respectively.

[0138] (2) Add 50 μL of E. coli Nova1 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, mix by flicking, then perform electroporation using a voltage of 1800 V. Immediately after electroporation, add 950 μL of sterile LB liquid medium without antibiotics, mix by flicking, then transfer to a centrifuge tube and incubate at 30°C on a shaker at 150 rpm for 2 h to recover the E. coli.

[0139] (3) Mix the bacterial solution in the centrifuge tube, and take 50 μL of the bacterial solution to MacConkey solid agar medium containing 34 μg / mL chloramphenicol, 100 μg / mL ampicillin, and 0.2% galactose, and spread the bacterial solution evenly on the medium with a sterile spreader. After the bacterial solution is absorbed by the medium, incubate the plate at 30°C for 16 h.

[0140] 6. Calculate the 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 E. coli in the colonies has not been edited (negative); if the colonies are white, it indicates that the E. coli in the colonies has been edited (positive).

[0142] Count the number of red and white colonies on the 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) x 100%

[0144] After calculation, the gene editing efficiency of the gene editing system NovaCas-1 (NovaCas-1 protein + sgRNA1) provided by the present application in E. coli is 98.42%, and the gene editing efficiency of the existing CRISPR / Cas12a gene editing system (AsCas12a protein + sgRNA2) is 48.13%. The comparison chart of the gene editing efficiencies of the two gene editing systems is shown in Figure 3 .

[0145] In summary, compared with the existing CRISPR / Cas12a gene editing system (AsCas12a protein + sgRNA2), the gene editing system NovaCas-1 (NovaCas-1 protein + sgRNA1) provided by the present application exhibits higher gene editing efficiency in E. coli.

[0146] Five, verify the gene editing activity of the gene editing system NovaCas-1 outside the cell

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

[0148] (1) Obtain E. coli genome: inoculate E. coli MG1655 into 3 mL LB liquid medium at an inoculation amount of 1%, and cultivate at 37°C in a shaker at 200 rpm for 12 h to amplify the E. coli, then extract the genome using a bacterial genome extraction kit, and then elute with ultrapure water to obtain the E. coli MG1655 genome. Finally, dilute the E. coli genome with ultrapure water to 500 ng / μL for standby (store in a -20°C refrigerator).

[0149] (2) Obtain in vitro cleaved double-stranded DNA substrate by PCR: use the forward primer and reverse primer shown in SEQ ID NO: 25 and SEQ ID NO: 26 to perform PCR amplification on the E. coli MG1655 genome. The PCR reaction system is as follows: 25 μL Q5High-Fidelity 2xMaster 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 make up to 50 μL. After shaking and mixing, place in a PCR instrument to run the amplification program: 98°C for 30 s, 30 cycles of 98°C for 10 s, 65°C for 30 s, and 72°C for 30 s, then 72°C for 2 min, and 4°C storage.

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

[0151] (4) Recover linear double-stranded DNA substrate: cut off the 3124 bp DNA fragment on the gel, and recover the sample DNA using a gel recovery kit, and finally elute with ultrapure water to obtain the linear double-stranded DNA substrate (template), which is diluted to a concentration of 225 ng / μL for standby (store in a -20°C refrigerator).

[0152] After preparing the linear double-stranded DNA substrate, verify the gene editing activity of the gene editing system NovaCas-1 outside the cell. The method for verifying the gene editing activity of the gene editing system NovaCas-1 outside the cell is as follows:

[0153] (1) Experimental group: mix 20 μL DEPC water, 3 μL buffer solution, 1 μL 1 μM NovaCas-1 protein, and 3 μL 300 nM gRNA1, and then place in a PCR instrument to run the incubation program: 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 mixed and then placed in a PCR instrument to run the incubation program: 25°C for 10 min.

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

[0156] (4) 1 μL proteinase K was added to the system, mixed and incubated at room temperature for 10 min, and then placed in a PCR instrument to run the proteinase inactivation program: 70°C for 30 min.

[0157] (5) 6 μL 6x loading buffer solution was further added to the system, mixed by blowing and electrophoresed on a 1% agarose gel at 120V for 30 min.

[0158] (6) The electrophoresis result was observed using a gel imaging instrument to preliminarily judge 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 is not cut, and the corresponding gene editing system has no activity;

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

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

[0162] The electrophoresis results of the experimental group (NovaCas-1 protein + sgRNA1), the control group (AsCas12a protein + sgRNA2) and the negative control group are shown in Figure 4 . It can be preliminarily judged that: Figure 4 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 not high.

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

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

[0165] It is calculated that the gene editing efficiency of the gene editing system (NovaCas-1 protein + sgRNA1) of the experimental group is 100%, and the gene editing efficiency of the gene editing system (AsCas12a protein + sgRNA2) of the control group is 47.2%. The comparison chart of the gene editing efficiencies of the two gene editing systems is as follows: Figure 5 .

[0166] In summary, compared with the existing CRISPR / Cas12a gene editing system (AsCas12a protein + sgRNA2), the gene editing system NovaCas-1 (NovaCas-1 protein + sgRNA1) provided by the present application shows higher gene editing efficiency in extracellular.

[0167] Six, verify the gene editing activity of the gene editing system NovaCas-1 in cells

[0168] The HEK293 cell line was plated on a 6-well plate and cultured to a cell density of 30% before verifying the gene editing activity. The HEK293 cell line was transfected with Cas protein and sgRNA before verifying the gene editing activity, and the AsCas12a protein and sgRNA4 complex were used as a control. The method for transfecting the HEK293 cell line with Cas protein and sgRNA is as follows:

[0169] (1) Plate the HEK293 cell line on a 6-well plate and culture to a cell density of 30%.

[0170] (2) Experimental group: Take 20 μg of the NovaCas-1 protein prepared in the foregoing and 2 μg of the sgRNA3 prepared in the foregoing and add them to 450 μL of Nucleofector culture medium, gently mix, and incubate the transfection solution at room temperature for 10 min.

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

[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 is sucked into the electroporation well plate, and the mixed transfection solution (experimental group transfection solution, control group transfection solution, negative control group transfection solution) is added respectively.

[0174] (6) The electroporation well plate is placed in the cell electroporator for electroporation. After the electroporation is completed, the cells are all sucked out and added to 500 μL of 37°C preheated Nucleofector medium, and cultured at 37°C in a 5% CO2 incubator for 24 h.

[0175] After the HEK293 cells are transfected with Cas protein (NovaCas-1 protein, AsCas12a protein) and sgRNA (sgRNA3, sgRNA4), the endogenous target gene editing activity is verified. The method for verifying the endogenous target gene editing activity is as follows:

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

[0177] (2) PCR amplification is performed using the forward primer and reverse primer shown in SEQ ID NO: 17 and SEQ ID NO: 18, and the PCR reaction system is as follows: 25 μL Q5 High-Fidelity 2x Master Mix, 1.25 μL 10 μM forward primer, 1.25 μL 10 μM reverse primer, 2 μL genomic DNA, and sterile water is added to 50 μL. After shaking and mixing, the amplification program is run in the PCR instrument: 98°C for 30 s, 30 cycles of 98°C for 10 s, 65°C for 30 s, and 72°C for 10 s, and then 72°C for 2 min, and 4°C storage.

[0178] (3) The 776 bp DNA fragment is purified from the PCR product using a PCR product purification kit, and the DNA concentration is determined using NanoDrop, and diluted to 200 ng / μL. The purified product is denatured and annealed. The reaction system is as follows: 1 μL purified product, 2 μL buffer solution, and sterile water is added to 20 μL. After shaking and mixing, the program (cooling rate 0.3°C / s) is run in the PCR instrument: 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 4°C storage (template).

[0179] (4) The template prepared in the foregoing is subjected to enzyme digestion using T7 EI enzyme, and the enzyme digestion product is subjected to electrophoresis using 2% agarose gel at 120 V for 30 min.

[0180] (5) Using a gel imager to observe the electrophoresis result, and preliminarily judging the gene editing activity of the gene editing system, specifically:

[0181] If there is only one band and the size is 776bp, it indicates that the template is not cut, and the corresponding gene editing system has no activity;

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

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

[0184] The electrophoresis results of the experimental group (NovaCas-1 protein + sgRNA3), the control group (AsCas12a protein + sgRNA4) and the negative control group are shown in Figure 6 . It can be preliminarily judged that: Figure 6 The gene editing system of the experimental group and the control group has gene editing activity, and the gene editing activity of the experimental group is slightly higher than that of the control group.

[0185] (6) The gray value of the template band on the gel can reflect the amount of template cutting, and the gene editing efficiency is calculated according to the gray value of the template band of each group by using the same method as before.

[0186] After calculation, the gene editing efficiency of the gene editing system (NovaCas-1 protein + sgRNA3) of the experimental group is 51.0%, and the gene editing efficiency of the gene editing system (AsCas12a protein + sgRNA4) of the control group is 31.7%. The comparison chart of the gene editing efficiencies 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-1 (NovaCas-1 protein + sgRNA3) provided by the present application shows higher gene editing efficiency.

[0188] Seven, other applications of the gene editing system NovaCas-1

[0189] The gene editing system NovaCas-1 provided by the present application can be used not only for gene editing, but also for detecting whether there is a target DNA sequence in double-stranded DNA.

[0190] When detecting whether there is a target DNA sequence in double-stranded DNA, a single-stranded DNA fluorescent reporter probe is needed, and the detection method is as follows:

[0191] The single-stranded DNA fluorescent reporter probe is contacted with the NovaCas-1 protein and sgRNA complex. When there is a target DNA sequence in the double-stranded DNA, the NovaCas-1 protein and gRNA complex accurately locates the target DNA sequence and produces a cleavage to cause a double-stranded break damage to the DNA. After completing the double-stranded DNA gene editing, the conformation of the NovaCas-1 protein changes, and the single-stranded DNA cleavage activity (“trans-cleavage” activity) is activated. The NovaCas-1 protein after the single-stranded DNA cleavage activity is activated will cut (degrade) the single-stranded DNA fluorescent reporter probe, thereby releasing the fluorescent group, and a strong fluorescence value can be detected using an enzyme marker instrument. When there is no target DNA sequence in the double-stranded DNA, the conformation of the NovaCas-1 protein will not change, the single-stranded DNA cleavage activity will not be activated, and the single-stranded DNA fluorescent reporter probe will not be cut (degraded), and the fluorescence signal detected using the enzyme marker instrument will not change (the fluorescence value will still be the basal value).

[0192] The method for verifying whether the NovaCas-1 gene editing system provided by the application can detect whether there is a target DNA sequence in double-stranded DNA is specifically as follows:

[0193] (1) Experimental group: 19 μL of DEPC water, 3 μL of buffer solution, 1 μL of 1 μM NovaCas-1 protein, 3 μL of 300 nsgRNA1, and 1 μL of single-stranded DNA fluorescent reporter probe (Guangzhou Meigene Biotechnology Limited, product number C009S, concentration 2 μM) are oscillated and mixed, and are transferred to a 96-well black enzyme marker plate;

[0194] (2) Control group: 19 μL of DEPC water, 3 μL of buffer solution, 1 μL of 1 μM AsCas12a protein, 3 μL of 300 nsgRNA2, and 1 μL of single-stranded DNA fluorescent reporter probe are oscillated and mixed, and are transferred to a 96-well black enzyme marker plate;

[0195] (3) 3 μL of the linear double-stranded DNA substrate prepared in the foregoing is added to the above system, oscillated and mixed, and then placed in an enzyme marker instrument for fluorescence detection. The reaction program is 37°C, 25 min, and fluorescence is collected every 0.5 min.

[0196] (4) Whether there is a target DNA sequence in the double-stranded DNA is judged according to the fluorescence signal change, specifically:

[0197] If the fluorescence intensity does not change (still the basal value) or does not change significantly with time, it indicates that the single-stranded DNA fluorescent reporter probe is not cut, and it indicates that there is no target 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 has been cleaved, suggesting that there is a target DNA sequence in the double-stranded DNA.

[0199] The fluorescence intensity curves of the experimental group (NovaCas-1 protein + sgRNA1) and the control group (AsCas12a protein + sgRNA2) as a function of cycle number (time) are shown in the figure. Figure 8 .Depend on Figure 8 It can be determined that the double-stranded DNA contains a target DNA sequence. The gene editing system in the experimental group can determine the presence of the target DNA sequence in the double-stranded DNA within 4 cycles (2 min), while the gene editing system in the control group can only determine the presence of the target DNA sequence in the double-stranded DNA after 20 cycles (10 min). Compared with the gene editing system in the control group, the gene editing system in 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 implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of this invention are still within the scope of protection of this invention.

Claims

1. A multi-PAM selected gene editing system NovaCas-1, characterized in that, The gene editing system NovaCas-1 is a NovaCas-1 protein and sgRNA complex, which can accurately locate and target a DNA sequence and produce a cut to cause a double-strand break damage to the DNA, wherein the amino acid sequence of the NovaCas-1 protein is shown as SEQ ID NO: 1, and the nucleotide sequence of the sgRNA is shown as SEQ ID NO: 7 or SEQ ID NO:

9.

2. The multiplex PAM selection gene editing system NovaCas-1 according to claim 1, characterized in that, The NovaCas-1 protein and sgRNA complex can accurately locate and target a DNA sequence, which means that the NovaCas-1 protein and sgRNA complex can accurately recognize and target a DNA sequence, or other proteins fused with the NovaCas-1 protein or proteins specifically recognizing the sgRNA are brought to the target DNA position.

3. The multiplex PAM selection gene editing system NovaCas-1 according to claim 2, characterized in that, The PAM sequence is ACTA, CCTA, CCTG, TCCA, TCCC, TCTA, TCTC, TCTG, ATTA, ATTG, CTCA, CTCC, CTTA, CTTC, CTTG, GTTA, GTTG, TTCA, TTCC, TTCG, TTCT, TTGC, TTTA, TTTC, TTTC, and TTTG.

4. The multiplex PAM selection gene editing system NovaCas-1 according to claim 3, characterized in that, The PAM sequence is CCTA, TCTA, TCTG, ATTA, ATTG, CTTA, CTTG, GTTA, GTTG, TTCA, TTCC, TTCT, TTT, TTTC, TTTC, and TTTG.

5. The multiplex PAM selection based gene editing system NovaCas-1 of claim 1, wherein, The NovaCas-1 protein is obtained by transcription and translation after codon optimization treatment of the nucleotide sequence of the original NovaCas-1 protein, wherein the nucleotide sequence of the original NovaCas-1 protein is shown as SEQ ID NO: 2, and the nucleotide sequence of the NovaCas-1 protein after optimization treatment is shown as SEQ ID NO:

3.

6. The multiplex PAM selection based gene editing system NovaCas-1 of claim 1, wherein, The NovaCas-1 protein and sgRNA complex can accurately locate and target a DNA sequence, which means that the NovaCas-1 protein and sgRNA complex can accurately recognize and target a DNA sequence, or other proteins fused with the NovaCas-1 protein or proteins specifically recognizing the sgRNA are brought to the target DNA position.

7. The multiplex PAM selection gene editing system NovaCas-1 according to claim 6, characterized in that, The other proteins fused with the NovaCas-1 protein or the proteins specifically recognizing the sgRNA can modify or regulate the target DNA sequence, and the modification or regulation includes regulation of gene transcription level and single base conversion.

8. The multiplex PAM selection gene editing system NovaCas-1 according to claim 7, characterized in that, The single base conversion includes conversion between adenine to guanine, cytosine to thymine or other bases.

9. Use of the multiplex PAM selection gene editing system NovaCas-1 of 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 or treatment.

10. Use of the multiplex PAM selection gene editing system NovaCas-1 of claim 1 in rapid detection of double-stranded DNA for the presence or absence of a targeted DNA sequence, characterized in that, The single-stranded DNA fluorescent reporter probe is contacted with the NovaCas-1 protein and sgRNA complex, and whether the double-stranded DNA has the target DNA sequence is judged according to the change of the fluorescent signal, and the specific steps are as follows: When the double-stranded DNA has the target DNA sequence, the single-stranded DNA cleavage activity of the NovaCas-1 protein is activated, the single-stranded DNA fluorescent reporter probe is degraded to release the fluorescent group, and the fluorescent signal is enhanced. When the double-stranded DNA does not have the target DNA sequence, the single-stranded DNA cleavage activity of the NovaCas-1 protein is not activated, and the fluorescent signal does not change.

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