CN112391367A

CN112391367A - Preparation method of Cas9 protein for gene editing of human primary cells

Info

Publication number: CN112391367A
Application number: CN201910745370.0A
Authority: CN
Inventors: 龙飞; 张亚莉; 岳江嫚; 弓蒙蒙; 胡金芳; 张丽琴
Original assignee: Xi'an Yufan Biotechnologies Co ltd
Current assignee: Xi'an Yufan Biotechnologies Co ltd
Priority date: 2019-08-13
Filing date: 2019-08-13
Publication date: 2021-02-23

Abstract

The invention discloses a preparation method of Cas9 protein for gene editing of human primary cells, in particular to a method for purifying Cas9 protein by anion exchange chromatography, and the Cas9 protein purified by the method has in vitro activity and can specifically cut DNA sequence as proved by in vitro cutting test; meanwhile, the N end and the C end of the modified Cas9 protein are provided with nuclear positioning sequences, and specific genes can be knocked out after the modified Cas9 protein enters human primary cells through electric transformation, so that the purified Cas9 protein is proved to have in vivo activity.

Description

Preparation method of Cas9 protein for gene editing of human primary cells

Technical Field

The invention relates to the technical field of protein purification, and in particular relates to a preparation method of a Cas9 protein for gene knockout of human primary cells.

Background

The CRISPR/Cas is a novel nuclease system developed from an adaptive immune system of bacteria and archaea against foreign viruses or plasmids, and comprises three different types, wherein the DNA endonuclease Cas9 of the CRISPR/Cas system of Type II only has one subunit, the structure is simplest, and the application is most extensive. The CRISPR/Cas system of Type II consists of two elements of a guide sequence sgRNA and a nuclease Cas 9. The sgRNA is used for recognizing a PAM motif with a nucleotide sequence of 5 '-NGG-3' in a genome, combining with a target sequence according to a base complementary pairing principle, and the Cas9 specifically cuts the target sequence under the guidance of the sgRNA to realize the insertion and knockout of genes. Compared with ZFN and TALEN, the CRISPR/Cas9 system has the advantages of strong controllability, high specificity, wide application and the like.

At present, a CRISPR/Cas system can be used for gene editing of specific sites in vivo and in vitro, and gene functional research at a cellular level is realized by utilizing a sgRNA library in a plurality of literature reports. Plasmids or RNAs encoding Cas9 and sgRNAs are transduced into embryonic stem cells, fertilized eggs or embryos, and various gene knockout organisms such as drosophila, nematodes, zebrafish, mice, rats, cynomolgus monkeys, pigs and the like can be rapidly prepared. In addition, studies have shown that the CRISPR/Cas system can be used for gene repair treatment of genetic diseases in germ stem cells and induced pluripotent stem cells (ipscs). Although there is a debate on CRISPR off-target effect at present, as the research and practice of gene therapy are deepened, the application value of the CRISPR off-target effect is more and more concerned.

The CRISPR/Cas system has 3 common application modes, the first is to construct a Cas9 protein gene sequence and sgRNA onto a plasmid vector, then introduce the vector into a host cell, and express a Cas9 protein and sgRNA by using the host cell itself. However, the efficiency of plasmid transfection is limited, and thus the gene editing process cannot be performed quickly and efficiently. The second is co-injection of mRNA encoding Cas9 protein and sgRNA into host cells, but there are also problems of low efficiency of gene editing or inconsistent gene modification in the same animal due to delayed mRNA translation. The third is also the current ideal way to introduce Cas9 into host cells together with sgRNA in protein form, the process not only reduces the difficulty of introducing CRISPR/Cas system and greatly improves the gene editing efficiency, but also the Cas9 protein and sgRNA function immediately after entering cells or animal bodies in the form of complex and shortens the action time.

However, the current research on Cas9 focuses on gene editing using CRISPR/Cas platform technology, but in practical application, obtaining a purified Cas9 protein is a key technical means for subsequent research and application. The existing protein purification means are still more complicated, and host cell protein, host cell DNA, endotoxin and other related impurities introduced in the protein expression and purification process need to be removed one by one, so that high-purity protein is not easy to obtain to meet the requirements of animal and even human cell tests. Particularly, endotoxin is widely present in cell walls of gram-negative bacteria, and is likely to be produced in a large amount in the process of expressing recombinant protein by escherichia coli, and as a pyrogen, the endotoxin can cause inflammatory fever reaction of organisms, and can cause endotoxin shock, disseminated intravascular coagulation and the like in severe cases. Meanwhile, the fusion protein label used in the purification process can bring some uncertain factors in later application. Therefore, a simple and efficient preparation method of the Cas9 protein is urgently needed.

Disclosure of Invention

The invention aims to provide a simple and efficient Cas9 protein preparation method, wherein a GST fusion protein label of a Cas9 protein is cut off in a purification process, host cell DNA, endotoxin, impurity protein and the like are removed by using an anion column according to the positive charge characteristic of the Cas9 protein, and the Cas9 protein with high purity and concentration is obtained and the in vitro and in vivo activity of the protein is verified.

In order to achieve the above object, the present invention provides the following technical solutions:

the preparation method of the Cas9 protein for removing endotoxin is characterized by comprising the following steps

1) The Cas9 gene (type II CRISPR RNA-regulated endonuclease Cas9, Streptococcus pyogenes; NCBI Reference Sequence: WP _010922251.1) is cloned on a pGEX-4T-1 vector, a GST tag cutting site is inserted, and the vector is transformed into a prokaryotic expression strain to induce expression;

2) crushing thalli, collecting protein supernatant, purifying by a GST tag combined column, and carrying out enzyme digestion on the GST tag;

3) collecting the flow-through liquid obtained in the step 2), adjusting the pH value and the ionic strength of the flow-through liquid, and removing impurities by using an anion exchange column;

4) collecting the Cas9 protein obtained in the step 3), and concentrating by an ultrafiltration tube to obtain the high-concentration endotoxin-removed Cas9 protein.

Specifically, the GST tag cleavage site inserted in step 1) includes, but is not limited to, Leu-Glu-Val-Leu-Phe-Gln T.X-Gly-Pro (SEQ ID No.1, recognition sequence of PreScission Protease), Leu-Val-Pro-Arg-Gly-Ser (SEQ ID No.2, recognition sequence of Thrombin), Ile-Glu/Asp-Gly-Arg-XX (recognition sequences of SEQ ID No.3 and SEQ ID No.4, Factor Xa) or Asp-Asp-Asp-Asp-Lys (SEQ ID No.5, recognition sequence of Enterokinase).

Wherein no excess amino acid residues remain on the Cas9 protein after excision using the Factor Xa and Enterokinase enzymes, preferably inserted into the Factor Xa or Enterokinase cleavage sites.

Step 1) the conditions for inducible expression of the Cas9 protein are: according to the inoculation amount of 1:100, the expressed strain is inoculated in an LB culture medium containing benzyl amine resistance, the strain is cultured at 37 ℃ until the OD600 is about 0.8, a shaking table is cooled to 16 ℃, the strain is continuously cultured for half an hour, and then inducer IPTG is added into the culture solution until the final concentration is 0.5mmol/L, the strain is cultured overnight at 16 ℃ and 200 rpm.

Specifically, the method for crushing the thallus in the step 2) comprises the following steps: 140mM NaCl, 2.7mM KCl, 10mM Na were used₂HPO₄，1.8mM KH₂PO₄The cells were lysed and disrupted with PBS (pH 8.0).

The GST tag binding column is GSTSep Glutathione Agarose Resin.

The purification of the GST tag binding column comprises the following specific steps:

collecting induced bacteria: centrifuging at 4000rpm for 5min, and removing supernatant; resuspending the thalli into PBS buffer solution, crushing the thalli under ultrasonic wave or high pressure, and centrifuging the crushed suspension at low temperature and high speed to take supernatant; purifying the protein by using a GST column, balancing the GST column by using PBS with 5 times of the volume of the resin, washing impurities by using PBS with 3 times of the volume of the column, then eluting the resin by using a GSH elution buffer solution (PBS +5mM GSH), and measuring the concentration of the protein estimated by A280.

The enzyme digestion of the GST tag binding column comprises the following specific steps: and (3) dialyzing the collected GST-Cas9 protein in a digestion buffer solution, digesting with a proper protease, removing the cleaved GST tag and the GST-Cas9 protein without the cleaved tag by using a GST protein purification column, and collecting the flow-through solution.

The enzyme used for the GST tag enzyme digestion includes, but is not limited to, PreScission Protease, Thrombin, Factor Xa, Enterokinase, and the like. Preferably, the protease is Factor Xa or Enterokinase.

Specifically, a liquid changing step can be added before the step 3), and the specific method comprises the following steps: the GST-tagged Cas9 protein was eluted using desalting column eluent for the next purification of the anion column.

Specifically, the step 3) of removing impurities by using an anion exchange column comprises the following steps:

s1, activating and balancing resin: placing the anion exchange column in a chromatography system, adjusting the flow rate of the system to be 1mL/min, washing 5 column volumes of a balance solution pH8.0, 20mM Tris +50mM NaCl;

s2, sample loading: pushing 10mL of loading protein into a loading ring at a constant speed, wherein the flow rate is 1 mL/min;

s3, collecting a sample: observing the change of UV280, and collecting the flow peak protein when the UV280 is in an ascending trend;

s4, eluting impurities and endotoxin: washing the column with an eluent of 20mM Tris +1M NaCl until impurities and endotoxin are washed out, and collecting an elution peak;

s5, cylinder regeneration and preservation: and after the elution peak is finished, performing CIP on the column, flushing to be neutral, flushing the 20% ethanol protected column, and taking down the column for storage at normal temperature.

Specifically, the Cas9 protein is concentrated by the ultrafiltration tube in the step 4), and the protein concentration is more than 2 mg/mL.

Therefore, the application provides a preparation method of Cas9 protein for gene editing, which comprises the steps of cloning Cas9 gene to pGEX-4T-1 vector, inserting a proper GST tag cleavage site at the 5' end of the Cas9 gene if necessary, transforming the Cas9 gene to prokaryotic expression strain, breaking the strain after induction expression, collecting protein supernatant, and purifying. The GST tag was enzymatically cleaved during purification using GST tag-bound column Glutathione Sehpase, and the flow-through was collected and its pH and ionic strength were adjusted. And (3) passing through an anion exchange column to enable impurities such as most of host cell protein residues, host cell DNA residues, endotoxin and the like to be combined on the anion exchange column, and the positively charged Cas9 protein is not combined with the anion exchange column or the membrane, so that the purpose of protein purification is achieved, and finally the high-concentration Cas9 protein is obtained by concentration through an ultrafiltration tube.

The method comprises the following steps:

(1) to enable the expressed Cas9 protein to exert a cleavage function within mammalian cells, we added a nuclear localization sequence across the bacterial Cas9 cDNA.

(2) Cas9 expression plasmid construction: the Cas9 gene with the nuclear localization sequence is cloned to a pGEX-4T-1 vector, then a proper enzyme cutting site is inserted into the N end, and the N end is transformed into a prokaryotic cell to express the Cas9 recombinant protein. The inserted enzyme cleavage site includes, but is not limited to, any one of Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro (SEQ ID No.1, recognition sequence of PreScission Protease), Leu-Val-Pro-Arg-XXX-Gly-Ser (SEQ ID No.2, recognition sequence of Thrombin), Ile-Glu/Asp-Gly-Arg (SEQ ID No.3 and SEQ ID No.4, recognition sequence of Factor Xa) or Asp-Asp-Asp-Asp-Lys (SEQ ID No.5, recognition sequence of Enterokinase).

(3) Preferably, the cleavage site is Ile-Glu/Asp-Gly-Arg XX (recognition sequences of SEQ ID No.3 and SEQ ID No.4, Factor Xa) or Asp-Asp-Asp-Lys XX (recognition sequence of SEQ ID No.5, Enterokinase).

(4) The conditions for induction of Cas9 protein expression were: according to the inoculation amount of 1:100, the expressed strain is inoculated in an LB culture medium, cultured at 37 ℃ for 8h until the OD600 is about 0.8, added with IPTG with the final concentration of 0.5mM and induced to express at 16 ℃ overnight.

(5) GST column purification step: collecting the induced thallus: centrifuging at 4000rpm for 5min, and discarding the supernatant. The cells were resuspended in PBS buffer and disrupted by sonication or high pressure. And centrifuging the crushed suspension at low temperature and high speed to obtain a supernatant. The protein was purified using a GST column. The GST column was equilibrated with 5 column volumes of PBS, the impurities were washed with 3 column volumes of PBS, and then the resin was subjected to protein elution with GSH elution buffer (PBS +5mM GSH), and the protein concentration was estimated by A280 assay.

(6) GST tag enzyme digestion: the collected GST-Cas9 protein was dialyzed against digestion buffer and digested with the appropriate protease. And removing the cut GST tag and GST-Cas9 protein without the cut tag by using a GST protein purification column, and collecting the flow-through liquid.

(7) Buffer for desalting column replacement protein: the protein background buffer was replaced using a desalting column, and the protein was replaced into 20mM Tris +50mM NaCl buffer at pH8.0, at a flow rate of 2mL/min for the chromatographic system. Observing the change of the system A280, and collecting the protein.

(8) Removing impurity protein and endotoxin by using an anion exchange column: an anion exchange column was used to remove foreign proteins and endotoxins other than the target protein, the equilibrium buffer was pH8.0, 20mM Tris +50mM NaCl, the elution buffer was pH8.0, 20mM Tris +1M NaCl, and the system flow rate was 1 mL/min. Collecting flow-through peak and high-salt elution peak, wherein the flow-through peak is target protein Cas9, and the elution peak is impurity protein and endotoxin.

(9) Concentration: filtering the Cas9 protein with impurity protein and endotoxin removed by a filter membrane of 0.22 mu m, concentrating by a 10kDa ultrafiltration tube to obtain Cas9 protein with the concentration of 2mg/mL, subpackaging with liquid nitrogen, quick-freezing and storing at-80 ℃.

The Cas9 protein purified by the method has in vitro activity and can specifically cut a DNA sequence as proved by an in vitro cutting test; meanwhile, the N end and the C end of the modified Cas9 protein are provided with nuclear positioning sequences, and specific genes can be knocked out after the modified Cas9 protein enters human primary cells through electric transformation, so that the purified Cas9 protein is proved to have in vivo activity and can be used for gene editing of the primary cells.

Due to gene editing in animals and particularity of human primary cells, high-purity test materials are needed and no exogenous impurities are contained, the Cas9 protein prepared by the method removes fusion tags, reduces the influence of the protein on the structure and function of the protein, simultaneously utilizes the characteristic that most of impurity proteins and endotoxin are negatively charged and Cas9 protein is positively charged in the purification process, utilizes an anion column to remove impurity proteins and endotoxin, and reduces the content of host cell proteins, host cell DNA and endotoxin in the Cas9 protein to the level allowed by clinical tests through tests (the specific test standard is < Chinese pharmacopoeia > 2015 third department general rule 3407 and < Chinese pharmacopoeia > 2015 third department general rule 1143). In addition, the test standard and in vitro and in vivo activity detection method of the Cas9 protein obtained by the preparation method are also within the protection scope of the invention.

Compared with the prior art, the invention has the beneficial effects that:

1, cutting off a GST fusion protein tag in a Cas9 protein purification process, reducing the influence of the tag on the structure and function of the protein, and reducing the immunogenicity of the protein; wherein no excess amino acid residues remain on the Cas9 protein after excision using the Factor Xa and Enterokinase enzymes, preferably inserted into the Factor Xa or Enterokinase cleavage sites.

2. In the purification process, the impurity proteins and endotoxin are removed in one step by using the difference of the electric charge of the impurities such as Cas9 protein and endotoxin, the impurity removal step is simplified, the protein recovery rate is improved, and the production cost is reduced;

3. the Cas9 protein purified by the method has in vitro activity and can specifically cut a DNA sequence as proved by an in vitro cutting test; after entering human primary cells through electrical transformation, specific genes can be knocked out, and the purified Cas9 protein is proved to have in vivo activity and can be used for gene editing of primary cells.

Drawings

FIG. 1 is a vector map of recombinant plasmid pGEX-4T-1-Cas 9.

FIG. 2 is a protein glue pattern verification before and after the enzyme digestion of GST tag, wherein,

m: marker; 1: GST-Cas9 protein; 2: an enzymatically cleaved Cas9 and GST tag mixture; 3: the GST-tagged Cas9 protein was removed.

FIG. 3 is a protein glue diagram before and after Cas9 protein passes through a desalting column, wherein,

m: marker; 1: loading protein on a desalting column; 2-3: after passing through a desalting column, Cas9 protein was collected.

FIG. 4 is a protein gel diagram before and after Cas9 protein passes through an anion exchange column, wherein,

m: marker; 1: an anion loading protein; 2: anion flux peak punch (Cas9 protein); 3: anion elution peak.

Fig. 5 is an in vitro activity assay of Cas9 protein, wherein;

m: marker; 1: negative control (no Cas9 protein); 2: positive control (commercially available 0.5ug of Cas9 protein); 3. 4: experimental group (0.5ug Cas9 protein purified according to this method).

FIG. 6 is an in vivo activity assay, wherein;

NC: samples without electrotransfer; 1-5: samples subjected to electrotransformation.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

Example 1: prokaryotic expression for constructing recombinant plasmids pGEX-4T-1-Cas9 and Cas9 protein

1.1, construction of recombinant plasmid pGEX-4T-1-Cas9 vector

The C end of the GST sequence of the vector pGEX-4T-1 is connected with a Factor Xa restriction site, and the C end of the restriction site is connected with a Cas9 fragment with a nuclear localization sequence.

The plasmid map is shown in FIG. 1. Wherein the base sequence of the Thrombin enzyme recognition site is SEQ ID No. 6: CTGGTTCCGCGTGGATCC (SEQ ID No. 2: Leu-Val-Pro-Arg-Gly-Ser); the base sequence of the Factor Xa recognition site is SEQ ID No. 7: ATCGAAGGTCGT (SEQ ID No. 3: Ile-Glu-Gly-Arg). Since the cleavage site of Factor Xa is at the last position of the recognition sequence Ile-Glu-Gly-Arg, Cas9 protein does not contain any extra amino acid residues after cleavage by Factor Xa.

1.2 prokaryotic expression of recombinant plasmid pGEX-4T-1-Cas9

1) Transforming plasmid pGEX-4T-1-Cas9 into the DE3 expression strain;

2) selecting a recombinant bacterium monoclonal to be cultured in 25mL LB culture medium at 37 ℃ overnight, inoculating an overnight strain to 1L LB culture medium according to the inoculation amount of 1:100, culturing at 37 ℃ until OD600 is about 0.8, continuing culturing at 16 ℃ for 30min, adding IPTG until the final concentration is 0.5mM, and culturing at 16 ℃ overnight;

3) collecting thalli: centrifuging at 4000rpm for 5min, and removing supernatant;

4) breaking the cells: suspending the thallus into a certain amount of PBS buffer solution, and then carrying out ultrasonic crushing;

5) collection of protein extract: 12000g of thallus suspension after ultrasonic disruption is centrifuged for 20min, and the supernatant is taken, namely the protein extracting solution.

Example 2: purification of Cas9 protein

2.1 purification of GST-Cas9 protein

1) GST column treatment: taking out the bottom cap of the GST column, dripping redundant liquid, vertically fixing the column with the top upward, washing the column with distilled water once, and balancing the column with PBS (phosphate buffer solution) with 5 times of the volume of the resin;

2) mediator binding to protein: protein extract was added from the top of the column until the addition was complete. Collecting and washing the flow-through liquid: washing the GST column with PBS 3 times the volume of the gel column;

3) sample collection: adding GSH elution buffer solution into resin, and collecting eluate. Measuring A280, and estimating the amount of eluted GST-Cas9 protein;

4) column washing: the column was washed with 3 resin volumes of PBS and then stored with 10 column volumes of 20% ethanol.

2.2 enzyme digestion of GST-Cas9 protein

The collected GST-Cas9 protein was dialyzed into dialysate (pH 8.0, 20mM Tris-HCl, 100mM NaCl and 2mM CaCl)₂) Middle, 4 ℃ overnight. The next day, the enzyme digestion was carried out with Factor Xa under the conditions described in the product instructions.

2.3 GST column elution method

The tagged Cas9 protein was mixed with the tag, the cleaved GST tag and the non-tagged GST-Cas9 protein were removed using a GST protein purification column, and the flow-through was collected. The label cutting effect is as shown in figure 2.

2.4 buffer solution for replacing Cas9 protein by desalting column

1) Activated, equilibrated resin: adjusting the flow rate of the system to be 2mL/min, adjusting the base line of the system to be 0, and flushing 5 column volumes of equilibrium solution with pH of 8.0 and 20mM Tris +50mM NaCl;

2) loading: suspending the chromatography system, sucking 3mL of loading protein by using a 5mL syringe, pushing the protein into a loading ring at a constant speed, starting the chromatography system by continuously using 2mL/min, and observing the change of UV280 and UV 230;

3) collecting the protein: collecting protein peaks, and reserving samples and running gel;

4) regeneration and preservation of the column: after sample collection was complete, the column was equilibrated with 5 column volumes of equilibration buffer, pH8.0, 20mM Tris +50mM NaCl. Protecting the column with 20% ethanol, and storing at room temperature.

The protein condition before and after passing through the desalting column is shown in figure 3.

2.5, removing impurities and endotoxin in Cas9 protein

1) Activated, equilibrated resin: adjusting the flow rate of the system to be 1mL/min, adjusting the base line of the system to be 0, and flushing 5 column volumes of equilibrium solution with pH of 8.0 and 20mM Tris +50mM NaCl;

2) loading: suspending the chromatography system, sucking 10mL of loading protein by using a 10mL syringe, pushing the protein into a loading ring at a constant speed, and continuously starting the chromatography system by using a flow rate of 1 mL/min;

3) sample collection: observing the change of UV280 and UV230, and collecting flow-through peak proteins;

4) impurity and endotoxin elution: after collecting the flow-through peak protein, washing the column with an eluent of pH8.0, 20mM Tris +1M NaCl until impurities and endotoxin are washed out, collecting the elution peak and carrying out gel running;

5) regeneration and preservation of the column: after the end of the peak elution, the column was equilibrated with 5 column volumes of equilibration buffer pH8.0, 20mM Tris +50mM NaCl, and protected with 20% ethanol, and removed for storage at ambient temperature.

2.6, concentration: filtering the Cas9 protein with impurity protein and endotoxin removed by a filter membrane of 0.22 mu m, concentrating by a 10kDa ultrafiltration tube to obtain Cas9 protein with the concentration of 2mg/mL, subpackaging with liquid nitrogen, quick-freezing and storing at-80 ℃.

2.7 detection of protein purification result of Cas9

The target band of the Cas9 protein is about 162kD (containing N segment and C terminal nuclear localization sequence), as shown in fig. 4, the finally obtained Cas9 protein band is clear, and the purity of the protein is more than 95% as shown by scanning gray scale with software.

Example 3: activity detection of Cas9 protein

3.1 detection of in vitro Cas9 protein Activity

The Cas9 protein and sgRNA work together to cut the target DNA sequence in vitro. We selected the TP53 gene as a substrate for testing Cas9 protein activity, and designed sgRNA (SEQ ID No.8) against the TP53 gene: GAGCGCTGCTCAGATAGCGATGG, and carrying out in vitro transcription, adding the Cas9 protein and sgRNA compound into a TP53 gene fragment amplified by PCR (F: CCAACTCTCTCTAGCTCGC (SEQ ID No. 9); R: ACGGGGTTTCACCGTTAGCC (SEQ ID No.10)), and electrophoretically cutting the fragment into two fragment fragments with the fragment sizes of 270bp and 530bp respectively, thereby determining that the Cas9 protein has in vitro activity.

The specific method for detecting the in vitro activity comprises the following steps: 100ng of target DNA, 100ng of sgRNA and 0.5ug of Cas9 protein were added to a 10uL reaction system, reacted at 37 ℃ for 1h, denatured at 65 ℃ for 10min, and the cleavage effect was analyzed using 0.8% agarose gel. When only sgRNA was added, the target DNA could not be cut; the Cas9 protein and sgRNA complex is added into a TP53 fragment amplified by PCR, and a target DNA fragment of 750bp is cut into fragments of 270bp and 530bp, so that the Cas9 protein obtained after purification has good in vitro activity, and is shown in figure 5.

3.2 detection of in vivo Cas9 protein Activity

Cas9 protein (16ug) and sgRNA (8ug) were mixed in proportion to a 10uL system and incubated at 37 ℃ for 10 min; sucking and mixing the sorted and activated human primary T cells, centrifuging at room temperature at 1000rpm for 5min to precipitate the cells, removing the supernatant culture medium, resuspending the cells with an electric transfer buffer R, and using

The electric transfer system performs electric transfer. Western Blot detection is carried out on T cell TP53 protein 72h after electrotransformation, and the gray scale ratio of the target gene and the reference gene of each sample is calculated, so that the knockout effect of the Cas9 protein-gRNA complex on TP53 is calculated. As can be seen from fig. 6, the purified Cas9 protein has high in vivo activity.

Example 4: endotoxin detection of Cas9 protein

The method for detecting the endotoxin content of the Cas9 protein by using a limulus reagent endotoxin detection kit comprises the following basic principles: the limulus reagent endotoxin detection kit contains factor C, factor B, proclotting enzyme, coagulogen and the like, and under proper conditions (temperature, pH value and non-interference substances), bacterial endotoxin activates the factor C to cause a series of enzymatic reactions, so that the limulus reagent generates agglutination reaction to form gel. If the product to be tested is negative, no gel can be formed.

The specific detection method comprises the following steps: substituting the concentration of Cas9 protein into a formula (0.25Eu/mL 0.2mL)/(2.1 μ g/μ L0.5 Eu/μ g) to calculate the adding amount of the protein, taking a corresponding amount of Cas9 protein, diluting the Cas9 protein to a volume to be measured by using sterile water for injection, and transferring the same volume of sterile water for injection into a limulus reagent tube to serve as negative control; placing the limulus reagent detection tube rack in a suspension float, reacting for 1h in a water bath kettle at 37 ℃, and observing whether the limulus reagent detection tube rack is coagulated; if the coagulation is not satisfactory, and if the coagulation is still in a liquid state, the coagulation is satisfactory.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.

In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.

SEQUENCE LISTING

<110> Xian Yuan Sheng Biotechnology Limited liability company

<120> preparation method of Cas9 protein capable of being used for human primary cell gene editing

<130> 2019

<160> 10

<170> PatentIn version 3.5

<210> 1

<211> 8

<212> PRT

<213> Artificial sequence

<400> 1

Leu Glu Val Leu Phe Gln Gly Pro

1 5

<210> 2

<211> 6

<212> PRT

<213> Artificial sequence

<400> 2

Leu Val Pro Arg Gly Ser

1 5

<210> 3

<211> 4

<212> PRT

<213> Artificial sequence

<400> 3

Ile Glu Gly Arg

1

<210> 4

<211> 4

<212> PRT

<213> Artificial sequence

<400> 4

Ile Asp Gly Arg

1

<210> 5

<211> 5

<212> PRT

<213> Artificial sequence

<400> 5

Asp Asp Asp Asp Lys

1 5

<210> 6

<211> 18

<212> DNA

<213> Artificial sequence

<400> 6

ctggttccgc gtggatcc 18

<210> 7

<211> 12

<212> DNA

<213> Artificial sequence

<400> 7

atcgaaggtc gt 12

<210> 8

<211> 23

<212> DNA

<213> Artificial sequence

<400> 8

gagcgctgct cagatagcga tgg 23

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<212> DNA

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ccaactctct ctagctcgc 19

<210> 10

<211> 20

<212> DNA

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acggggtttc accgttagcc 20

Claims

1. The preparation method of the Cas9 protein for removing endotoxin is characterized by comprising the following steps

1) The Cas9 gene is cloned to a pGEX-4T-1 vector, a GST tag cutting site is inserted, and the vector is transformed to a prokaryotic expression strain for induced expression;

4) collecting the Cas9 protein obtained in the step 3), and concentrating by an ultrafiltration tube to obtain the Cas9 protein with high concentration and endotoxin removal.

2. The method of claim 1, wherein the step 3) of removing impurities using an anion exchange column comprises the steps of:

s1, activating and balancing resin: placing the anion exchange column in a chromatography system, adjusting the flow rate of the system to be 1mL/min, and washing 5 column volumes of equilibrium solution 20mM Tris +50mM NaCl, with the pH value of 8.0;

3. The method of claim 1, wherein the inserted GST tag cleavage site of step 1) comprises Leu-Glu-Val-Leu-Phe-Gln t-Gly-Pro, a recognition sequence of PreScission Protease; Leu-Val-Pro-Arg t-Gly-Ser, a recognition sequence of Thrombin; Ile-Glu/Asp-Gly-Arg T, i.e., Factor Xa recognition sequence, or Asp-Asp-Asp-Asp-Lys T, i.e., Enterokinase recognition sequence.

4. The preparation method according to claim 1 or 2, wherein the conditions for inducible expression of the Cas9 protein in step 1) are as follows: according to the inoculation amount of 1:100, the expressed strain is inoculated in an LB culture medium containing benzyl amine resistance, the strain is cultured at 37 ℃ until the OD600 is about 0.8, a shaking table is cooled to 16 ℃, the strain is continuously cultured for half an hour, and then inducer IPTG is added into the culture solution until the final concentration is 0.5mmol/L, the strain is cultured overnight at 16 ℃ and 200 rpm.

5. The method according to claim 1, wherein the step 2) comprises the steps of: 140mM NaCl, 2.7mM KCl, 10mM Na were used₂HPO₄，1.8mM KH₂PO₄The cells were lysed and disrupted with PBS (composition) at pH 8.0.

6. The method according to claim 1, wherein the GST tag-binding column of step 2) is GSTSep Glutathione Agarose Resin.

7. The preparation method of claim 5 or 6, wherein the step 2) of purifying the GST-tag-bound column comprises the following specific steps:

collecting induced bacteria: centrifuging at 4000rpm for 5min, and removing supernatant; resuspending the thalli into PBS buffer solution, crushing the thalli under ultrasonic wave or high pressure, and centrifuging the crushed suspension at low temperature and high speed to take supernatant;

purifying the protein by using a GST column, balancing the GST column by using PBS with 5 times of the volume of the resin, washing impurities by using PBS with 3 times of the volume of the column, then eluting the protein solution by using GSH elution buffer PBS +5mM GSH, and measuring A280 to estimate the concentration of the protein.

8. The preparation method of claim 5 or 6, wherein the GST tag-binding column enzyme in step 2) is prepared by the following steps: and (3) dialyzing the collected GST-Cas9 protein in a digestion buffer solution, digesting with a proper protease, removing the cleaved GST tag and the GST-Cas9 protein without the cleaved tag by using a GST protein purification column, and collecting the flow-through solution.

9. The method of claim 7, wherein the GST tag enzyme in step 2) is selected from the group consisting of PreScission protease, Thrombin, Factor Xa and Enterokinase.

10. The preparation method according to claim 1, wherein a liquid changing step can be added before the step 3), and the method comprises the following steps: the GST-tagged Cas9 protein was washed out using desalting column buffer for further purification on the anion column.