CN115786303A - In vitro cleavage efficiency kit and application of Cas9 H840A mutant based on CRISPR-Cas9 - Google Patents

Abstract

The invention discloses an in-vitro shearing efficiency kit based on CRISPR-Cas9 and application thereof. The kit comprises a Cas9 nuclease mutant H840A, a target gene, sgRNA-1, sgRNA-2 and 10X reaction buffer. Two sgRNA sequences respectively complementary to the target gene can be combined with Cas9H840A, and the Cas9H840A-sgRNA compound can be matched with the target DNA for cutting to form double-strand break of the target gene. By the test method, the efficiency of the Cas9H840A-sgRNA compound on target gene in-vitro cutting can be rapidly detected, and the success rate of subsequent intracellular gene editing is improved.

Description

CRISPR-Cas9-based in vitro cleavage efficiency kit for Cas9 H840A mutant and applications

technical field

The invention belongs to the field of genetic engineering and relates to a Cas9H840A mutant in vitro shearing efficiency detection kit based on CRISPR-Cas9 technology. In addition, the invention also discloses the application of the kit.

Background technique

Genetic engineering is a powerful tool widely used in biological science research. The development of CRISPR (clustered regularly interspaced short palindromic repeats/cas, clustered, regularly interspaced short palindromic repeats/cas) technology, also known as CRISPR-Cas9 (CRISPR Associated Protein 9, CRISPR-associated protein 9), has revolutionized the field of gene editing.

CRISPR-Cas9 can facilitate gene editing by directional cutting DNA, triggering DNA double-strand break (DSB), which can transform almost any organism and cell type. In addition, CRISPR-Cas9 technology has been successfully applied to many other applications, including regulation of endogenous gene expression, epigenome editing, live cell labeling of chromosomal loci, single-stranded RNA editing, and high-throughput genetic screening, among others.

The CRISPR-Cas9 system consists of two parts: a Cas9 protein with nuclease properties and a single-stranded guide RNA (sgRNA). The Cas9 protein is an RNA-dependent endonuclease that contains two nuclease domains: the HNH domain and the RuvC domain, which are responsible for cleaving the complementary and non-complementary strands, respectively, by pairing the sgRNA with the targeting DNA sequence. The Cas9 H840A mutant inactivates the HNH domain of the Cas9 nuclease, so Cas9H840A has only the RuvC domain with cleavage activity, and can only generate a nick on the DNA strand that is non-complementary to the sgRNA target sequence, thereby forming a functional single-strand break, Unintended off-target genetic modifications can be reduced.

sgRNA, also called gRNA (single guide RNA), consists of crRNA (CRISPR RNA) sequence and trRNA (trans-activating crRNA) sequence. The sgRNA guides the Cas9 nuclease to the target DNA for cutting through complementary pairing with the target DNA sequence. PAM (proto-spacer adjacent motif, protein auxiliary motif) is a plurality of nucleotides adjacent to the target site, which is a unique and key component of the Cas9 nuclease directional cutting target DNA, 5'-NGG-3' Can be recognized by Cas9 nuclease as PAM. Under the cooperative action of the two domains of HNH and RuvC, the Cas9 nuclease cleaves the target DNA about three bases upstream of the PAM sequence NGG to generate a DNA double-strand break (DSB).

Under the guidance of sgRNA, the Cas9 nuclease can site-specifically cut the target sequence of genomic DNA of prokaryotic and eukaryotic organisms, and then use error-prone repair (error-prone repair) such as non-homologous end joining Nucleotide insertion, deletion or Replacement, which may generate frameshift mutations, resulting in deletion mutations of the target gene.

With the development of CRISPR technology, this technology can not only realize gene knockout, but also realize gene point mutation, insertion mutation and other mutation methods, especially in clinical application, it can be used to repair bad mutations.

Contents of the invention

One of the technical problems solved by the present invention is to provide a Cas9H840A mutant in vitro cleavage efficiency detection kit based on CRISPR-Cas9 technology, which enables the Cas9 nuclease H840A mutant to perform fixed-point cleavage on the coding sequence of the target gene, and can quickly Detect the efficiency of the Cas9H840A-sgRNA complex for in vitro cleavage of target DNA, and improve the success rate of subsequent intracellular gene editing.

The second technical problem solved by the present invention is to provide the application of the kit, which contains Cas9H840A, sgRNA-1, sgRNA-2, target gene and 10X reaction buffer, through the technical scheme of the kit, can detect the Cas9H840A Splicing activity or off-target effects of sgRNA can improve the success rate of gene editing in subsequent cells.

In order to achieve the above object, technical solution of the present invention is achieved in that way:

The Cas9 H840A mutant in vitro shearing efficiency kit based on CRISPR-Cas9, the kit includes a combination of Cas9 H840A nuclease, target gene, sgRNA-1, sgRNA-2 and 10X reaction buffer; the sgRNA-1 contains Two key sequences, namely crRNA sequence and trRNA sequence, wherein the crRNA sequence can specifically recognize the sgRNA of the target gene, and the trRNA sequence can recognize and bind to the Cas9 H840A nuclease, so as to guide the Cas9 H840A nuclease to specifically cut the corresponding sequence of the target gene; sgRNA-2 contains two key sequences, namely crRNA sequence and trRNA sequence, in which the crRNA sequence can specifically recognize the sgRNA of the target gene, and the trRNA sequence can recognize and bind to the Cas9 H840A nuclease, so as to guide the Cas9 H840A nuclease to specifically cut the corresponding target gene sequence.

Further, the sgRNA-1 sequence is shown in SEQ ID NO.1, and the sgRNA-2 sequence is shown in SEQ ID NO.2.

Further, the target gene sequence is shown in SEQ ID NO.3.

Further, the Cas9 H840A nuclease is expressed and purified through prokaryotic or eukaryotic protein expression systems (including but not limited to Escherichia coli protein expression systems, yeast protein expression systems, insect protein expression systems and cell protein expression systems).

Further, Cas9 nuclease mutants include but are not limited to H840A mutants or D10A mutants.

Further, the 10X reaction buffer formulation includes: 0.5M-3M NaCl, 100mM-1M Tris-HCl, pH7.0-9.0, 50mM-500mM MgCl ₂ , 0mg/mL-10mg/mL recombinant albumin; preferably , the 10X reaction buffer formulation includes: 1M NaCl, 500mM Tris-HCl, pH 7.9, 100mM MgCl ₂ , 1mg/mL recombinant albumin.

Further, it is applied through the following steps:

Step 1: Add Cas9 H840A nuclease, target gene, sgRNA-1, sgRNA-2 and 10X reaction buffer into 10 μl reaction system in proportion;

Step 2, constant temperature reaction, heat inactivation, low temperature storage;

Step 3: Add 2 μl of 6×DNA sample buffer to the reaction system, and perform electrophoresis analysis using agarose nucleic acid gel of appropriate concentration.

Further, in step 1, the reaction system is: 10X reaction buffer 1μl, sgRNA-1:sgRNA-2=1:1 total 10ng-1000ng, target gene 10ng-1000ng, Cas9 H840A nuclease 10ng-1000ng, replenish water to 10μl Preferably, in step 1, the reaction system is: 10X reaction buffer 1 μl, sgRNA-1:sgRNA-2=1:1 total 100ng, target gene 50ng, Cas9 H840A nuclease 100ng, water up to 10μl.

Further, in step 2, the reaction conditions are: 30°C-45°C reaction for 5 minutes-2 hours, 65°C-100°C heat inactivation for 1 minute-1 hour, and stored at 1-10°C; preferably, the reaction The conditions are: react at 37°C for 15 minutes, heat inactivate at 65°C for 5 minutes, and store at 4°C.

Application of the kit in the preparation of Cas9 H840A mutant in vitro cleavage efficiency kit products based on CRISPR-Cas9 technology.

Compared with prior art, the present invention has following advantage:

1. This kit contains 2 kinds of sgRNA, which can meet the in vitro shearing efficiency detection of Cas9 nuclease and Cas9 nuclease mutants (including but not limited to: D10A or H840A).

2. This kit quickly and efficiently detects the shearing efficiency of Cas9H840A-sgRNA.

Description of drawings

The drawings constituting a part of the present invention are used to provide a further understanding of the present invention, and the schematic embodiments and descriptions of the present invention are used to explain the present invention, and do not constitute an improper limitation of the present invention. In the attached picture:

Figure 1 shows the recognition position of sgRNA-1 in the target gene in the present invention, and the position where sgRNA-1 specifically binds to the target gene lies in 20 bases from 3320 to 3339 of the target gene.

Figure 2 is the recognition position of sgRNA-2 in the target gene in the present invention, and the position where sgRNA-2 specifically binds to the target gene lies in 20 bases from 3351 to 3370 of the target gene.

Fig. 3 is a schematic diagram of the result of obtaining the target gene by double digestion of the plasmid in Example 1 of the present invention.

Fig. 4 is a schematic diagram of the result of obtaining the target gene from the plasmid in Example 1 of the present invention by PCR technology.

Fig. 5 is a schematic diagram of the results of detecting the in vitro cleavage efficiency of Cas9H840A-sgRNA-1+sgRNA-2 in Example 1 of the present invention.

Detailed ways

It should be noted that, in the case of no conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other.

The present invention will be described in detail below with reference to the accompanying drawings and examples.

CRISPR-Cas9-based Cas9 H840A mutant in vitro shearing efficiency detection kit, the kit includes a sgRNA combination consisting of sgRNA-1 and sgRNA-2; the sgRNA-1, whose sequence is shown in SEQ ID NO.1, the The crRNA in the sequence can specifically recognize the target gene and the specific recognition structure composed of trRNA in the sequence, can bind to Cas9H840A, guide Cas9H840A to specifically cut the target gene, and obtain it through in vitro synthesis; the sgRNA-2 has a sequence as shown in SEQ ID NO.2 The results show that the crRNA in this sequence can specifically recognize the target gene and the specific recognition structure composed of trRNA in this sequence, can bind to Cas9H840A, guide Cas9H840A to specifically cut the target gene, and obtain it through in vitro synthesis.

The kit also includes a target gene whose sequence is shown in SEQ ID NO.3. The target gene is obtained by using the pCANTAB 5E vector, including the following two methods:

Method 1: Plasmid double enzyme digestion to obtain the target gene, the steps are as follows:

Step 1: Carry out double enzyme digestion of the pCANTAB 5E vector, first digest with NotI restriction endonuclease at 37°C for 15 minutes, then react at 65°C for 20 minutes to inactivate NotI restriction endonuclease, and then Add SfiI restriction endonuclease and digest at 50°C for 30 minutes.

Step 2, carry out nucleic acid electrophoresis and product recovery of the target gene after double digestion to obtain the target gene (results shown in Figure 3), wherein, passages 1-5 are pCANTAB 5E vector NotI restriction endonuclease and SfiI restriction positive endonuclease double digestion results; channel M is the DNA molecular weight standard. Compared with channel M, the band size of channel 1-5 is between 3000bp-5000bp of channel M DNA molecular weight standard.

Method 2, the plasmid obtains the target gene by PCR technology, and the steps are as follows:

Step 1, use the pCANTAB 5E vector as a template, use the forward primer F, whose sequence is shown in SEQ ID NO.4; the reverse primer R, whose sequence is shown in SEQ ID NO.5, amplify the target gene by PCR technology, and its sequence As shown in SEQ ID NO.3.

Step 2, the PCR product is subjected to nucleic acid electrophoresis and product recovery to obtain the target gene (results are shown in Figure 4), wherein, channels 1-5 are the positive results of the target gene obtained by PCR technology for the pCANTAB 5E carrier; channel M is the DNA molecular weight standard . Compared with channel M, the band size of channel 1-5 is between 5000bp-8000bp of the channel M DNA molecular weight standard.

The kit also includes Cas9H840A. Cas9H840A is expressed and purified through prokaryotic or eukaryotic protein expression systems (including but not limited to Escherichia coli protein expression systems, yeast protein expression systems, insect protein expression systems and cell protein expression systems).

The kit also includes 10X reaction buffer, the 10X reaction buffer formula includes: 1M NaCl, 500mM Tris-HCl, pH 7.9, 100mM MgCl ₂ , 1mg/ml recombinant albumin.

Another aspect of the present invention provides the application of the above-mentioned kit in the in vitro shearing efficiency based on CRISPR-Cas9, the main technical route is as follows (all have been carried out under the optimal reaction conditions in this kit):

Step 1: Put Cas9H840A, sgRNA-1:sgRNA-2 = 1:1 mixture, and target gene on an ice bath, dilute sgRNA-1 or sgRNA-2 to 100ng, and target gene to 100ng with RNase-free water.

Step 2, prepare the reaction system according to the following table (take 10 μl system as an example):

Step 3: Use a pipette to mix evenly, centrifuge at room temperature, react at 37°C for 15 minutes, and stop the reaction at 65°C for 5 minutes. The reaction time can be appropriately increased or decreased according to the actual situation, for example, 5 minutes to 120 minutes.

Step 4: Add 2 μl of 6×DNA loading buffer to each reaction system, and use an appropriate concentration of agarose nucleic acid gel for electrophoresis analysis (results shown in Figure 5), where channel 1 is without adding sgRNA-1 and sgRNA- 2 is the negative control; Channel 2-4 is the positive result of Cas9H840A-sgRNA-1+sgRNA-2 shearing efficiency in vitro; Channel M is the DNA molecular weight standard. As shown in the figure: No sgRNA was added in channel 1, so the target gene was not cleaved; after the sgRNA-1+sgRNA-2 mixture was added in channel 2-4, the target gene was cleaved, and the target gene that was not cleaved was removed. , resulting in 2 sheared products with sizes of 3322bp and 1104bp respectively.

In the present invention, two sgRNAs are preferably selected as the target sequence of Cas9H840A through software design of the target gene, and then synthesized in vitro respectively. The target gene is obtained by two methods: method 1, plasmid double enzyme digestion; method 2, plasmid as template PCR technology; and purification by nucleic acid electrophoresis and DNA purification technology. Cas9H840A is expressed and purified through prokaryotic or eukaryotic protein expression systems (including but not limited to Escherichia coli protein expression systems, yeast protein expression systems, insect protein expression systems and cell protein expression systems). The sgRNA can form a specific recognition structure, so as to guide Cas9H840A to specifically cut the target gene. At 37°C, Cas9H840A, the target gene and sgRNA were co-incubated in 10X reaction buffer for 15 minutes, and then reacted at 65°C for 5 minutes to heat inactivate Cas9H840A, followed by nucleic acid electrophoresis to detect the cutting efficiency.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of the present invention. within the scope of protection.

Claims (10)

1. The Cas9 H840A mutant in vitro shearing efficiency kit based on CRISPR-Cas9 is characterized in that the kit includes a combination of Cas9 H840A nuclease, target gene, sgRNA-1, sgRNA-2 and 10X reaction buffer; The sgRNA-1 contains two key sequences, namely crRNA sequence and trRNA sequence, wherein the crRNA sequence can specifically recognize the sgRNA of the target gene, and the trRNA sequence can recognize and bind Cas9 H840A nuclease, so as to guide Cas9 H840A nuclease to specifically cut the target gene. Gene corresponding sequence; the sgRNA-2 contains two key sequences, namely crRNA sequence and trRNA sequence, wherein the crRNA sequence can specifically recognize the sgRNA of the target gene, and the trRNA sequence can recognize and bind Cas9 H840A nuclease, so as to guide Cas9 H840A nuclease Specifically cut the corresponding sequence of the target gene. 2. The kit according to claim 1, wherein the sgRNA-1 sequence is as shown in SEQ ID NO.1, and the sgRNA-2 sequence is as shown in SEQ ID NO.2. 3. The kit according to claim 1, wherein the target gene sequence is as shown in SEQ ID NO.3. 4. The kit according to claim 1, wherein the Cas9 H840A nuclease is expressed and purified by a prokaryotic or eukaryotic protein expression system. 5. The kit of claim 1, wherein the Cas9 nuclease mutants include but are not limited to H840A mutants or D10A mutants. 6. The kit according to claim 1, wherein the 10X reaction buffer formulation comprises: 0.5M-3M NaCl, 100mM-1M Tris-HCl, pH 7.0-9.0, 50mM-500mM MgCl ₂ , 0mg/ mL-10mg/mL recombinant albumin; preferably, the 10X reaction buffer formulation includes: 1M NaCl, 500mM Tris-HCl, pH 7.9, 100mM MgCl ₂ , 1mg/mL recombinant albumin. 7. test kit as claimed in claim 1, is characterized in that, is applied by the following steps: Step 1: Add Cas9 H840A nuclease, target gene, sgRNA-1, sgRNA-2 and 10X reaction buffer into 10 μl reaction system in proportion; Step 2, constant temperature reaction, heat inactivation, low temperature storage; Step 3: Add 2 μl of 6×DNA sample buffer to the reaction system, and perform electrophoresis analysis using agarose nucleic acid gel of appropriate concentration. 8. The kit according to claim 7, wherein in step 1, the reaction system is: 1 μl of 10X reaction buffer, sgRNA-1:sgRNA-2=1:1 total 10ng-1000ng, target gene 10ng- 1000ng, Cas9 H840A nuclease 10ng-1000ng, water to 10μl; preferably, in step 1, the reaction system is: 10X reaction buffer 1μl, sgRNA-1:sgRNA-2=1:1 total 100ng, target gene 50ng, Cas9 H840A nuclease 100ng, add water to 10μl. 9. The kit according to claim 7, wherein in step 2, the reaction conditions are: 30°C-45°C reaction for 5 minutes-2 hours, 65°C-100°C heat inactivation for 1 minute-1 hour , stored at 1-10°C; preferably, the reaction conditions are: react at 37°C for 15 minutes, heat inactivate at 65°C for 5 minutes, and store at 4°C. 10. The application of the kit according to any one of claims 1-9 in the preparation of the Cas9 H840A mutant in vitro cleavage efficiency kit product based on CRISPR-Cas9 technology.

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