CN114410608B

CN114410608B - Method for efficiently expressing and purifying Cas9 protein and application thereof

Info

Publication number: CN114410608B
Application number: CN202210054940.3A
Authority: CN
Inventors: 潘力; 廖清; 王斌
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2022-01-18
Filing date: 2022-01-18
Publication date: 2023-08-22
Anticipated expiration: 2042-01-18
Also published as: CN114410608A

Abstract

The invention discloses a method for efficiently expressing and purifying Cas9 protein and application thereof. The invention mainly comprises construction of a Cas9 expression vector, transformation of escherichia coli, induction expression of Cas9 protein, purification of Cas9 protein and application in vitro and in vivo. The GB1 is used as a dissolution promoting tag, so that the stability and the solubility of the SpCas9 can be increased, the expression quantity of the SpCas9 is primarily improved, the function of the SpCas9 is not affected by the GB1, and the SpCas9 is not required to be removed later, so that the method is simple, convenient and feasible. And by using a 10 XHis tag, the high affinity of the fusion protein and Ni-NTA resin is facilitated, the high salt washing is facilitated, the polluted nucleic acid is removed, and the purification effect is improved. The tandem T7 promoter is used for obviously improving the expression quantity of the SpCas 9. Through in vivo and in vitro application, the purified SpCas9-GB1 protein is proved to be correctly folded, has the capability of cutting DNA and can enter a nucleus for gene editing.

Description

Method for efficiently expressing and purifying Cas9 protein and application thereof

Technical Field

The invention belongs to the technical fields of biotechnology and genetic engineering, relates to a nuclease Cas9-GB1 protein expression vector driven by multiple promoters and a protein expression and purification method, and in particular relates to a method for efficiently expressing and purifying Cas9 protein and application thereof.

Background

The use of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) -related RNA-guided Cas9 endonuclease activity derived from prokaryotes makes gene editing in multiple species simple and rapid. The CRISPR system mainly comprises two parts, namely Cas9 protein and single guide RNA (sgRNA). Cas9 proteins are nucleases containing two domains with cleavage activity: HNH domain and RuvC domain, which can be directed to specific locations to cleave target sites in the presence of gRNA, resulting in DNA double strand breaks. CRISPR systems have been applied to gene editing of multiple species.

Riboprotein complexes (RNPs) formed by in vitro assembly of Cas9 protein and sgrnas also have gene editing capabilities and have also been used in a variety of organisms for gene editing. Gene editing of RNP has several advantages: if RNP avoids translation expression in a host body, is easy to degrade, and can reduce off-target effect by performing instantaneous cleavage on a host genome; different sites can be targeted only by replacing sgRNA, so that the complicated process of constructing plasmids is omitted, and the flux gene editing is convenient; meanwhile, RNP can also be used for in vitro cutting of templates to verify the efficiency of sgRNA.

At present, in the process of expression and purification of Cas9 protein, insoluble protein in the form of inclusion bodies is easy to form, the problems of incorrect protein folding, low yield and the like caused by high endotoxin content and overlarge protein are easy to occur, and in addition, cas9 protein in the current market is expensive, so that the popularity of in-vitro assembly and in-vivo editing of a Cas9 system is limited.

Disclosure of Invention

In order to overcome the disadvantages and shortcomings of the prior art, the present invention is directed to a method for efficiently expressing and purifying Cas9 protein. The method is a Cas9 protein expression purification method capable of performing gene editing and remarkably improving the in vitro cutting expression quantity. The invention mainly comprises construction of a Cas9 expression vector, transformation of escherichia coli, induction expression of Cas9 protein, purification of Cas9 protein and application in vitro and in vivo. In the constructed pet28a+ -SpCas9-GB1-10 xHis-3T 7 vector, GB1 is used as a dissolution promoting tag, so that the stability and the solubility of the SpCas9 can be increased, the expression quantity of the SpCas9 is primarily improved, and the function of the SpCas9 is not influenced by GB1, so that additional removal is not needed in the follow-up process, and the method is simple, convenient and feasible. And by using a 10 XHis tag, the high affinity of the fusion protein and Ni-NTA resin is facilitated, the high salt washing is facilitated, the polluted nucleic acid is removed, and the purification effect is improved. The method of using the tandem T7 promoter obviously improves the expression quantity of the SpCas 9. Through in vivo and in vitro application, the purified SpCas9-GB1 protein is proved to be correctly folded, has the capability of cutting DNA, and can enter a nucleus for gene editing.

The aim of the invention is achieved by the following technical scheme:

a method for efficiently expressing and purifying Cas9 protein, comprising the steps of:

step 1, constructing a nuclease Cas9 expression vector pet28a+ -SpCas9-GB1-10 xHis-3T 7 driven by multiple promoters;

step 2, expression of the cas9 expression vector pep28a+ -SpCas9-GB1-10 xhis-3T 7: transferring the constructed pet28a+ -SpCas9-GB1-3T7 into a host E.coli Rosetta (DE 3) to obtain a recombinant strain; transferring and activating the recombinant strain, performing induction expression by using IPTG, and centrifugally collecting thalli to obtain thalli expressing the SpCas9-GB1 protein;

step 3, purification of SpCas9-GB1 protein: suspending the strain in the step 2 by using a buffer solution, performing ultrasonic crushing, centrifuging and collecting the supernatant to obtain soluble SpCas9-GB1 protein in positive clones, filtering to remove cell fragments and particles, directly loading the filtered liquid to a Ni+ affinity chromatography column for purification, and collecting eluted protein to obtain SpCas9-GB1 protein solution; and carrying out ultrafiltration concentration on the obtained SpCas9-GB1 protein solution to obtain the SpCas9-GB1 protein.

In order to better implement the present invention, the method further includes step 4:

step 4, application of SpCas9-GB1 protein: the SpCas9-GB1 and the sgRNA are assembled and then used for in-vitro cutting of templates and gene editing by transferring into a fungus host, and whether the SpCas9-GB1 is correctly folded or not is determined to have the capability of cutting DNA and performing gene editing by nuclear insertion.

Further, the vector construction in step 1: the 6 Xthioredoxin-histidine tag on the pet28a+ plasmid was replaced with a 10 Xthioredoxin-histidine tag, the number of T7 promoters was increased to 3, while the 56-residue B1 immunoglobulin binding domain of streptococcal protein G (Streptococcal protein G, GB 1) was used as fusion tag to ligate with Cas9 protein through linker (ggtggagcaggtggcagtggcgcagggggagccgga), the entire Cas9-GB1 gene sequence, which was codon optimized for easy expression in E.coli, was ligated with the 10 Xthioredoxin-histidine tag of pet28 a+. The optimization replaces the rare codon in the original GB1 sequence and the base which is not easy to translate protein with the base which is easy to translate and express in host bacteria.

Furthermore, the SpCas9-GB1-10 xHis has a sequence shown in SEQ ID NO:19.

Further, the recombinant strain in the step 2 is selected in LB liquid medium of Kan+ and CHL+, and cultured for 8-16 h activation at 37+ -1 ℃ and 200-220 rpm (preferably, the activation is performed at 37+ -1 ℃ and 220 rpm); after activation, according to (1-5): 100 transferring ratio, taking activated bacterial liquid in LB liquid culture medium of Kan+ and CHL+, culturing at 37+ -1deg.C and 200-220 rpm to OD ₆₀₀ At 0.6 to 0.8, IPTG is added to a final concentration of 0.3 to 0.8mM (preferably 0.5 mM) to induce expression.

Further, the conditions for inducing expression in the step 2 are 16-20 ℃ and 200-220 rpm; the fermentation time is 18-22 h.

Further, the centrifugation condition in the step 2 is 4-8 ℃, 8000-10000 rpm for 5-10 minutes; further, the mixture was centrifuged at 8000rpm at 4℃for 5 minutes.

Further, the conditions of ultrasonic disruption in step 3 are: the ultrasonic switch is turned on for 3s, the ultrasonic switch is turned off for 3s, the power is 30-40% (preferably 30%), and the ultrasonic switch is used for 20-30 min (preferably 20 min) on ice.

Further, the centrifugation condition in the step 3 is 4-8 ℃, and centrifugation is carried out at 8000-10000 rpm for 30-40 min; further, the mixture was centrifuged at 10000rpm at 4℃for 30min.

Further, in step 3, the SpCas9-GB1 protein was eluted using a gradient elution with Buffer A (500mM NaCl,20mM Tris-HCl, pH=8.0) at an imidazole concentration of 0mM and Buffer B (500mM NaCl,500mM Imidazole,20mM Tris-HCl, pH=8.0) at an imidazole concentration of 500mM. The gradient was 5%, 10%, 20%, 30%, 50% buffer B.

Further, in step 4, all sgrnas were T7 transcribed in vitro. The in vitro cleavage template was the Niad site of A.oryzae and the in vivo gene editing was the pyrg site of A.niger. The Buffer used is a commercial Cas9 protein matched Buffer.

The method for efficiently expressing and purifying the Cas9 protein is applied to efficiently expressing and purifying the Cas9 protein.

Compared with the prior art, the invention has the following advantages and effects:

(1) The invention is thatThe GB1 is used as a dissolution promoting tag and a 10 XHis tag to fusion express the SpCas9, so that the stability of the SpCas9 is improved, the nucleic acid pollution is avoided, and the purification effect is improved. By Ni ²⁺ The high-purity SpCas9-GB1 protein can be effectively obtained through affinity chromatography column purification, and the obtained SpCas9-GB1 protein is correctly folded. And GB1 does not have influence on the SpCas9 function, does not need to be removed later, and is very convenient.

(2) The invention utilizes a tandem T7 promoter, and under the condition of a triple T7 promoter, the expression quantity of the SpCas9-GB1 protein is further increased.

(3) Through in-vitro and in-vivo application of the purified SpCas9-GB1 protein, the invention verifies that a complex formed by the purified Cas9-GB1 and sgRNA can effectively cut in-vitro DNA fragments and in-vivo targeted genes. The SpCas9-GB1 protein related to the invention is easy to purify, has high purification degree and high cleavage activity, and can be used for industrial production of Cas9 protein and laboratory Cas9 protein sources.

(4) The invention fuses NLS nuclear localization signal peptide at the C end of Cas9 protein. The NLS nuclear localization signal peptide can enable the protein to be used for in vitro cutting and gene editing of prokaryotes, and is also suitable for genome editing of eukaryotes.

Drawings

FIG. 1 is an agarose gel electrophoresis of the results of digestion of the pet28a+ plasmid.

FIG. 2 is a graph of the sequencing results of the pet28a+ multiple promoter vector.

FIG. 3 is a agarose gel electrophoresis chart (A, B) of the PCR results of SpCas9, spCas9-N, spCas9-C, GB1-C, GB1-N and a agarose gel electrophoresis chart (B) of the digestion results of pet28a+ plasmid.

FIG. 4 is an agarose gel electrophoresis of the pet28a+ -2T7 plasmid using both NcoI and XhoI digestion.

FIG. 5 is a map of the pet28a+ -SpCas9-GB1-10 XHis-3T 7 plasmid.

FIG. 6 is a SDS-PAGE analysis of the effect of GB1 on SpCas9 expression; wherein His-SpCas9 refers to SpCas9; NGB1-SpCas9 refers to GB1-SpCas9; spCas9-CGB1 refers to SpCas9-GB1.

FIG. 7 is a graph showing the effect of SDS-PAGE analysis of multiplex promoters on SpCas9 expression; wherein A is the influence of multiple promoters on the expression quantity of NGB1-SpCas9 (namely GB1-SpCas 9); b is the influence of multiple promoters on the expression quantity of the SpCas9-CGB1 (namely, the SpCas9-GB 1).

FIG. 8 is a SDS-PAGE analysis gel of SpCas9-GB1 protein after AKTA pure Ni-NTA column purification.

FIG. 9 is a diagram of agarose electrophoresis gel of sgRNA transcription template PCR.

FIG. 10 is a diagram showing an example of the application of the purified SpCas9-GB1 protein; wherein, cas9-GB1 refers to SpCas9-GB1.

FIG. 11 is a diagram of an example in vivo editing application of the purified SpCas9-GB1 protein; wherein A is a positive control plate and a negative control plate, wherein the positive plate is an RNP complex-free plate and no screening substances (5-fluoroorotic acid and uridine) are added; the negative plate is not added with RNP complex, and screening substances are applied; b is a transformation plate, which is an RNP complex added, and a screening substance is applied.

FIG. 12 is a schematic (A) and a result chart (B) of the purified SpCas9-GB1 protein gene editing.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.

The test methods for specific experimental conditions are not noted in the examples below, and are generally performed under conventional experimental conditions or under experimental conditions recommended by the manufacturer. The materials, reagents and the like used, unless otherwise specified, are those obtained commercially.

The materials used in the examples are as follows:

1. cell source: e.coli Mach 1T 1, E.coli Rosetta (DE 3), are conventional commercial strains.

2. Plasmid source: the pET-28a (+) plasmid is a conventional commercial product.

3. Primer source primer: are synthesized in the Shanghai stock of biological engineering, guangzhou Tian Yihui Yuan Gene technology Co.

4. The main reagent comprises: all are commercial products.

Example 1 construction of pet28a+ -SpCas9-GB1-10 XHis-3T 7 plasmid

Construction of Pet28a+ multiple promoter vector

According to the pET-28a (+) plasmid, a pair of primers F-1/R-1 (SEQ ID NO: 1-2) are designed, and the pET-28a (+) plasmid is used as a template, and a pET28a+ linearization skeleton fragment is obtained through PCR reaction. The PCR reaction system is shown in Table 1.

TABLE 1 PCR reaction System

Reaction components	Volume of
		2×Primer Star	25μL
F-1～F-6(10μM)	0.5μL
		R-1～R-6(10μM)	0.5μL
pET-28a (+) plasmid	1μL(10ng)
		Ultrapure water	Up to 50μL

Linear amplification reaction procedure:

reaction temperature	Time
		98℃	2min
98℃	30s
		62 ℃ (tm value)	30s
72℃	1Kb/min
		72℃	7min
25℃	1s

The PCR results are shown in FIG. 1. Lane 1 is pet28a+ PCR backbone fragment, lane 2 is DNA 1Kb marker. Recovering the fragment.

Multiple promoter primers (Table below) were designed and 2T7-F/2T7-R, 3T7-F/3T7-R, 4T7-F/4T7-R (SEQ ID NOS: 3-8) were annealed at 98℃for 10min, respectively, to form multiple promoter 2T7 fragments, 3T7 fragments, 4T7 fragments. Recovering the fragment.

Pet28a+ linearized fragments 0.5. Mu.L, 2T7 fragment, 3T7 fragment or 4T7 fragment 4.5. Mu.L, infusion enzyme 5. Mu.L were added to PCR tubes and ligated at 50℃for 1 h.

Transferring the above connection products into E.coli Mach 1T 1 competent cells by 42 ℃ heat shock method, adding 890 μl of LB liquid medium, placing in a shaking table at 37 ℃ for culturing at 200rpm for 1h, coating 200 μl on Kan+ resistant LB solid plate, and culturing in a 37 ℃ incubator overnight.

Microcolonies on the plates were picked, amplified in small amounts, subjected to bacterial liquid electrophoresis, and observed for correct plasmid size. Randomly picking plasmids with correct band sizes, carrying out sequencing, and obtaining the pet28a+ -2T7, pet28a+ -3T7 and pet28a+ -4T7 plasmids after the correct sequencing is identified. The sequencing results are shown in FIG. 2.

Construction of pet28a+ -6×his-SpCas9, pet28a+ -10×His-GB1-SpCas9, pet28a+ -SpCas9-GB1-10×His plasmids

The pET-28a (+) plasmid was digested with NcoI and XhoI and digested with BamHI, and subjected to agarose gel electrophoresis, as shown in FIG. 3B, lane 3 was a single digested fragment, lane 4 was a double digested fragment, and lane 5 was a control plasmid.

Primers were designed using pCas plasmid (conventional commercial product) as a template, and the SpCas9 gene was amplified by PCR using F-2/R-2 (SEQ ID NOS: 9-10), F-3/R-3 (SEQ ID NOS: 11-12) and F-4/R-4 (SEQ ID NOS: 13-14), respectively, to obtain SpCas9-N and SpCas9-C, spCas fragment. The synthesized GB1 and linker (see SEQ ID NO: 25) templates are used, and the designed primers, F-5/R-5 (SEQ ID NO: 15-16) and F-6/R-6 (SEQ ID NO: 17-18) are used for PCR amplification of GB1-N, GB 1-C. The amplified systems are shown in Table 1. The PCR results are shown in FIG. 3, A, B. FIG. 3A, lane 1, spCas9-N, lane 2, spCas9-C, lane 3, GB1-N, lane 4, GB1-C fragment, lane 5, 200bp marker, recovery fragment; in FIG. 3B, the first lane is a SpCas9 fragment, and the second lane is a 200bp marker.

Linearizing the pet28a+ vector with 0.5 mu L, spCas9 1 mu L of the pet28a+ vector and 0.5 mu L, spCas-N/SpCas 9-C1 mu L, GB1-N/GB 1-C0.5 mu L, ddH ₂ O3. Mu.L of the infusion enzyme was added to each of the PCR tubes at 50℃for 1 hour. And carrying out heat shock transformation according to the method, screening transformants, and verifying by sequencing to obtain correct pet28a+ -6×his-SpCas9, pet28a+ -10×His-GB1-SpCas9 and pet28a+ -SpCas9-GB1-10×His plasmids. The complete SpCas9-GB1-10 XHis sequence is shown in SEQ ID NO:19.

construction of pet28a+ -SpCas9-GB1-10 XHis triple promoter plasmid

The correctly sequenced pet28a+ multiple promoter plasmid (pet 28a+ -2T7 plasmid) was subjected to double digestion with NcoI and XhoI, and agarose gel electrophoresis was performed after digestion, as shown in FIG. 4, lanes 1 and 3 were digested fragments, and lanes 2 and 4 were control plasmids. The same applies to pet28a+ -3T7 and pet28a+ -4T7 plasmids.

Linearizing the pet28a+ -3T7 vector with fragments 0.5 μ L, spCas 9-C1 μL, GB 1-C0.5 μL, ddH ₂ O3. Mu.L of the ligation enzyme (5. Mu.L) was added to the PCR tube, and ligation was performed at 50℃for 1 hour. Heat shock transformation was performed as described previously, the correct transformants were selected, and sequencing verified for the correct plasmid pet28a+ -SpCas9-GB1-10 xhis-3T 7 (i.e., pet28a+ -SpCas9-CGB1-3T 7). The plasmid map of successful construction is shown in FIG. 5.

Similarly, the pet28a+ -2T7, pet28a+ -3T7 or pet28a+ -4T7 vector was linearized with 0.5. Mu. L, spCas 9-N1. Mu.L, GB 1-N0.5. Mu.L, ddH ₂ O3. Mu.L of the ligation enzyme (5. Mu.L) was added to the PCR tube, and ligation was performed at 50℃for 1 hour. Heat shock transformation is carried out according to the method, correct transformants are screened, and sequencing is carried out to verify correct plasmids pet28a+ -10 xHis-GB 1-SpCas9-2T7, pet28a+ -10 xHis-GB 1-SpCas9-3T7 and pet28a+ -10 xHis-GB 1-SpCas9-4T7.

Example 2 expression of fusion protein SpCas9-GB1

1. Induction expression of fusion protein SpCas9-GB1

Transferring the identified correct pet28a+ -SpCas9-GB1-10 xHis-3T 7 plasmid into an E.coli expression host E.coli Rosetta (DE 3) to obtain a single colony with stable transformation; inoculating single colonies to a strain containing 100. Mu.g/mL Kan ⁺ CHL (CHL) ⁺ Culturing overnight at 220rpm and 37 ℃ in LB liquid medium of (chloramphenicol) to obtain overnight bacteria. Overnight bacteria were inoculated into 500mL of LB medium at an inoculation ratio of 1:100, cultured at 200rpm,37℃to OD ₆₀₀ Reaching 0.6-0.8, adding IPTG (isopropyl thiogalactoside) with the final concentration of 0.5mM, and then culturing for 18-22 hours at the temperature of 16-18 ℃. After the culture, the cells were collected by centrifugation at 8000rpm for 5 minutes at 4 ℃. Then using 30mL TE Buffer to re-suspend the bacteria, and centrifuging at 8000rpm for 5 minutes at 4 ℃ to obtain the fusion expression protein SpCas9-GB1 containing nuclease Cas9-10 xhis cells.

Comparison of the expression level of SpCas9 protein and fusion protein GB1-SpCas9, spCas9-GB1

Plasmids of pet28a-6 XHis-SpCas 9, pet28a+ -SpCas9-GB1-10 XHis-1T 7 (i.e., pet28a+ -SpCas9-GB1-10 XHis), pet28a+ -10 XHis-GB 1-SpCas9-1T7 (i.e., pet28a+ -10 XHis-GB 1-SpCas 9) were induced to be expressed. Respectively centrifuging 2mL of bacterial liquid to obtain thalli, washing twice by using TE Buffer, and obtaining the thalli according to the final OD ₆₀₀ Adding PBS buffer solution into the solution at the ratio of 10OD/1mL PBS for resuspension, then using ultrasonic disruption, taking 40 mu L of whole bacteria sample, centrifuging the rest liquid at 4 ℃ and 10000rpm, taking 40 mu L of supernatant, removing the supernatant, and then using PBS for resuspension precipitation, thus preparing a precipitation sample. SDS-PAGE running analysis was performed and the results are shown in FIG. 6. Gray scale comparison is performed by using Image J, the expression quantity of the SpCas9-GB1 protein is highest and is 1.68 times of that of the SpCas9 protein without the GB1 fusion. SpCas9-GB1 was subsequently selected for further optimization.

3. Effect of multiple promoters on expression level

The E.coli Rosetta (DE 3) strain transformed by the pet28a+ -10 xHis-GB 1-SpCas9-1T7, 2T7, 3T7 and 4T7 plasmids was induced to be expressed, and the sample was prepared according to the method described above, and the result of SDS-PAGE gel running analysis is shown in FIG. 7A. In the comparison of multiple promoters, the 3, 4-fold T7 promoter has little effect on the expression amount of the target protein, so that the 3-fold T7 promoter is selected later to improve the expression amount.

The plasmids of pet28a+ -SpCas9-GB1-10 xHis-1T 7 and pet28a+ -SpCas9-GB1-10 xHis-3T 7 are induced to be expressed, and the sample preparation and SDS-PAGE running analysis are carried out according to the method. The results are shown in FIG. 7B. The 3-fold T7 promoter obviously improves the expression quantity of the SpCas9-GB1 protein, and the expression quantity of the SpCas9-GB1 driven by the 3-fold T7 promoter is 1.26 times compared by using Image J for gray level analysis.

EXAMPLE 3 purification of fusion protein SpCas9-GB1

Collecting the above induced thallus, mixing with PBS buffer solution, re-suspending, ultrasonic crushing (under the conditions of ultrasonic on for 3s, ultrasonic off for 3s, power of 30%, ultrasonic on ice for 20 min), centrifuging at 4deg.C for 30min at 10000rpm to collect supernatant, and processingFiltering with 0.22 μm filter membrane, directly loading sample, and collecting penetrating fluid; warp Ni ²⁺ Affinity column purification was performed using Buffer a (500mM NaCl,20mM Tris-HCl, ph=8.0) as the equilibration solution, while phase B (500mM NaCl,500mM Imidazole,20mM Tris-HCl, ph=8.0) was mixed with phase a in a gradient to perform gradient elution (gradient 5%, 10%, 20%, 30%, 50% Buffer B), eluting the target protein, and the protein size was 165KD, and the experimental results are shown in fig. 8.

Ni is added with ²⁺ The fusion protein SpCas9 solution purified by the affinity chromatography column was subjected to ultrafiltration (500mM NaCl,20mM Tris-HCl,3mM DTT (dithiothreitol), ph=8.0) to remove imidazole and hetero-proteins. Cas9 protein was stored in 50% glycerol after concentration.

Example 4 Activity detection of fusion protein SpCas9-GB1

The sgRNA (Niad-sgRNA) is designed according to the Niad gene (AO 090012001035) locus of Aspergillus oryzae, and the specific sequence is shown in SEQ ID NO:23 from base 21 to 40. Primers were designed, F-7/R-7 (SEQ ID NO: 20-21) was used, and any plasmid containing the fragment of the sgRNA backbone was used as a template to amplify the backbone containing the sgRNA (see SEQ ID NO:23 for specific sequences), followed by agarose gel electrophoresis and fragment recovery. As a result, FIG. 9 shows that lanes 1 and 2 are Niad-sgRNA templates (amplified skeletons containing sgRNA), and lane 5 is DL5000 DNA marker.

In vitro transcription using T7 RNA polymerase and alcohol precipitation extraction using isopropanol were performed as described in Table 2 to give purified Niad-sgRNA.

Table 2: RNA in vitro transcription system

Template DNA	1μg
		T7 RNA pol/50% glycerol	2μL
NTP	10μL
		RNAase free water	Up to 30μL

SpCas9-GB1 in vitro cleavage system was configured according to the system of Table 3. A template negative control was set up and Cas9 purchased on the market was set up as a positive control. The system was placed in an incubator at 37℃for 8 hours, and then subjected to agarose gel electrophoresis. The results are shown in FIG. 10, wherein lane 1 is a positive control, lane 2 is a SpCas9-GB1 experimental group, and lanes 3 and 4 are negative controls. The template was sheared in the positive control and experimental groups, indicating that the purified SpCas9-GB1 protein had the correct protein activity and was able to be subjected to gene editing.

Table 3: cas9 protein in-vitro cutting template system

Example 5 application example of fusion protein SpCas9-GB1

The sgRNA (pyrg-sgRNA) was designed according to the Aspergillus niger pyrg gene (An 12g 03570) site, and the specific sequence is shown in SEQ ID NO:24 from base 21 to 40. Primers were designed and F-8/R-7 (SEQ ID NO:22, SEQ ID NO: 21) was used to amplify the backbone containing the sgRNA using any plasmid containing the fragment of the backbone of the sgRNA as template (see SEQ ID NO:24 for specific sequences).

In vitro transcription using T7 RNA polymerase and alcohol precipitation extraction using isopropanol was performed as described in Table 2 to give purified pyrg-sgRNA.

The in vitro assembled RNP complex was transformed into protoplasts of A.niger CBS513.88 (a conventional commercially available product) according to the Aspergillus transformation method of conventional PEG, and observed for the presence of transformants. The results are shown in FIG. 11. The positive plate showed colony growth, the negative plate showed no colony growth, and the transformation plate showed colony growth. Wherein, the positive plate is not added with RNP complex and screening substance (5-fluoroorotic acid and uridine), the negative plate is not added with RNP complex but added with screening substance (5-fluoroorotic acid and uridine), and the conversion plate is added with RNP complex and screening substance (5-fluoroorotic acid and uridine).

Transformants were picked on plates and the genome was extracted in 24 well plates for PCR amplification of the pyrg gene. Sequencing identified a deletion at pyrg at the sgRNA position, as shown in FIG. 12. SpCas9-GB1 can be seen to have the ability to enter the nucleus for gene editing.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

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<400> 10

cagtggtggt ggtggtggtg ttacaccttc ctcttcttct tgggg 45

<210> 11

<211> 45

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> F-3

<400> 11

ctttaagaag gagatatacc atggataaga aatactcaat aggct 45

<210> 12

<211> 42

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> R-3

<400> 12

ccactgccac ctgctccacc caccttcctc ttcttcttgg gg 42

<210> 13

<211> 45

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> F-4

<400> 13

gtggacagca aatgggtcgc ggaatggata agaaatactc aatag 45

<210> 14

<211> 65

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> R-4

<400> 14

gtcgacggag ctcgaattcg tcacaccttc ctcttcttct tggggtcacc tcctagctga 60

ctcaa 65

<210> 15

<211> 83

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> F-5

<400> 15

aactttaaga aggagatata ccatgggcag cagccatcat catcatcatc accaccacca 60

tcatgccgtg ggcggatccg gtg 83

<210> 16

<211> 39

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> R-5

<400> 16

attgagtatt tcttatccat gctgcctccg ccacccgaa 39

<210> 17

<211> 61

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> F-6

<400> 17

ggtggagcag gtggcagtgg cgcaggggga gccggatata aattgatcct gaacggcaaa 60

a 61

<210> 18

<211> 97

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> R-6

<400> 18

cagtggtggt ggtggtggtg gtgatgatga tgagagcctc ccccaccacc cccggcaccg 60

cttgcccctc cggcttctgt gacagtgaag gttttgg 97

<210> 19

<211> 4395

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> SpCas9-GB1-10×His

<400> 19

atggataaga aatactcaat aggcttagat atcggcacaa atagcgtcgg atgggcggtg 60

atcactgatg aatataaggt tccgtctaaa aagttcaagg ttctgggaaa tacagaccgc 120

cacagtatca aaaaaaatct tataggggct cttttatttg acagtggaga gacagcggaa 180

gcgactcgtc tcaaacggac agctcgtaga aggtatacac gtcggaagaa tcgtatttgt 240

tatctacagg agattttttc aaatgagatg gcgaaagtag atgatagttt ctttcatcga 300

cttgaagagt cttttttggt ggaagaagac aagaagcatg aacgtcatcc tatttttgga 360

aatatagtag atgaagttgc ttatcatgag aaatatccaa ctatctatca tctgcgaaaa 420

aaattggtag attctactga taaagcggat ttgcgcttaa tctatttggc cttagcgcat 480

atgattaaat ttcgtggtca ttttttgatt gagggagatt taaatcctga taatagtgat 540

gtggacaaac tatttatcca gttggtacaa acctacaatc aattatttga agaaaaccct 600

attaacgcaa gtggagtaga tgctaaagcg attctttctg cacgattgag taaatcaaga 660

cgattagaaa atctcattgc tcagctcccc ggtgagaaga aaaatggctt atttgggaat 720

ctcattgctt tgtcattggg tttgacccct aattttaaat caaattttga tttggcagaa 780

gatgctaaat tacagctttc aaaagatact tacgatgatg atttagataa tttattggcg 840

caaattggag atcaatatgc tgatttgttt ttggcagcta agaatttatc agatgctatt 900

ttactttcag atatcctaag agtaaatact gaaataacta aggctcccct atcagcttca 960

atgattaaac gctacgatga acatcatcaa gacttgactc ttttaaaagc tttagttcga 1020

caacaacttc cagaaaagta taaagaaatc ttttttgatc aatcaaaaaa cggatatgca 1080

ggttatattg atgggggagc tagccaagaa gaattttata aatttatcaa accaatttta 1140

gaaaaaatgg atggtactga ggaattattg gtgaaactaa atcgtgaaga tttgctgcgc 1200

aagcaacgga cctttgacaa cggctctatt ccccatcaaa ttcacttggg tgagctgcat 1260

gctattttga gaagacaaga agacttttat ccatttttaa aagacaatcg tgagaagatt 1320

gaaaaaatct tgacttttcg aattccttat tatgttggtc cattggcgcg tggcaatagt 1380

cgttttgcat ggatgactcg gaagtctgaa gaaacaatta ccccatggaa ttttgaagaa 1440

gttgtcgata aaggtgcttc agctcaatca tttattgaac gcatgacaaa ctttgataaa 1500

aatcttccaa atgaaaaagt actaccaaaa catagtttgc tttatgagta ttttacggtt 1560

tataacgaat tgacaaaggt caaatatgtt actgaaggaa tgcgaaaacc agcatttctt 1620

tcaggtgaac agaagaaagc cattgttgat ttactcttca aaacaaatcg aaaagtaacc 1680

gttaagcaat taaaagaaga ttatttcaaa aaaatagaat gttttgatag tgttgaaatt 1740

tcaggagttg aagatagatt taatgcttca ttaggtacct accatgattt gctaaaaatt 1800

attaaagata aagatttttt ggataatgaa gaaaatgaag atatcttaga ggatattgtt 1860

ttaacattga ccttatttga agatagggag atgattgagg aaagacttaa aacatatgct 1920

cacctctttg atgataaggt gatgaaacag cttaaacgtc gccgttatac tggttgggga 1980

cgtttgtctc gaaaattgat taatggtatt agggataagc aatctggcaa aacaatatta 2040

gattttttga aatcagatgg ttttgccaat cgcaatttta tgcagctgat ccatgatgat 2100

agtttgacat ttaaagaaga cattcaaaaa gcacaagtgt ctggacaagg cgatagttta 2160

catgaacata ttgcaaattt agctggtagc cctgctatta aaaaaggtat tttacagact 2220

gtaaaagttg ttgatgaatt ggtcaaagta atggggcggc ataagccaga aaatatcgtt 2280

attgaaatgg cacgtgaaaa tcagacaact caaaagggcc agaaaaattc gcgagagcgt 2340

atgaaacgaa tcgaagaagg tatcaaagaa ttaggaagtc agattcttaa agagcatcct 2400

gttgaaaata ctcaattgca aaatgaaaag ctctatctct attatctcca aaatggaaga 2460

gacatgtatg tggaccaaga attagatatt aatcgtttaa gtgattatga tgtcgatcac 2520

attgttccac aaagtttcct taaagacgat tcaatagaca ataaggtctt aacgcgttct 2580

gataaaaatc gtggtaaatc ggataacgtt ccaagtgaag aagtagtcaa aaagatgaaa 2640

aactattgga gacaacttct aaacgccaag ttaatcactc aacgtaagtt tgataattta 2700

acgaaagctg aacgtggagg tttgagtgaa cttgataaag ctggttttat caaacgccaa 2760

ttggttgaaa ctcgccaaat cactaagcat gtggcacaaa ttttggatag tcgcatgaat 2820

actaaatacg atgaaaatga taaacttatt cgagaggtta aagtgattac cttaaaatct 2880

aaattagttt ctgacttccg aaaagatttc caattctata aagtacgtga gattaacaat 2940

taccatcatg cccatgatgc gtatctaaat gccgtcgttg gaactgcttt gattaagaaa 3000

tatccaaaac ttgaatcgga gtttgtctat ggtgattata aagtttatga tgttcgtaaa 3060

atgattgcta agtctgagca agaaataggc aaagcaaccg caaaatattt cttttactct 3120

aatatcatga acttcttcaa aacagaaatt acacttgcaa atggagagat tcgcaaacgc 3180

cctctaatcg aaactaatgg ggaaactgga gaaattgtct gggataaagg gcgagatttt 3240

gccacagtgc gcaaagtatt gtccatgccc caagtcaata ttgtcaagaa aacagaagta 3300

cagacaggcg gattctccaa ggagtcaatt ttaccaaaaa gaaattcgga caagcttatt 3360

gctcgtaaaa aagactggga tccaaaaaaa tatggtggtt ttgatagtcc aacggtagct 3420

tattcagtcc tagtggttgc taaggtggaa aaagggaaat cgaagaagtt aaaatccgtt 3480

aaagagttac tagggatcac aattatggaa agaagttcct ttgaaaaaaa tccgattgac 3540

tttttagaag ctaaaggata taaggaagtt aaaaaagact taatcattaa actacctaaa 3600

tatagtcttt ttgagttaga aaacggtcgt aaacggatgc tggctagtgc cggagaatta 3660

caaaaaggaa atgagctggc tctgccaagc aaatatgtga attttttata tttagctagt 3720

cattatgaaa agttgaaggg tagtccagaa gataacgaac aaaaacaatt gtttgtggag 3780

cagcataagc attatttaga tgagattatt gagcaaatca gtgaattttc taagcgtgtt 3840

attttagcag atgccaattt agataaagtt cttagtgcat ataacaaaca tagagacaaa 3900

ccaatacgtg aacaagcaga aaatattatt catttattta cgttgacgaa tcttggagct 3960

cccgctgctt ttaaatattt tgatacaaca attgatcgta aacgatatac gtctacaaaa 4020

gaagttttag atgccactct tatccatcaa tccatcactg gtctttatga aacacgcatt 4080

gatttgagtc agctaggagg tgaccccaag aagaagagga aggtgggtgg agcaggtggc 4140

agtggcgcag ggggagccgg atataaattg atcctgaacg gcaaaacttt gaagggcgaa 4200

acaacaacag aagctgttga cgctgctacc gctgaaaaag tatttaaaca gtatgcaaac 4260

gacaacggtg ttgatggaga atggacatac gatgacgcaa ccaaaacctt cactgtcaca 4320

gaagccggag gggcaagcgg tgccgggggt ggtgggggag gctctcatca tcatcaccac 4380

caccaccacc accac 4395

<210> 20

<211> 65

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> F-7

<400> 20

taatacgact cactataggg gaacgcgtca aaaaggctgg gttttagagc tagaaatagc 60

aagtt 65

<210> 21

<211> 19

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> R-7

<400> 21

gcaccgactc ggtgccact 19

<210> 22

<211> 65

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> F-8

<400> 22

taatacgact cactataggg ggtcagcagt accagacgcc gttttagagc tagaaatagc 60

aagtt 65

<210> 23

<211> 116

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> scaffold containing niad-sgRNA

<400> 23

taatacgact cactataggg gaacgcgtca aaaaggctgg gttttagagc tagaaatagc 60

aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgc 116

<210> 24

<211> 116

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> pyrg-sgRNA containing backbone

<400> 24

taatacgact cactataggg ggtcagcagt accagacgcc gttttagagc tagaaatagc 60

aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgc 116

<210> 25

<211> 282

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> synthesized GB1 and linker

<400> 25

gccgtgggcg gatccggtgg ttcaggcggt agttataaat tgatcctgaa cggcaaaact 60

ttgaagggcg aaacaacaac agaagctgtt gacgctgcta ccgctgaaaa agtatttaaa 120

cagtatgcaa acgacaacgg tgttgatgga gaatggacat acgatgacgc aaccaaaacc 180

ttcactgtca cagaaggtgg tgggagcgga ggcggtggca gcggtggcgg cggtagtggc 240

ggtggcggtt cgggcggcgg tggttcgggt ggcggaggca gc 282

Claims

1. A method for efficiently expressing and purifying a Cas9 protein, which is characterized by comprising the steps of: the method comprises the following steps:

step 1, constructing a nuclease Cas9 expression vector pet28a+ -SpCas9-GB1-10 xHis-3T 7 driven by multiple promoters; the SpCas9-GB1-10 xHis sequence is shown in SEQ ID NO: 19; 3T7 refers to the 3-fold T7 promoter;

step 2, expression of the cas9 expression vector pep28a+ -SpCas9-GB1-10 xhis-3T 7: transferring the constructed pet28a+ -SpCas9-GB1-3T7 into a hostE.coli Rosetta (DE 3), a recombinant strain is obtained; transferring and activating the recombinant strain, performing induction expression by using IPTG, and centrifugally collecting thalli to obtain thalli expressing the SpCas9-GB1 protein;

2. The method according to claim 1, characterized in that: further comprising step 4:

3. The method according to claim 1 or 2, characterized in that:

the recombinant strain in the step 2 is selected in LB liquid medium of Kan+ and CHL+, and cultured at 37+ -1 ℃ and 200-220 rpm for 8-16 h activation; after activation, according to (1-5): 100 transferring ratio, taking activated bacterial liquid in LB liquid culture medium of Kan+ and CHL+, culturing at 37+ -1deg.C and 200-220 rpm to OD ₆₀₀ When the concentration is 0.6-0.8, IPTG is added to the final concentration of 0.3-0.8 mM for induction expression.

4. The method according to claim 1 or 2, characterized in that:

the conditions for inducing expression in the step 2 are 16-20 ℃ and 200-220 rpm; the fermentation time is 18-22 h.

5. The method according to claim 1 or 2, characterized in that:

the centrifugation condition in the step 2 is 4-8 ℃, and the centrifugation is carried out at 8000-10000 rpm for 5-10 minutes.

6. The method according to claim 1 or 2, characterized in that:

the conditions of ultrasonic disruption in step 3 are: ultrasonic switch 3s, ultrasonic switch 3s, power 30-40%, ultrasonic on ice for 20-30 min.

7. The method according to claim 1 or 2, characterized in that:

the centrifugation condition in the step 3 is 4-8 ℃, and the centrifugation is carried out at 8000-10000 rpm for 30-40 min.

8. The method according to claim 1 or 2, characterized in that:

in the step 3, the SpCas9-GB1 protein is eluted in a gradient way, the imidazole concentration of Buffer A is 0mM, and the imidazole concentration of Buffer B is 500 and mM; gradient 5%, 10%, 20%, 30%, 50% Buffer B;

Buffer A：500 mM NaCl，20 mM Tris-HCl，PH=8.0；

Buffer B：500 mM NaCl，500 mM Imidazole，20 mM Tris-HCl，PH=8.0。

9. use of the method of high-efficiency expression and purification of Cas9 protein according to any one of claims 1 to 8 for high-efficiency expression and purification of Cas9 protein.