Application of fusion protein KSa-SspCas9 and mutant thereof in gene editing

Abstract

The invention discloses application of a fusion protein KSa-SspCas9 and a mutant thereof in gene editing, and belongs to the field of genetic engineering. SspCas9 protein in Staphyloccus sp.17KM0847 bacteria is excavated through analysis of bioinformatics, the protein has PAM recognition capability of NNG, a PI structural domain of the SspCas9 protein is fused with a non-PI structural domain of the SaCas9 protein to obtain the fusion protein KSa-SspCas9, and the fusion protein KSa-SspCas9 is used for gene editing. The fusion protein has higher base editing efficiency. Meanwhile, the specific site of the fusion protein is mutated, and the PAM recognition range is greatly widened through PAM sequences of NRN and NYN which can be recognized by mutants. Through discovery of the Cas9 protein and the gene editing system, the variety of gene editing tools is expanded, and the Cas9 protein and the gene editing system have wide application prospects in the aspects of disease treatment, animal model preparation, plant breeding and the like.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and in particular to the application of a fusion protein KSa-SspCas9 and a mutant thereof in gene editing. Background Art

[0002] The CRISPR / Cas9 system (Clustered Regularly Interspaced Palindromic Repeats / CRISPR-associated proteins system) is a natural immune system from archaea and bacteria that protects bacteria and archaea from invasion by foreign viruses or plasmids. Its working principle is: when a virus invades certain bacteria, the bacteria can integrate a small segment of the virus's gene into its own DNA. When the virus invades again, the bacteria can recognize the viral gene and cut its gene to silence its expression, thereby achieving the effect of resisting the virus.

[0003] The three stages of the CRISPR / Cas9 system function mainly include the adaptation stage (Adaptation), expression and maturity stage (Expression) and interference stage (Interference). In the adaptation stage, bacteria recognize the PAM sequence in the invading nucleic acid through specific Cas proteins and integrate the DNA fragment adjacent to the PAM sequence into their own CRISPR sequence; in the expression and maturity stage, the CRISPR array is transcribed into pre-crRNA, which is further processed into mature crRNA. The crRNA sequence is complementary to the exogenous DNA fragment, and the Cas protein is expressed at the same time; in the interference stage, crRNA and Cas9 protein form a ribonucleoprotein complex (RNP), the crRNA-Cas9 complex recognizes and binds to the PAM site of the target DNA, and the Cas9 protein cuts the target DNA to produce DBS, which triggers the repair mechanism in the cell, non-homologous end-joining (NHEJ) and homologous recombination (homologous end-joining).

[0004] NHEJ repair can cause deletion or insertion of bases, which can be used for gene knockout; when a homologous template is provided, HR repair can be used for site-specific insertion of genes and precise replacement of bases.

[0005] As a third-generation editing tool, compared with the first-generation ZFNs and the second-generation TALENs, it has the advantages of simpler design, higher editing efficiency and wider application range, becoming the most mainstream gene editing system today. Among them, the most widely used is the type II CRISPR / Cas gene editing system. In the prior art, the type II system derived from Streptococcus pyogene (SpCas9) and Staphylococcus aureus (SaCas9) has a higher cutting efficiency. However, SaCas9 (1053 amino acid residues) is significantly smaller than SpCas9 (1368 amino acid residues), so it is easier to be delivered to somatic tissues for genome editing. In addition, Cas9 needs to recognize a specific PAM, among which SpCas9 recognizes the PAM of NGG and SaCas9 recognizes the PAM of NNGRRT. The specificity of PAM recognition also limits the application of Cas9. Therefore, based on the existing type II CRISPR / Cas9 system, it is urgent to develop a new CRISPR / Cas gene editing system with stronger editing ability and smaller molecular weight. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a Cas protein and CRISPR / Cas gene editing system with stronger editing ability.

[0007] The technical solution of the present invention is: a fusion protein KSa-SspCas9, whose amino acid sequence is shown in SEQ ID No.3.

[0008] A gene encoding the fusion protein KSa-SspCas9 described above.

[0009] A mutant of the fusion protein KSa-SspCas9, whose amino acid sequence is shown in SEQ ID No.4.

[0010] A gene encoding the mutant described above.

[0011] A vector containing a gene encoding a fusion protein KSa-SspCas9 or a mutant thereof.

[0012] A cell comprising the above-mentioned vector.

[0013] The application of the above-mentioned fusion protein KSa-SspCas9 or mutant or protein SspCas9 with the amino acid sequence shown in SEQ ID No.1 in the CRISPR-Cas9 gene editing system.

[0014] Furthermore, the CRISPR-Cas9 gene editing system is a single base editor.

[0015] A conjugate, comprising the fusion protein KSa-SspCas9 or mutant or protein SspCas9 with the amino acid sequence shown in SEQ ID No. 1 as described above.

[0016] The conjugate described above may also include a) a modification portion; for example, the modification portion is selected from another protein or polypeptide, a detectable label or a combination thereof; for example, the other protein or polypeptide is selected from one or more of cytosine deaminase (CBE), adenine deaminase (ABE), cytosine methyltransferase DNMT3A and MQ1, cytosine demethylase Tet1, transcription activator protein VP64, p65 and RTA, and transcription repressor protein KRAB; or / and b) an optional linker or non-covalent coupling system for connecting the Cas9 protein to the modification portion; for example, the linker is a linker with a length of 1-50 amino acids; the non-covalent coupling system includes RNA / polypeptide interaction systems based on MS2, PP7, boxB, etc. and polypeptide / polypeptide interaction systems based on GCN4 / scFV, etc.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) A type II CRISPR / Cas9 gene editing system of the present invention has a smaller molecular weight than SpCas9 (1368 amino acid residues), which is 1052 amino acid residues; at the same time, the type II CRISPR / Cas9 gene editing system of the present invention can recognize a specific NNG PAM sequence, greatly increasing the targetable range and overcoming the limitations of SpCas9.

[0019] (2) The KSa-SspCas9 fusion protein and its variants of the present invention have stronger editing efficiency than SspCas9 in base editing. At the same time, the Sa-SspCas9-KKH variant recognizes a wider range of NNN PAM sequences and has a higher editing efficiency for NRN than for NYN.

[0020] (3) Type II CRISPR / Cas9 and its variant base editors using the Cas9 protein of the present invention can perform gene editing functions in mammalian cells under the guidance of sgRNA. The discovery of the Cas9 protein and its variant gene editing system of the present invention has expanded the types of gene editing tools and played an important role in promoting the application of gene therapy in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the AIphaFold2 result prediction diagram of the SspCas9 protein of the present invention.

[0022] Figure 2Schematic diagram of the structure of the fusion protein mutant Sa-SspCas9-KKH.

[0023] Figure 3 PAM requirements for the CRISPR / Cas9 gene editing system composed of SspCas9.

[0024] Figure 4 PAM requirements for the CRISPR / Cas9 gene editing system composed of KSa-Sspcas9.

[0025] Figure 5 PAM requirements for the CRISPR / Cas9 gene editing system composed of Sa-SspCas9-KKH.

[0026] Figure 6 This is a test of the editing efficiency of the fusion protein variant Sa-SspCas9-KKH base editor in the PAM sequence NNA.

[0027] Figure 7 The editing efficiency of the fusion protein variant Sa-SspCas9-KKH base editor in the present invention was tested on the PAM sequence NNG.

[0028] Figure 8 The editing efficiency of the fusion protein variant Sa-SspCas9-KKH base editor in the present invention was tested on the PAM sequence NNT.

[0029] Fig. 9 The editing efficiency of the fusion protein variant Sa-SspCas9-KKH base editor in the present invention is tested on the PAM sequence NNC.

[0030] Fig.10 This is a comparison of the efficiency of the SspCas9 and its fusion protein KSa-SspCas9 base editors described in the present invention at the endogenous target CT.

[0031] Fig.11 This is a comparison of the efficiency of the SspCas9 and its fusion protein KSa-SspCas9 base editors described in the present invention at the endogenous target site AG.

[0032] Fig.12 Plasmid map of pCMV_ABE8e. DETAILED DESCRIPTION

[0033] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0034] The Cas9 endonuclease described in the present invention is a DNA endonuclease. In the base sequence described in the present invention, N represents any one of A, G, C, and T. The Cas9 protein described in the present invention is a multi-domain DNA endonuclease, including a REC domain, a Ruv domain, an HNH domain, and a PI domain, which can recognize a DNA sequence complementary to sgRNA at the 5' end of PAM, and cut the DNA chain complementary to sgRNA through the HNH domain and the RuvC domain cuts the non-complementary chain DNA.

[0035] Example 1

[0036] (1) Construction of CBE / ABE-SspCas9 expression plasmid

[0037] Bioinformatics methods were used to analyze and screen proteins related to the type II CRISPR / Cas9 gene editing system. Through BLAST analysis of annotated genomes in the NCBI database, a Cas9 nuclease with high homology to Sacas9 was obtained. It comes from the bacterium Staphylococcus sp.17KM0847 and is named Sspcas9. The Sspcas9 protein consists of 1052 amino acids (SEQ ID No. 1). The alphaford predicted structure is as follows Figure 1 shown.

[0038] pCMV-hA3A-BE3 (addgene #113410) / pCMV_ABE8e (plasmid map as shown Fig.12 The sequence on the pCMV-hA3A-BE3 / pCMV_ABE8e plasmid (excluding the SpCas9 part) was amplified by PCR using the DNA sequence as shown in SEQ ID No. 8 and SspCas9 DNA as templates. The PCR primers are shown in Tables 1 and 2.

[0039] Table 1 PCR primers for constructing CBE-SspCas9 expression plasmid

[0040]

[0041] Table 2 PCR primers for constructing ABE-SspCas9 expression plasmid

[0042]

[0043] Table 3 Max Super-Fidelity DNA Polymerase Reaction System

[0044]

[0045] The PCR run procedure is as follows:

[0046] Table 4 Max Super-Fidelity DNA Polymerase Reaction Procedure

[0047]

[0048] The PCR product was electrophoresed on a 1% agarose gel at 130 V for 35 min, and the target DNA fragment was purified using a gel recovery kit according to the steps provided by the manufacturer. The DNA concentration was determined using a NanoDropTM Lite spectrophotometer (Thermo Scientific). The three fragments were recombined and connected using a homologous recombination kit using the following reaction system and reaction conditions.

[0049] The reaction system is as follows:

[0050] Table 5 ClonExpress II One Step Cloning Reaction System

[0051]

[0052] Prepare the above reaction system on ice, use a pipette to gently mix (do not oscillate), and centrifuge briefly to collect the reaction solution to the bottom of the tube. React at 37℃ for 30min; reduce to 4℃ or immediately cool on ice, take 3μL of the ligation product and add it to 100μL of E. coli DH5α competent cells (purchased from Shanghai Weidi Biotechnology Co., Ltd.), incubate on ice for 30min, heat shock at 42℃ for 1min, incubate on ice for 2min, add 900μL LB medium, and culture at 37℃ for 1 hour. Centrifuge at 5000 rpm for 1 minute, keep about 100μL of bacterial solution and spread it on the LB solid plate containing ampicillin, incubate it upside down in a 37℃ incubator for 16 hours, and pick a single clone for Sanger sequencing verification.

[0053] The clones with correct connection verified by sequencing were shaken and the plasmids were extracted to obtain CBE / ABE-SspCas9 expression plasmids, which were set aside or stored at -20°C for long-term storage.

[0054] (2) Construction of CBE / ABE-KSa-SspCas9 expression plasmid

[0055] The non-PI domain from SaCas9 (nucleotide sequence as SEQ ID No. 2) was fused with the PI domain of SspCas9, that is, using the CBE / ABE-SspCas9 plasmid as a template, the SspCas9 1-2721bp sequence was removed and replaced with the SaCas9 1-2724bp sequence to obtain the fusion protein KSa-SspCas9, whose amino acid sequence is shown in SEQ ID No. 3.

[0056] Table 6 PCR primers for constructing CBE-KSa-SspCas9 expression plasmid

[0057]

[0058] Table 7 PCR primers for constructing ABE-KSa-SspCas9 expression plasmid

[0059]

[0060] Table 8 Max Super-Fidelity DNA Polymerase Reaction System

[0061]

[0062] The PCR run procedure is as follows:

[0063] Table 9 Max Super-Fidelity DNA Polymerase Reaction Procedure

[0064]

[0065] (3) Construction of CBE / ABE-Sa-SspCas9-KKH mutant plasmid

[0066] Using CBE-KSa-SspCas9 plasmid and ABE-KSa-SspCas9 plasmid as templates, PCR reaction was performed on the two sites to obtain expression plasmids containing E782K, T966K, and R1012H mutations ( Figure 2 )

[0067] Table 10 PCR primers for constructing CBE / ABE-Sa-SspCas9-KKH expression plasmid

[0068]

[0069] Table 11 Max Super-Fidelity DNA Polymerase Reaction System

[0070]

[0071]

[0072] The PCR run procedure is as follows:

[0073] Table 12 Max Super-Fidelity DNA Polymerase Reaction Procedure

[0074]

[0075] The PCR product was sequentially run on gel, purified, concentration determined, homologous recombination ligated (single recombination fragments were up to 3), DH5α transformed, single clone sequenced and identified, and plasmid extracted and stored. The specific steps were the same as the construction of CBE / ABE-SspCas9 expression plasmid in (1), thereby obtaining the mutant expression plasmid CBE / ABE-Sa-SspCas9-KKH expression plasmid (the amino acid sequence of the mutant protein is shown in SEQ ID No. 4).

[0076] By constructing a cytosine base editor and editing the PAM library experiment, we explored the PAM sequences required for SspCas9, fusion protein KSa-SspCas9 and mutant Sa-SspCas9-KKH to recognize spacer sequences.

[0077] The specific operations are as follows:

[0078] (1) Eight random bases NNNNNNNN were added to the 3' end of the PAM library spacer sequence (whose sequence is shown in SEQ ID No. 5, 6, and 7) (a total of 65,536 insertion fragments), and the library was constructed into the pUC19 backbone vector by restriction digestion and ligation to obtain a PAM library plasmid with 65,536 different PAM combinations, but the 5' end spacer sequence was the same as the spacer-PAM mixed plasmid. The second-generation sequencing detected that the Gini value of the random base abundance at the 8 positions was less than 0.1, indicating that the random bases at the 8 positions were distributed relatively evenly and the PAM library plasmid had an Amp resistance gene.

[0079] (2) CBE-SspCas9, CBE-KSa-SspCas9, CBE-Sa-SspCas9-KKH and PAM library plasmids were co-transfected into HEK293T cells, and the cell genome was collected.

[0080] (3) Appropriate primers library-NGS-F and M13 R were designed upstream of the random bases, and PCR amplification and purification were performed at the position containing the spacer sequence and PAM combination. Deep sequencing was performed, and eight random bases were analyzed using ladder to obtain the PAM sequences recognized by the above three Cas9 proteins.

[0081] Table 13 PCR primers for high-throughput sequencing

[0082]

[0083] (4) PAM analysis results are as follows Figure 2 As shown, SspCas9 and KSa-SspCas9 recognize the same NNG PAM, while the mutant Sa-SspCas9-KKH recognizes a wider range of NRN and NYN PAMs, but is more inclined to recognize NRN PAM sites, while the recognition and targeting ability of NYN PAM sites is weaker (N = any base, R = A or G, Y = C or T) ( Figure 3 , Figure 4 , Figure 5 ).

[0084] The above transfection process is as follows:

[0085] (1) Inoculate an appropriate amount of cells one day before transfection to achieve a cell density of 80-90% for transfection the next day, such as 1-4×10 4 .

[0086] (2) Prepare the DNA-Buffer Transeasy complex by mixing as follows

[0087] i. Dilute the appropriate amount of DNA in 25 μL Opti-MEM serum-free medium and mix well. (For base editing experiments, the amount of transfected DNA is 300 ng editor plasmid, 100 ng sgRNA plasmid and 30 ng puromycin selection plasmid. For experiments to determine PAM, the amount of transfected DNA is 300 ng editor plasmid, 100 ng sgRNA plasmid and 45 ng PAM plasmid library.)

[0088] ii. Gently mix Buffer Transeasy, pipette 0.75 μL of the solution and dilute it in 25 μL of Opti-MEM serum-free medium, mix well and place at room temperature for 5 minutes.

[0089] iii. After 5 minutes, mix the diluted DNA with Buffer Transeasy, a total of 50 μL, mix well and leave at room temperature for 20 minutes to form a complex.

[0090] iv. Add 50 μL DNA-Buffer Transeasy complex to the cell culture dish and gently shake the dish to mix.

[0091] v. 24 hours after transfection, the culture medium was replaced with fresh complete medium containing 3 μg / mL puromycin, and the cell genome was collected after further culture for 48 hours.

[0092] (3) After transfection, remove the culture medium from the cells and add 50 μL 1× PBS to each well. Gently shake to wash away excess culture medium and dead cells. Then remove and discard the PBS. Repeat the washing process three times.

[0093] (4) Add 100 μL 1× PBS to each well, blow the cells off with a pipette, place them in a centrifuge tube, and centrifuge at 12,000 rpm for 3 minutes at room temperature.

[0094] (5) Discard the supernatant, add 30 μL of cell lysis working solution to the cell pellet, pipette and mix evenly, then incubate in a 55°C metal bath for 20 minutes and inactivate at 95°C for 10 minutes. The cell genome is stored in a -20°C refrigerator for subsequent experiments.

[0095] Example 2: Sa-SspCas9-KKH endogenous target verification

[0096] (1) To verify the specificity of mutant Sa-SspCas9-KKH in endogenous target recognition, in this example, endogenous genes were selected in mammalian HEK293T cells, and the PAM sequence identified in this example 1 was used as a reference to find a suitable target site, and the CBE-Sa-SspCas9-KKH cytosine base editor and sgRNA constructed above were transfected into HEK293T cells. The experiment was carried out using the same transfection steps as in Example 1. The genome was collected for sequencing detection.

[0097] (2) Editing of endogenous targets by mutant CBE-Sa-SspCas9-KKH Figure 6 , 7 , 8 and 9, NNAG, NNAC, NNAA, NNAT, NNGG, NNGC, NNGT, and NNGA all showed good editing efficiency.

[0098] Example 3 Endogenous target CT editing efficiency test

[0099] This example verifies the editing efficiency of the gene editing system composed of SspCas9 and KSa-SspCas9 proteins in mammalian cells by performing CT editing efficiency tests on endogenous targets.

[0100] (1) In mammalian HEK293T cells, endogenous genes were selected, and the PAM sequence identified in Example 1 was used as a reference to find a suitable target site, and the CBE-SspCas9, CBE-KSa-SspCas9 cytosine base editor and sgRNA constructed above were transfected into HEK293T cells. The experiment was carried out using the same transfection steps as in Example 1. The genome was collected for sequencing detection.

[0101] (2) CT editing results analysis results are as follows Fig.10 As shown, in HEK239T cells, both CBE-SspCas9 and CBE-KSa-SspCas9 editors showed good editing ability in the target of NNG, among which the editing ability of CBE-KSa-SspCas9 in the targets with PAM of NNGT, NNGA, and NNGC was significantly higher than that of CBE-SspCas9.

[0102] Example 4: Endogenous target AG editing efficiency test

[0103] In this example, the editing efficiency of the gene editing system composed of SspCas9 and KSa-SspCas9 proteins in mammalian cells was verified by performing AG editing efficiency tests on endogenous targets.

[0104] (1) In mammalian HEK293T cells, endogenous genes were selected, and the PAM sequence identified in Example 1 was used as a reference to find a suitable target site, and the ABE-SspCas9, ABE-KSa-SspCas9 and ABE-Sa-SspCas9-KKH cytosine base editors and sgRNA constructed above were transfected into HEK293T cells. The experiment was carried out using the same transfection steps as in Example 1. The genome was collected for sequencing detection.

[0105] (2) AG editing results analysis results are as follows Fig.11 As shown, in HEK239T cells, both ABE-SspCas9 and ABE-KSa-SspCas9 editors showed good editing ability in the target of NNG, among which the editing ability of ABE-KSa-SspCas9 was significantly higher than that of ABE-SspCas9 in the targets with PAM of NNGT, NNGA, and NNGC.

[0106] In summary, the present invention has identified for the first time a new type II CRISPR / Cas9 gene editing system, named SspCas9, which is 1052 amino acids in size, smaller than SpCas9. The above examples demonstrate that a type II CRISPR / Cas9 gene editing system of the present invention can specifically recognize the PAM of NNG. The fusion protein of SspCas9 and SaCas9 has a stronger base editing efficiency, and its KH variant can specifically recognize the PAM sequence of NNN, but has a higher editing efficiency for NRN than NYN. It can perform gene editing functions in mammalian cells under the guidance of sgRNA, expanding the gene editing toolbox.

[0107] The present invention verifies the editing ability of a type II CRISPR / Cas9 gene editing system and its fusion proteins and variants through eukaryotic DNA. The Cas9 protein recognizes the target sequence, which requires a nucleotide sequence complementary to the sgRNA spacer sequence and a recognizable PAM sequence. First, through the base editing PAM Library, the PAM sequence recognized by the Cas9 protein and its variants was obtained. Then, through the mammalian endogenous target verification, the correctness of the PAM was verified, and the base editing ability of the Cas9 protein and its variants was analyzed at the same time.

[0108] The discovery of a type II CRISPR / Cas9 gene editing system of the present invention further expands the types of gene editing tools, provides a powerful research tool for disease treatment and plant breeding, and plays an important role in promoting the application of gene editing in multiple fields.

Claims

1. A fusion protein KSa-SspCas9, whose amino acid sequence is shown in SEQ ID No.

3.

2. A gene encoding the fusion protein KSa-SspCas9 according to claim 1.

3. A mutant of the fusion protein KSa-SspCas9, whose amino acid sequence is shown in SEQ ID No.

4.

4. A gene encoding the mutant according to claim 3.

5. A vector comprising the gene according to claim 2 or claim 4. A cell comprising the vector according to claim 5.

7. Use of the fusion protein KSa-SspCas9 according to claim 1, or the mutant according to claim 3, or the protein SspCas9 with the amino acid sequence shown in SEQ ID No. 1 in the CRISPR-Cas9 gene editing system.

8. The use according to claim 7, characterized in that: The CRISPR-Cas9 gene editing system is a single base editor.

9. A conjugate, characterized in that The conjugate contains the fusion protein KSa-SspCas9 described in claim 1 or the mutant described in claim 3 or the protein SspCas9 with the amino acid sequence shown in SEQ ID No.1.

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