CN111394422A - Cas9 protein functional activity detection method and application thereof - Google Patents

Cas9 protein functional activity detection method and application thereof Download PDF

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CN111394422A
CN111394422A CN202010351091.9A CN202010351091A CN111394422A CN 111394422 A CN111394422 A CN 111394422A CN 202010351091 A CN202010351091 A CN 202010351091A CN 111394422 A CN111394422 A CN 111394422A
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cas9 protein
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sgrna
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岑山
田东芳
张永欣
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Institute of Medicinal Biotechnology of CAMS
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Abstract

The invention relates to the technical field of biology, and particularly discloses a method for detecting functional activity of Cas9 protein and application thereof. In the method for detecting functional activity of Cas9 protein, Cas9 protein is derived from Streptococcus pyogenes of II-A type CRISPR system, and the method comprises the following steps: (1) respectively marking two ends of a double chain of a target DNA with a fluorescent group and biotin to obtain a substrate; the target DNA is recognizable by the sgRNA; the sgRNA is an RNA that can direct the type II-A CRISPR system to perform a function; (2) binding the substrate to the reaction vessel by binding biotin to avidin; (3) further reacting the substrate, Cas9 protein, and sgRNA in a reaction vessel; after the reaction is finished, cleaning a reaction vessel; (4) and (5) carrying out fluorescence signal detection. The method is rapid and effective, and can be applied to screening of inhibitors of Cas9 protein.

Description

Cas9 protein functional activity detection method and application thereof
Technical Field
The invention relates to the technical field of biology, in particular to a method for detecting functional activity of Cas9 protein and application thereof.
Background
The CRISPR system is an adaptive immune system for most bacteria and archaea, designed to combat exogenously invading nucleic acids. The CRISPR immune system performs a function, requiring 3 stages: adaptation, expression and interference. During the adaptation phase, the bacteria integrate nucleic acid sequences derived from the phage or foreign plasmid as spacer sequences between the repeated sequences of the CRISPR site. These spacer sequences perform a sequence memory function to ensure a targeted defense during subsequent invasion of the corresponding phage or plasmid. In the interference stage, the memory RNA is used as a guide, when homologous nucleic acid invades again, the RNA can quickly recognize the homologous sequence, and the nucleic acid which is invaded and recognized by the guide RNA is degraded by related protein, so that the aim of immunosuppression is fulfilled.
CRISPR systems are currently divided into two classes (class 1 and 2) and 19 subtypes, based on the sequence similarity between Cas genetic makeup, Cas proteins and gene structures, with 3 types (type I, III and IV) in class 1 and 3 types (type II, V and VI) in class 2. During the interference phase of the application of the CRISPR system of streptococcus pyogenes belonging to type II-a, Cas9 is involved as an important protein, comprising two important domains, HNH and RuvC. The RNA obtained in the first two stages is used as guide RNA (crRNA) to identify the target site. The target DNA has a PAM site (the PAM site of the II type CRISPR system is NGG) nearby the complementary strand of the target DNA, and the Cas9 can specifically recognize the PAM site. In addition, a trans-activation crRNA is needed for targeting a target sequence, and the two crRNAs are artificially fused to form a sgRNA with a guiding function. The sgRNA forms an R-loop structure when it binds to the target DNA strand. After structure formation, the HNH domain of Cas9 begins to cleave the complementary strand of the sgRNA and RuvC begins to cleave the non-complementary strand. Thereby degrading the exogenous invasive nucleic acid.
The study on the activity of the Cas9 protein is beneficial to the study on the mechanism of a bacterial CRISPR system, and the activity of the Cas9 protein can be controlled to influence the immune system of bacteria. Therefore, there is a need to provide a method for detecting functional activity of Cas9 protein and application thereof.
Disclosure of Invention
In order to solve the above problems, the present invention aims to provide a rapid and effective method for detecting functional activity of Cas9 protein.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a method for detecting functional activity of Cas9 protein, wherein the Cas9 protein is derived from streptococcus pyogenes of type II-a CRISPR system, comprising:
(1) respectively marking two ends of a double chain of a target DNA with a fluorescent group and biotin to obtain a substrate; the target DNA has a sequence complementary to the sgRNA and comprises a PAM site of a type II-a CRISPR system, recognized by the sgRNA; the sgRNA is an RNA that can direct the type II-A CRISPR system to perform a function;
(2) binding said substrate to a reaction vessel by binding said biotin to avidin;
(3) further reacting the substrate, the Cas9 protein, and the sgRNA in the reaction vessel; after the reaction is finished, cleaning the reaction vessel;
(4) and (5) carrying out fluorescence signal detection.
Compared with the traditional E L ISA, the invention has the advantages that:
(1) compared with direct E L ISA, the invention marks signal on nucleic acid, not protein, and the nucleic acid is easier to operate and lower in cost than protein.
(2) Compared with the indelect E L ISA, the invention does not need to combine a secondary antibody any more, thereby avoiding the occurrence of interaction reaction.
(3) The present invention does not require more than two binding sites for the antigen, as compared to the sandwich E L ISA.
(4) Compared to the competitive E L ISA, the present invention does not require two antigen competitions.
(5) Compared with the cell-based E L ISA, the invention does not need to be based on cells and can directly carry out detection at a cell-free level.
In the invention, the Cas9 protein sequence is an amino acid sequence shown as SEQ ID NO. 1; or an amino acid sequence which is obtained by replacing, inserting or deleting one or more amino acids in the amino acid sequence shown as SEQ ID NO.1 and has the same function with the Cas9 protein.
In the present invention, a fluorophore and biotin are labeled upstream of a complementary strand (a strand complementary to a sgRNA) and a non-complementary strand of a target DNA, respectively, or downstream of the complementary strand and the non-complementary strand of the target DNA, respectively, and then annealed to form a DNA double strand as a substrate. Also included in the target DNA are protective bases located upstream and downstream of the sequence complementary to the sgRNA, the number of protective bases being unlimited and can be designed according to common general knowledge in the art, the PAM site (TGG) being located downstream of the target sequence.
As a specific embodiment, the sequence of sgRNA used in the scheme of the invention is shown in SEQ ID NO.2 (the sequence is disclosed in Nature journal, Dong, Guo M, Wang S, et al. structural basis of CRISPR-spyCas9 inhibition by an anti-CRISPR protein. Nature.2017,546(7658): 436-439.). The sequences of two single-stranded nucleotides in the substrate adopted by the scheme of the invention are shown as SEQ ID NO.3 and SEQ ID NO.4, and the 3' ends of the two single-stranded nucleotides are respectively marked with a fluorescent group and Biotin (Biotin). In the invention, the avidin is streptavidin, and the fluorescent group is FAM.
SEQ ID NO.3：
5’-CTAGAGGATCCCAATCCCAGCCAAGCGCACCTAATTTCCGAATTCGTAATCATGGTCAT-FAM-3’；
SEQ ID NO.4：
5’-ATGACCATGATTACGAATTCGGAAATTAGGTGCGCTTGGCTGGGATTGGG ATCCTCTAG-Biotin-3’。
20bp of base in the sequence SEQ ID NO.3 and sgRNA can be complementarily paired (the 20bp of base is GCCAAGCGCACCTAATTTCC), 19bp of protective base and 20bp of protective base are respectively added at the upstream and downstream, and finally SEQ ID NO.3 is formed, wherein the PAM site is TGG.
In the step (2), streptavidin can be adopted to coat the reaction vessel, and then the substrate is added to be fixed on the reaction vessel through the combination of biotin and streptavidin.
PBS is used as a washing solution when washing the reaction vessel in step (3), and the main purpose of this step is to thoroughly wash out DNA fragments sheared off in the function of Cas 9.
In the step (4), a certain amount of PBS is added, and then fluorescence signal detection is carried out.
The method of the present invention is to test the functional activity of Cas9 protein according to a method similar to that of E L ISA.A streptavidin-coated 384-well plate can be used as a screening well plate.A substrate is a double-stranded structure formed by annealing two DNA strands, FAM fluorophore and Biotin are labeled at the 3' ends of complementary strand and non-complementary strand, respectively, as described above, and then after annealing to form double strands, a double-stranded DNA structure is formed, both ends of which are labeled with fluorophore and Biotin, Biotin can strongly interact with streptavidin, so that the substrate with the Biotin can be attached to the well plate by binding with streptavidin.A fluorescence labeled end is cleaved when Cas9 is functional, and then the fluorescence signal is washed away by washing the well plate with PBS, whereas when Cas9 is not functional or functionally inhibited, the fluorescence signal at the Biotin-labeled end is bound to the streptavidin-coated well plate, and the fluorescence signal at the other end is not washed away, and finally the fluorescence signal value of the fluorescence signal is detected by an enzyme immunoassay 9.
The method of the invention is schematically shown in figure 1. The fluorescently labeled substrate shown in the figure, when Cas9 is functional, the end with the FAM group will be cleaved off and the fluorescence will be washed away when the well plate is washed with PBS, and the fluorescence signal will decrease.
In the invention, the reaction system in the step (3) comprises a Cas9 protein solution 2 mu L, a substrate solution 2 mu L with the concentration of 1 mu M, a reaction solution 2 mu L, a sgRNA solution 2 mu L with the concentration of 400 ng/mu L and water 12 mu L, wherein the concentration of HEPES in the reaction solution is 200mM, and the concentration of KCl in the reaction solution is 1M, MaCl2Is 20mM, the concentration of the Cas9 protein in the solution of the Cas9 protein is 0.8-1.2mg/μ L (preferably 1mg/μ L), the concentration of Tris-HCl is 20mM, the concentration of NaCl is 500mM, and the concentration of glycerol by volume is 40%.
In the reaction system, the solvents in the solution of Cas9 protein, the solution of substrate, the reaction solution and the solution of sgRNA are all water.
In the invention, the reaction time of the step (3) is 2-3 hours, the reaction temperature is 36-38 ℃, preferably the reaction time is 2 hours, and the reaction temperature is 37 ℃, so that the full reaction is facilitated and the time is saved.
The invention also provides a kit for detecting the functional activity of the Cas9 protein, which comprises a streptavidin-coated reaction vessel, a substrate, the Cas9 protein and sgRNA; the substrate is a target DNA with double-stranded ends respectively labeled with a fluorescent group and biotin, the target DNA has a sequence complementary with the sgRNA and contains a PAM site of a II-A type CRISPR system, and the target DNA can be recognized by the sgRNA; the sgRNA is an RNA that can direct the type II-a CRISPR system to perform a function.
In the invention, a 20 mu L reaction system of the kit comprises a solution 2 mu L of the Cas9 protein, a solution 2 mu L of 1 mu M of the substrate, a solution 2 mu L of a reaction solution, a solution 2 mu L of the sgRNA of 400 ng/mu L of the reaction solution, and the balance of water, wherein the concentration of HEPES in the reaction solution is 200mM, and the concentration of KCl in the reaction solution is 1M, MaCl2Is 20mM, the concentration of the Cas9 protein in the solution of the Cas9 protein is 0.8-1.2 mg/mu L, the concentration of Tris-HCl is 20mM, the concentration of NaCl is 500mM, and the volume concentration of glycerol is 40%.
The invention further provides an application of the detection method or the kit in screening the inhibitor of the Cas9 protein.
In the invention, the reaction of the substrate, the Cas9 protein and the sgRNA is carried out while adding the inhibitor to be screened for reaction. The reaction conditions and time were the same as described above.
If the activity of the Cas9 protein is inhibited by the inhibitor to be screened, the fluorescence value detected by the microplate reader cannot be reduced compared with the initial value or the reduction fluctuation is small, so that the effect of the inhibitor to be screened can be judged according to the change of the fluorescence value.
In the present invention, the concentration of the inhibitor to be screened in the reaction system is not higher than 2mM (the lowest concentration is not set).
If the concentration of the inhibitor to be screened is too high, the streptavidin in a reaction vessel falls off, and false negative of an experimental result is caused.
As a specific embodiment, the reaction system for screening the inhibitor is 2 mu L of the Cas9 protein solution, 2 mu L of 1 mu M of the substrate solution, 2 mu L of the reaction solution, 2 mu L of the inhibitor with the concentration of 2mM, 2 mu L of the sgRNA with the concentration of 400 ng/mu L solution and 10 mu L of water, wherein the concentration of HEPES in the reaction solution is HEPES200mM (pH7.5), KCl concentration 1M, MaCl2Is 20mM, the concentration of the Cas9 protein in the solution of the Cas9 protein is 1mg/μ L, the concentration of Tris-HCl is 20mM, the concentration of NaCl is 500mM, and the volume concentration of glycerol is 40%.
Preferably, after the reaction is completed and the reaction vessel is thoroughly washed with PBS, 100. mu. L of PBS is added for detection of the fluorescence signal.
The method has high sensitivity, the reaction system only needs 20 mu L, and high-throughput screening can be carried out in a 384-well plate.
A specific screening process as a Cas9 protein inhibitor, comprising:
(1) respectively marking two ends of a double chain of a target DNA with a fluorescent group and biotin to obtain a substrate; the target DNA has a sequence complementary to the sgRNA and comprises a PAM site of a type II-a CRISPR system, recognized by the sgRNA; the sgRNA is an RNA that can direct the type II-A CRISPR system to perform a function;
(2) binding the substrate to a reaction vessel (384-well plate) by binding the biotin to avidin;
(3) further reacting the substrate, the Cas9 protein, the inhibitor to be screened, and the sgRNA in the reaction vessel; after the reaction is finished, cleaning the reaction vessel;
(4) and (5) carrying out fluorescence signal detection. The less the fluorescence signal is reduced from the initial fluorescence signal of the substrate, the more potent the inhibitor is.
The invention has the beneficial effects that:
the invention quantifies the cutting condition of double-stranded DNA by a fluorescence method, establishes a simple, sensitive and rapid quantification method, and further verifies the feasibility of the method as a Cas9 protein inhibitor high-throughput screening method. Through the screening of more than 2000 natural products, the screening model is evaluated, and the result proves that the model is suitable for high-throughput screening of the Cas9 protein.
The screening model can quickly, effectively and high-flux screen the inhibitor of the CRISPR system-associated protein Cas 9. The interaction relationship between biotin and streptavidin is fully utilized, so that the substrate can be combined on a reaction vessel. And the Z factor is calculated through experimental verification, which proves that the screening model is feasible. Compared with the traditional method for detecting the activity of the Cas9 by separating nucleic acid by using PAGE gel, the method shows the activity of the Cas9 through the strength of a fluorescence signal, and is more sensitive, quicker and more convenient.
Drawings
FIG. 1 is a schematic diagram of the method of the present invention;
FIG. 2 is a graph showing the results of the Z factor experiment in the screening method of the present invention;
FIG. 3 is a graph showing the results of factor Z of the inhibitor to be screened in example 2 of the present invention.
Detailed Description
Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1
In this embodiment, the stability of the Cas9 protein functional activity detection method provided by the present invention is verified (Z factor is calculated), and the specific method is as follows:
(1) constructing a substrate: adopting target DNA with two single-stranded nucleotide sequences shown as SEQ ID NO.3 and SEQ ID NO.4 as a substrate to be marked, respectively marking a fluorescent group and biotin at the 3' ends of the two single-stranded nucleotides, and then annealing to form a double strand as the substrate;
(2) a 384-well plate coated with streptavidin is used as a screening well plate (reaction vessel), and the substrate is added to be combined with the screening well plate;
(3) adding a reaction system consisting of Cas9 protein (Genebank: AII16589.1, derived from II-A type streptococcus pyogenes CRISPR system, with the sequence shown in SEQ ID NO. 1) and sgRNA (with the sequence shown in SEQ ID NO. 2) into 384-well plates combined with substrates respectively, and incubating at 37 ℃ for 2h to serve as a positive control;
the reaction system is specifically that the solution of Cas9 protein is 2 mu L, the solution of substrate is 1 mu M2 mu L, the reaction solution is 2 mu L, the solution of sgRNA is 400 ng/mu L2 mu L, water is added to make up 20 mu L, the concentration of HEPES in the reaction solution is 200mM (pH7.5), the concentration of KCl is 1M, MaCl2Is 20mM, the concentration of the Cas9 protein in the solution of the Cas9 protein is 1mg/μ L, the concentration of Tris-HCl is 20mM, the concentration of NaCl is 500mM, and the volume concentration of glycerol is 40%.
After the reaction, the well plate is washed by PBS to thoroughly wash off the DNA fragments sheared off in the reaction;
(3) adding a solution which is the same as the reaction system but does not contain Cas9 protein into a reaction hole, and incubating at 37 ℃ for 2h to serve as a negative control;
(4) 100 μ L PBS was added to each well, the fluorescence was measured in each well, and the Z factor was calculated as follows:
Figure BDA0002471954990000081
wherein SD represents the standard deviation of fluorescence values, and X represents the mean of fluorescence values.
Results referring to fig. 2, the number of each control sample was 48. The Z factor is an important index for judging whether a high-throughput screening mode has stability, the screening method is a better experiment by calculating that the Z factor (Z-factor) is 0.55 and is between 0.5 and 1.0, and meets the requirement of the stability of a high-throughput screening model in a credible interval of the Z factor.
Example 2
This example applies the Cas9 protein functional activity detection method of the present invention to screening Cas9 protein inhibitors. The specific method comprises the following steps:
(1) constructing a substrate: adopting target DNA with two single-stranded nucleotide sequences shown as SEQ ID NO.3 and SEQ ID NO.4 as a substrate to be marked, respectively marking a fluorescent group and biotin at the 3' ends of the two single-stranded nucleotides, and then annealing to form a double strand as the substrate;
(2) a 384-well plate coated with streptavidin is used as a screening well plate (reaction vessel), and the substrate is added to be combined with the screening well plate;
(3) adding a reaction system consisting of Cas9 protein (Genebank: AII16589.1, derived from II-A type streptococcus pyogenes CRISPR system, with the sequence shown in SEQ ID NO. 1), sgRNA (with the sequence shown in SEQ ID NO. 2) and each inhibitor to be screened into a 384-well plate combined with a substrate respectively, and incubating for 2h at 37 ℃;
the reaction system comprises a Cas9 protein solution 2 mu L, a substrate solution 2 mu L with the concentration of 1 mu M, a reaction liquid 2 mu L, an inhibitor to be screened with the concentration of 2mM 2 mu L, a sgRNA solution with the concentration of 400 ng/mu L2 mu L, and water with the concentration of 10 mu L, wherein the HEPES concentration in the reaction liquid is 200mM (pH7.5), and the KCl concentration is 1M, MaCl2The concentration of the Cas9 protein in the Cas9 protein solution is 20mM, the concentration of the Cas9 protein is 1mg/μ L, the concentration of Tris-HCl is 20mM, the concentration of NaCl is 500mM, and the volume concentration of glycerol is 40%. this example selects a total of 2056 compounds from natural product libraries, wherein the natural product library of ceramic biochemical technology limited (1440 compounds in total) and the natural product library of Selleck (616 compounds in total) are used as inhibitors to be screened;
after the reaction, the well plate is washed by PBS to thoroughly wash off the DNA fragments sheared off in the reaction;
(4) 100 μ L of PBS was added to each well, and the intensity of the fluorescence signal in each well was measured to calculate the deviation from the subject variance (Z value) by the following equation.
Z＝(Xi-XAverage)/SD
Where Xi is the measured fluorescence signal value of each well, XAverageThe mean fluorescence signal value of all samples and the SD standard deviation of the fluorescence signal values of all samples.
The test results are shown in FIG. 3, in which the number of compounds (having positive effects) screened in this example was 5 more than that of the subject, and the screening efficiency was high.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
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Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly
1090 1095 1100
Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile
1105 1110 1115 1120
Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser
1125 1130 1135
Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly
1140 1145 1150
Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile
1155 1160 1165
Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala
1170 1175 1180
Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys
1185 1190 1195 1200
Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser
1205 1210 1215
Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr
1220 1225 1230
Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser
1235 1240 1245
Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His
1250 1255 1260
Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val
1265 1270 1275 1280
Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys
1285 1290 1295
His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu
1300 1305 1310
Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp
1315 1320 1325
Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp
1330 1335 1340
Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile
1345 1350 1355 1360
Asp Leu Ser Gln Leu Gly Gly Asp
1365
<210>2
<211>98
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>2
ggaaauuagg ugcgcuuggc guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuu 98
<210>3
<211>59
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>3
ctagaggatc ccaatcccag ccaagcgcac ctaatttccg aattcgtaat catggtcat 59
<210>4
<211>59
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>4
atgaccatga ttacgaattc ggaaattagg tgcgcttggc tgggattggg atcctctag 59

Claims (8)

1. A method for detecting functional activity of Cas9 protein, wherein the Cas9 protein is derived from Streptococcus pyogenes of II-A type CRISPR system, and the method comprises the following steps:
(1) respectively marking two ends of a double chain of a target DNA with a fluorescent group and biotin to obtain a substrate; the target DNA has a sequence complementary to the sgRNA and comprises a PAM site of a type II-a CRISPR system, recognized by the sgRNA; the sgRNA is an RNA that can direct the type II-A CRISPR system to perform a function;
(2) binding said substrate to a reaction vessel by binding said biotin to avidin;
(3) further reacting the substrate, the Cas9 protein, and the sgRNA in the reaction vessel; after the reaction is finished, cleaning the reaction vessel;
(4) and (5) carrying out fluorescence signal detection.
2. The detection method according to claim 1, wherein the reaction system in step (3) comprises a solution of Cas9 protein 2 μ L, a solution of 1 μ M of the substrate 2 μ L, a reaction solution 2 μ L, a solution of 400ng/μ L of the sgRNA 2 μ L, and water 12 μ L, the concentration of HEPES in the reaction solution is 200mM, and the concentration of KCl in the reaction solution is 1M, MaCl mM2Is 20mM, the concentration of the Cas9 protein in the solution of the Cas9 protein is 0.8-1.2 mg/mu L, the concentration of Tris-HCl is 20mM, the concentration of NaCl is 500mM, and the volume concentration of glycerol is 40%.
3. The detection method according to claim 1 or 2, wherein the reaction time of step (3) is 2 to 3 hours, and the reaction temperature is 36 ℃ to 38 ℃.
4. A kit for detecting functional activity of Cas9 protein, which is characterized by comprising a reaction vessel coated by avidin, a substrate, a Cas9 protein and sgRNA; the substrate is a target DNA with double-stranded ends respectively labeled with a fluorescent group and biotin, the target DNA has a sequence complementary with the sgRNA and contains a PAM site of a II-A type CRISPR system, and the target DNA can be recognized by the sgRNA; the sgRNA is an RNA that can direct the type II-a CRISPR system to perform a function.
5. The kit of claim 4, wherein the 20 μ L reaction system of the kit comprises a solution of Cas9 protein 2 μ L, a solution of the substrate 2 μ L at 1 μ M, a reaction solution 2 μ L, a solution of the sgRNA 2 μ L at 400ng/μ L, and the balance of water, the concentration of HEPES in the reaction solution is 200mM, and the concentration of KCl in the reaction solution is 1M, MaCl mM2At a concentration of 20mM, the Cas9 protein in solutionThe concentration of Cas9 protein is 0.8-1.2 mg/mu L, the concentration of Tris-HCl is 20mM, the concentration of NaCl is 500mM, and the volume concentration of glycerol is 40%.
6. Use of the detection method of any one of claims 1-3 or the kit of claim 4 or 5 for screening for an inhibitor of the Cas9 protein.
7. The use of claim 6, wherein the substrate, Cas9 protein, and sgRNA reactions are performed simultaneously with the addition of an inhibitor to be screened for reaction.
8. The use according to claim 7, wherein the concentration of the inhibitor to be screened in the reaction system is not higher than 2 mM.
CN202010351091.9A 2020-04-28 2020-04-28 Cas9 protein functional activity detection method and application thereof Pending CN111394422A (en)

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Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BASUDEB MAJI等: "A High-Throughput Platform to Identify Small-Molecule Inhibitors of CRISPR-Cas9", 《CELL》 *
JARED CARLSON-STEVERMER等: "Assembly of CRISPR ribonucleoproteins with biotinylated oligonucleotides via an RNA aptamer for precise gene editing", 《NATURE COMMUNICATIONS》 *
PHILIP J.R. ROCHE等: "Efficient Homology Directed Repair by Cas9: Donor Localization and Cationic Polymeric Transfection in Mammalian Cells", 《BIORXIV PREPRINT DOI: HTTPS://DOI.ORG/10.1101/248179》 *
王思涵: "S.pyogenes Cas9的表达纯化及其活性抑制剂筛选的研究", 《中国优秀博硕士学位论文全文数据库(硕士)基础科学辑》 *

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