High-efficiency micro CRISPR / Cas9 gene editing system and application

Abstract

The application belongs to the technical field of genetic engineering, and discloses a high-efficiency micro CRISPR / Cas9 gene editing system and application. The amino acid sequence of the Cas9 protein is shown as SEQ ID NO. 1, or is a sequence in which at least one amino acid in the amino acid sequence shown as SEQ ID NO. 1 is replaced, deleted or inserted. The Cas9 protein of the application can maximize the simplification of the protein structure while maintaining the core catalytic activity, can improve the editing efficiency in different application scenarios, can effectively target the genomic region where the non-standard PAM sequence is located, and can improve the editing capacity of the CRISPR system in the complex genome. The application provides a new idea for the diversified development of gene editing technology.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and in particular to a high-efficiency micro CRISPR / Cas9 gene editing system and its application. Background Art

[0002] CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a natural immune system found in bacteria and archaea that has been widely used in the field of gene editing. Among them, the CRISPR / Cas9 system is one of the most widely used gene editing tools. The working principle of the CRISPR / Cas9 system involves several key steps: (1) Design and synthesis of single guide RNA (sgRNA): gRNA consists of two parts: CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). crRNA contains a guide sequence that can pair with the target gene sequence, and tracrRNA helps crRNA bind to the Cas9 nuclease; (2) Guidance of Cas9 nuclease: Cas9 nuclease can recognize and bind to the target gene sequence through the guide sequence in the gRNA; (3) Cutting of the target gene sequence: Cas9 bound to the target sequence will use its nuclease activity to cut the double-stranded DNA; (4) DNA repair process: The cut DNA can be repaired through non-homologous end joining (NHEJ) or homologous recombination (HR) mechanisms in the cell. Both mechanisms can lead to gene editing outcomes such as gene knockout or gene insertion.

[0003] The key advantages of the CRISPR / Cas9 system include strong targeting, ease of use, and low cost. Compared to previous gene-editing technologies, such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), CRISPR / Cas9 is more flexible and efficient. Since its initial application in human cell line gene editing in 2013, CRISPR / Cas9 has demonstrated tremendous potential in basic research, medical applications, and agricultural improvement. For example, CRISPR / Cas9 can precisely edit disease-causing genes, providing new treatment options for genetic diseases. It can also be used to create new crop varieties and improve agricultural production efficiency. CRISPR / Cas9 technology has become one of the most active and promising gene-editing tools. With continued advancements in the technology and expanded applications, CRISPR / Cas9 is poised to play an even more important role in the future.

[0004] However, the size limitation of the Cas9 protein is a key challenge in the application of the CRISPR / Cas9 system. As the most commonly used CRISPR nuclease, SpCas9's molecular weight of approximately 160 kDa means its coding sequence is 4.2 kb long, presenting significant challenges in gene therapy delivery systems. This is particularly true when using the most commonly used adeno-associated virus (AAV) vector. With a packaging capacity of only 4.7 kb, AAV is barely able to accommodate the SpCas9 gene, promoter, terminator, and necessary sgRNA expression components. This size limitation directly impacts the efficacy of in vivo treatments. For example, in the treatment of Duchenne muscular dystrophy, researchers have had to deliver SpCas9 separately using two AAV vectors, which not only increases treatment costs but also reduces overall efficacy. Furthermore, the large size of SpCas9 significantly impacts nuclear pore transport efficiency and cell transfection efficiency, a problem particularly acute in certain difficult-to-transfect cell types, such as primary neurons.

[0005] PAM sequence dependence is another significant limitation of Cas9. SpCas9 requires a PAM sequence (NGG) at the 3' end of the target sequence. This characteristic, originating from the need for bacterial defense systems to distinguish between native and foreign DNA, poses a significant limitation in genome editing applications. While NGG sequences occur on average every 8-12 bases in the human genome, this distribution is not uniform. In practical applications, researchers often encounter a lack of suitable PAMs near the ideal cleavage site. For example, when attempting to repair sickle cell anemia pathogenic mutations via homologous recombination, the NGG sequence closest to the mutation site may be located far from the mutation site, significantly reducing repair efficiency. To overcome this limitation, researchers have engineered various SpCas9 variants, such as SpCas9-NG, which recognizes the NG PAM, and xCas9, which recognizes a wider range of PAM variants. However, the increased flexibility of these variants often comes with reduced activity and the potential for increased off-target effects. For example, while SpCas9-NG significantly expands the range of editable sites, its editing efficiency is generally lower than that of wild-type SpCas9 and exhibits higher off-target rates in certain sequence contexts. This trade-off between efficiency, specificity, and flexibility has become a difficult problem that needs to be continuously optimized in the current development of CRISPR technology. Summary of the Invention

[0006] The purpose of the present invention is to overcome the shortcomings of the existing technology and provide an efficient micro CRISPR / Cas9 gene editing system and its application, so as to overcome the technical limitations of the existing CRISPR / Cas9 gene editing system in terms of Cas9 protein size and PAM dependence.

[0007] To achieve the above object, the technical scheme adopted by the present application is as follows:

[0008] In a first aspect, the present application provides a Cas9 protein, whose amino acid sequence is shown in SEQ ID NO. 1; or a sequence obtained by replacing, deleting or inserting at least one amino acid from the amino acid sequence shown in SEQ ID NO. 1.

[0009] The present application screens a novel Cas nuclease with a molecular weight of no more than 100 kDa from naturally occurring Cas family proteins, ensures that the length of its coding sequence is controlled within 3.5 kb, and ensures that its protein structure is simplified to the maximum extent while maintaining core catalytic activity. On the one hand, the present application further optimizes the function of small proteins and directs protein modification to improve editing activity and targeting specificity. On the other hand, the miniaturized Cas9 protein is more compatible with various delivery vectors, especially AAV, which has a limited packaging volume; it is also suitable for non-viral delivery systems (such as lipid nanoparticles), which can improve its editing efficiency in different application scenarios.

[0010] The Cas nuclease with diversified PAM recognition in the present application can effectively target the genomic region where the non-standard PAM sequence is located, improving the editing ability of the CRISPR system in complex genomes. This provides the possibility of covering more difficult-to-edit genomic regions and solves the limitations of target selection in traditional systems. This wide-targeting Cas nuclease system not only has important significance in medical fields such as gene therapy and functional gene research, but also can be widely applied in animal and plant genetic modification and synthetic biology fields, providing new ideas for the diversification of gene editing technology.

[0011] As a preferred embodiment of the Cas9 protein described in the present application, the replacement includes at least one of S48R, Y64R, Q74R, D79R, G96R, Y100R, Q125R, I176R, G178R, V179R, Q220R, Y464R, E476R, I766R, S767R, G768R, L770R, I775R, G777R, S779R, D806R, L840R, P849R, N851R, T852R, D870R, N873R, E1008R, D772R, P795R, K797R, E800R, D835R, V857R, P863R, D772K, P795K, E800K, D835K, V857K, P863K.

[0012] As a preferred embodiment of the Cas9 protein described in the present invention, the replacement is a double-site mutation; the double-site mutation is any one of N851R and S767R, N851R and D772R, N851R and P795R, N851R and K797R, N851R and E800R, N851R and D835R, N851R and V857R, N851R and P863R, N851R and D772K, N851R and P795K, N851R and E800K, N851R and D835K, N851R and V857K, and N851R and P863K.

[0013] As a preferred embodiment of the Cas9 protein described in the present invention, the PAM sequence specifically recognized by the Cas9 protein is NRRRAY; the N = A or C or G or T; the R = A or G; the Y = C or T; the A is adenine in DNA bases, the T is thymine in DNA bases, the C is cytosine in DNA bases, and the G is guanine in DNA bases.

[0014] In a second aspect, the present invention provides a nucleic acid molecule encoding the Cas9 protein.

[0015] In a third aspect, the present invention provides a highly efficient micro-CRISPR / Cas9 gene editing system, comprising the Cas9 protein.

[0016] As a preferred embodiment of the efficient micro-CRISPR / Cas9 gene editing system described in the present invention, it also includes a DR sequence with a nucleotide sequence as shown in SEQ ID NO.2.

[0017] As a preferred embodiment of the efficient miniature CRISPR / Cas9 gene editing system of the present invention, it also includes a tracrRNA sequence as shown in SEQ ID NO.3.

[0018] As a preferred embodiment of the efficient micro-CRISPR / Cas9 gene editing system described in the present invention, it also includes a scaffold sequence such as the nucleotide sequence shown in SEQ ID NO.5.

[0019] In a fourth aspect, the present invention applies the Cas9 protein, the nucleic acid molecule, and the efficient miniature CRISPR / Cas9 gene editing system to gene editing and / or gene delivery, including gene editing and / or gene delivery in prokaryotic and eukaryotic systems.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. Discovery and characterization of an innovative and efficient micro-CRISPR / Cas9 system

[0022] Using bioinformatics methods, this study successfully developed and validated a novel CRISPR / Cas9 gene editing system (28c19). The core components of this system include the Cas9 protein, the CRISPR array, and the tracrRNA element. Through sophisticated prokaryotic depletion and interference experiments, the system's specific PAM sequence recognition mechanism was precisely defined: 28c19 recognizes NR(A / G)RRAY(C / T). This system exhibits stable and efficient DNA cleavage in a prokaryotic environment.

[0023] 2. Accurate optimization of Scaffold sequence

[0024] The present invention systematically optimized the 28c19 scaffold, rigorously screening to determine the optimal DR length, tracrRNA range, and spacer length, ultimately designing a highly refined scaffold sequence. This optimization significantly improved the accuracy and overall performance of the gene editing system.

[0025] 3. Protein engineering drives a leap in editing efficiency

[0026] After validating the system's basic functionality in a eukaryotic environment, the researchers engineered the 28c19 protein using an arginine / lysine substitution strategy, significantly improving its editing efficiency. The optimized 28c19 variant achieved a 100% improvement in editing efficiency compared to the original protein, reaching twice that of the original system. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a protein domain partition diagram of 28c19 described in the present invention.

[0028] Figure 2 This is a prokaryotic PAM sequence identification diagram of the 28c19 gene editing system of the present invention; Figure 2 In the figure, A shows the prokaryotic PAM recognition sequence of the 28c19 gene editing system, which is NR(A / G)RRAY(C / T), showing a complex and broad PAM preference; B shows the prokaryotic interference experiment results of 28c19 on four PAM sequences.

[0029] Figure 3 Diagram showing the exploration of the scaffold (A), spacer (B) and DR (C) of the 28c19 gene editing system described in the present invention;

[0030] Figure 4 This is a diagram of the editing efficiency of the 28c19 protein after evolution according to the present invention; Figure 4In the figure, A is the editing efficiency diagram of a single point mutation; B is the editing efficiency diagram of a double point mutation. DETAILED DESCRIPTION

[0031] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. Those skilled in the art should understand that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0032] Unless otherwise specified, the experimental methods used in the examples are conventional methods; the materials, reagents, etc. used are all available from commercial sources unless otherwise specified.

[0033] Example 1: Bioinformatics mining of small CRISPR / Cas9 gene editing systems

[0034] (1) Data acquisition and preparation

[0035] Use public databases (e.g., NCBI, UniProt, PDB) to obtain sequence and structural information for known Cas9 systems. Collect unannotated sequences from prokaryotic microbial genomes, particularly metagenomic data from viruses, microorganisms, and small bacteria, to screen and discover novel and small CRISPR systems.

[0036] (2) Sequence analysis and comparison

[0037] Compile CRISPR-related sequences using tools such as BLAST or Clustal Omega to identify potential small Cas proteins, with sequence coverage ≥50% and sequence similarity ≥30%. Use hidden Markov model (HMM) tools such as HMMER to identify small proteins with similar functions, with a minimum contig length of ≥500 bp. Use CRISPRFinder with spacer lengths between 26 and 50 bp and DR lengths between 20 and 50 bp.

[0038] (3) Evolutionary analysis

[0039] By identifying key catalytic residues (such as active sites for DNA cleavage), an evolutionary tree of small CRISPR-associated proteins was constructed to understand their evolutionary relationships and infer their functions.

[0040] By setting the above parameters to search for new small CRISPR systems, we finally identified a miniaturized Cas9 editing system named 28c19. The 28c19 protein encodes 1008 amino acids (SEQ ID NO. 1), and its structural characteristics are as follows Figure 1The DR sequence of the 28c19 gene editing system is shown in SEQ ID NO.2; the tracrRNA sequence is shown in SEQ ID NO.3. Figure 1 As shown, like other Cas9s, 28c19 has HNH and RuvC catalytic domains, which cut the two strands of the target DNA respectively.

[0041] Example 2: Identification and Verification of the PAM Sequence of the 28c19 Gene Editing System

[0042] The identification and verification of PAM sequences are crucial for the function of the CRISPR / Cas system. In this example, the PAM sequence of the novel protein 28c19 was identified based on prokaryotic PAM depletion. The specific steps are as follows:

[0043] (1) Construction of prokaryotic codon-optimized 28c19 vector

[0044] Based on the amino acid sequence shown in SEQ ID NO. 1, the nucleotide sequence of 28c19 was codon-optimized for E. coli and constructed into the prokaryotic expression vector pET28a (Kana-resistant) for protein expression in E. coli. This vector also contained the DR and tracrRNA sequences necessary for 28c19's editing function, resulting in the recombinant vector pET28a-28c19.

[0045] (2) Synthesis and construction of PAM library

[0046] A random PAM library was designed, containing a six-base sequence (NNNNNN) with each site containing a random combination of four bases (A, T, C, G), covering all 4096 possible PAM sequence combinations (4*4*4*4*4*4). After amplification, the library was inserted into the pUC19 vector (Amp-resistant). This library contained a 30-bp spacer sequence (shown in SEQ ID NO. 4) at the 5' end of the six-base sequence. To verify the randomness and uniformity of the library, the distribution of PAM sequences was analyzed by high-throughput sequencing to ensure that all 4096 possible sequences were evenly distributed within the library. The Gini coefficient of the PAM library used in this example was less than 0.2, indicating good abundance.

[0047] (3) PAM depletion experiment

[0048] The pet28a-28c19 vector was transformed into DH5a competent cells to obtain a single clone strain containing the pet28a-28c19 sequence, and the competent cells were prepared by different gradients of glycerol. 200 ng of the 6N library was electroporated into the pet28a-28c19 competent cells, incubated at 30°C for 2 h, and 1.5 mL of the recovered culture medium was evenly coated on the Amp and Kana double-antibiotic plates and incubated at 30°C for 30 h. All the colonies were scraped and the mixed plasmids were extracted, and named 28c19-PAM.

[0049] (4) High-throughput sequencing and PAM analysis

[0050] NGS primers were designed upstream and downstream of the 6N position, and the 28c19-PAM plasmid was used as a template for amplification. After magnetic bead purification, Illumina sequencing was performed to ensure sufficient sequencing depth to cover all PAM sequences in the random library. The same primer amplification and sequencing were performed on the empty vector as a control group (without Cas system), and the empty vector group was used to correct the randomness bias of the experimental group. The relative abundance of each PAM sequence was calculated as the ratio of the experimental group abundance to the control group abundance. PAM sequences with significantly reduced relative abundance (highest depletion value, top 10%) were selected as candidate PAMs for Cas system recognition and cleavage. The high-depletion PAM sequences selected were subjected to multiple sequence alignment to extract conserved sequence features. The WebLogo 3 tool was used to generate a visual chart of the sequence, which intuitively showed the sequence preference of the PAM site.

[0051] (5) Verification of PAM sequences

[0052] The PAM results selected in step (4) were constructed into single PAM sequences, and the 28c19 sequences to be verified included NAGAAC, NGAAAC, NAGGAC, and NAGAAT. Referring to step (3), the pet28a-28c19 competent cells were prepared, and 100 ng of each of the four PAM plasmids was electroporated into the competent cells. After recovery, 100 μL of the bacterial solution was diluted by 10-fold (1:10, 1:100, 1:1000). The diluted solution was added dropwise to LB plates containing Amp and Kana double antibiotics, and incubated at 30°C for 30 h. The number of colonies at each dilution gradient was counted. The control group was the empty PAM plasmid, which could not be cleaved. The fewer the colonies, the higher the cleavage efficiency of Cas9 to the PAM sequence.

[0053] The results are shown in Table A. Figure 2 Figure 2 Table A shows that the prokaryotic PAM recognition sequence of the 28c19 gene editing system is NR (A / G) RRAY (C / T), which exhibits complex and broad PAM preference. Figure 2 ​Figure B shows the results of a prokaryotic interference experiment using 28c19 against four PAM sequences. In the control group, because Cas9 was unable to recognize and cleave DNA, the bacteria retained dual resistance, resulting in normal bacterial growth across all dilution gradients, ensuring the reliability of the interference results. In the experimental group, 28c19 recognized the four PAM sequences NAGAAC, NGAAAC, NAGGAC, and NAGAAT, exhibiting significant cleavage activity. The number of colonies in the dilution gradient was significantly reduced or completely absent, indicating that 28c19 has high cleavage activity against all four sequences. These results confirm the PAM recognition sequence of 28c19 and confirm its applicability in prokaryotic gene editing systems.

[0054] Example 3: Determination of mature scaffolds of the 28c19 gene editing system

[0055] In the CRISPR / Cas9 system, the sgRNA scaffold plays a key role in gene editing efficiency. This example aims to optimize the scaffold design of the 28c19 system and determine its optimal length and range to improve editing performance. The specific steps are as follows:

[0056] (1) DR sequence and length optimization

[0057] Based on the 35bp DR sequence identified in Example 1 (shown as SEQ ID NO. 2), a series of candidate truncated DR versions were designed and generated by progressively truncating nucleotides of varying lengths from its 3' end. For the 28c19 system, the designed truncated versions included 18 bp, 20 bp, 21 bp, 22 bp, 24 bp, 26 bp, 28 bp, 30 bp, and a complete 35 bp sequence. Each truncated DR version was constructed into a plasmid and combined with tracrRNA to form a complete sgRNA scaffold structure. Subsequently, the effects of these different sgRNA scaffold versions on gene editing efficiency were tested in cell-based experiments to identify the optimal DR length.

[0058] (2) Spacer sequence and length optimization

[0059] Using the 30 bp spacer sequence used in Example 2 as a reference, truncated versions were designed by gradually reducing the number of bases. Combined with the optimal DR and tracrRNA sequences, their effects on editing efficiency were evaluated. For the 28c19 system, truncated versions were 10 bp, 11 bp, 12 bp, 13 bp, and 15 bp. The binding of spacers of varying lengths to the target DNA was tested to identify the spacer length that exhibited the best editing efficiency.

[0060] (3) Construction of eukaryotic expression of 28c19

[0061] The amino acid sequence of 28c19 described in Example 1 was eukaryotic codon optimized and then constructed into the PX330 expression vector, driven by the CBh promoter, and named PX330-28c19.

[0062] (4) sacffold test

[0063] Combining length-optimized DR and tracrRNA, various candidate scaffolds were constructed, and their editing efficiency was assessed using a fluorescent reporter system (SSA-GFP). The SSA-GFP system induces restoration of green fluorescence by interrupting repair of the GFP gene. The intensity of green fluorescence directly reflects the cleavage efficiency of the CRISPR system. The optimized scaffolds were co-transfected with PX330-28c19 into cells, and the fluorescence signal was measured to quantify the editing efficiency.

[0064] (5) RNA secondary structure prediction

[0065] After determining the optimal DR and spacer sequences, the RNAfold tool was used to model and analyze the secondary structure of the mature sgRNA. The stability of the secondary structure was predicted to further verify the rationality of the optimized sgRNA scaffold design.

[0066] See the results Figure 3 :like Figure 3 As shown in Figure A, the RNA secondary structure model of the optimal scaffold of the 28c19 gene editing system. Figure 3 As shown in Figure B, the optimal spacer length for the 28c19 system is 20 bp. Figure 3 As shown in Figure C, the optimal DR length of the 28c19 system is 13 bp. The mature scaffold sequence of the 28c19 system is shown in SEQ ID NO.5

[0067] Example 4: Protein evolution of 28c19

[0068] To improve the performance of the 28c19 system in gene editing, a systematic protein engineering strategy was used to structurally optimize the Cas9 protein. The specific methods are as follows:

[0069] (1) Screening of key amino acid sites

[0070] By comparing the structure of the Cas9 protein with that of the homologous protein, the key amino acid sites for binding to the sgRNA and target DNA were precisely located. Twenty-eight sites in 28c19 were selected as targets for protein modification, including S48R, Y64R, Q74R, D79R, G96R, Y100R, Q125R, I176R, G178R, V179R, Q220R, Y464R, E476R, I766R, S767R, G768R, L770R, I775R, G777R, S779R, D806R, L840R, P849R, N851R, T852R, D870R, N873R, and E1008R.

[0071] (2) Single-point mutation strategy

[0072] At each selected site, the original amino acid codon was replaced with the arginine (R) coding sequence (AGG). The resulting single-point mutant vector was named PX330-28c19-R, and the original unmutated vector (WT) was used as a control.

[0073] (3) Construction of double-site mutation vector

[0074] Based on the screening results of single-point mutations, double-point combined mutations were performed at sites that significantly improved editing efficiency. In addition to arginine (R), lysine (K) mutations were also explored, including N851R and S767R, N851R and D772R, N851R and P795R, N851R and K797R, N851R and E800R, N851R and D835R, N851R and V857R, N851R and P863R, N851R and D772K, N851R and P795K, N851R and E800K, N851R and D835K, N851R and V857K, and N851R and P863K. Double-point mutation vectors PX330-28c19-2R or PX330-28c19-K / R were constructed. Designed to improve protein performance using positively charged amino acids.

[0075] (4) Editorial efficiency evaluation

[0076] The gene editing efficiency of each mutant was quantified by flow cytometry using the SSA-GFP fluorescent reporter system. This system evaluates editing performance by measuring the ratio of green fluorescence after repair of target DNA breaks, based on the expression of green fluorescent protein.

[0077] Result analysis:

[0078] (1) Single point mutation effect

[0079] Among the 28 sites in 28c19, 7 sites showed improved efficiency, and the editing efficiency of the 5 stronger mutation sites (S767R, P849R, N851R, T852R and N873R) increased by about 2 times.

[0080] (2) Multi-point mutation effect

[0081] The effect of single-point mutations on 28c19 was significantly improved. Based on these single-point mutations, various double-point mutation combinations were then tested. Experiments showed that some combinations (such as N851R / S767R, N851R / K797R, N851R / E800R, and N851R / E800K) further improved editing efficiency.

[0082] In summary, through protein evolution, 5 single mutants and 8 double arginine / lysine mutants with the strongest editing efficiency increased by about 2 times were evolved on 28c19.

[0083] The present invention screens naturally occurring Cas family proteins to screen for novel Cas nucleases with a molecular weight not exceeding 100 kDa, ensuring that the length of their coding sequences is controlled within 3.5 kb, ensuring that the protein structure is simplified to the greatest extent while maintaining its core catalytic activity. On the one hand, the present invention further optimizes the functions of these small proteins and conducts targeted protein modification to enhance editing activity and targeting specificity. On the other hand, miniaturized Cas9 proteins are more compatible with a variety of delivery vectors, especially AAV vectors with limited packaging volume; they are also suitable for non-viral delivery systems (such as lipid nanoparticles), which can improve their editing efficiency in different application scenarios.

[0084] The Cas nucleases of the present invention with diverse PAM recognition can effectively target genomic regions containing non-standard PAM sequences, enhancing the editing capabilities of CRISPR systems in complex genomes. This opens up the possibility of covering more difficult-to-edit genomic regions and overcomes the limitations of traditional systems in target selection. This broadly targeted Cas nuclease system is not only of great significance in medical fields such as gene therapy and functional gene research, but can also be widely applied in the fields of genetic modification of plants and animals and synthetic biology, providing new ideas for the diversified development of gene editing technology.

[0085] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A Cas9 protein, characterized in that The amino acid sequence is a sequence obtained by subjecting the amino acid sequence shown in SEQ ID NO. 1 to single-site mutation or double-site mutation; The single site mutation is any one of S767R, P849R, N851R, T852R and N873R; The double-site mutation is any one of N851R and S767R, N851R and K797R, N851R and E800R, and N851R and E800K.

2. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the Cas9 protein according to claim 1.

3. An efficient micro-CRISPR / Cas9 gene editing system, characterized in that: Comprising the Cas9 protein of claim 1.

4. The efficient micro CRISPR / Cas9 gene editing system according to claim 3, characterized in that It also includes the DR sequence shown as the nucleotide sequence of SEQ ID NO.

2.

5. The efficient micro CRISPR / Cas9 gene editing system according to claim 3, characterized in that It also includes a tracrRNA sequence such as the nucleotide sequence shown in SEQ ID NO.

3.

6. The efficient micro CRISPR / Cas9 gene editing system according to claim 3, characterized in that It also includes a scaffold sequence such as the nucleotide sequence shown in SEQ ID NO.

5.

7. Use of the Cas9 protein according to claim 1, the nucleic acid molecule according to claim 2, or the efficient miniature CRISPR / Cas9 gene editing system according to any one of claims 3 to 6 in gene editing and / or gene delivery for purposes other than diagnosis and treatment of diseases.

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