CN111471744A - Method for detecting gene editing target point cutting efficiency - Google Patents
Method for detecting gene editing target point cutting efficiency Download PDFInfo
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- CN111471744A CN111471744A CN202010408380.8A CN202010408380A CN111471744A CN 111471744 A CN111471744 A CN 111471744A CN 202010408380 A CN202010408380 A CN 202010408380A CN 111471744 A CN111471744 A CN 111471744A
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Abstract
The invention discloses a method for detecting the cutting efficiency of a gene editing target spot. Constructing a target detection vector containing a frame shift mutation reporter gene; then inserting the gene editing target sequence into the N end of the target detection vector of the frame shift mutation reporter gene to obtain a detection vector; then the detection vector is transferred into animal cells, when endonuclease successfully cuts the target, the cells repair the mutant reporter gene, and the cutting efficiency of the target is obtained by detecting the activity of the reporter gene. The method is sensitive and visual in detection and can quantify; the target point cutting occurs in cells, is very similar to the reaction conditions of actual gene editing, and can reflect the target point cutting effect in the actual gene editing.
Description
Technical Field
The invention belongs to the field of animal gene engineering and gene editing, and relates to a method for detecting the gene editing target cutting efficiency of specific nuclease in animal cells, in particular to a method for detecting the gene editing target cutting efficiency.
Background
The key steps in site-directed gene editing technology are site-directed cleavage of genomic DNA of an organism using specific DNA endonucleases that recognize and cleave specific sequences of DNA different gene editing technologies employ different nucleases such as Zinc Finger Nucleases (ZFNs), transcription activator-like effector nucleases (TA L ENs), and CRISPR/Cas9 nuclease regardless of which nuclease is employed, recognition and site-directed cleavage of specific sites in the genome by nucleases is the core step of gene editing.
Because it cannot be guaranteed that the designed gene editing target can be effectively recognized and cut by endonuclease, in the actual gene editing operation, a plurality of gene editing targets need to be designed simultaneously, and gene editing is carried out simultaneously, so that the target with high cutting efficiency can be ensured, and the gene editing can be successfully realized. This approach increases the cost and time of gene editing.
With the popularization of CRISPR/Cas9 technology, the application of gene editing is increasingly wide. The simple and effective gene target point verification method can reduce the experiment cost of gene editing, improve the success rate and has great application value and economic value.
Disclosure of Invention
The invention aims to provide a method for detecting the cutting efficiency of a gene editing target spot, which has the advantages of sensitive and visual detection and capability of quantification; the target point cutting occurs in cells, is very similar to the reaction conditions of actual gene editing, and can reflect the target point cutting effect in the actual gene editing.
In order to achieve the above object, the method for detecting the gene editing target cutting efficiency of the invention comprises the following steps:
(1) constructing a carrier for expressing the frame shift mutated reporter gene, namely constructing the frame shift mutated reporter gene containing a gene editing target;
(2) transferring the carrier of the reporter gene which is constructed in the step (1) and expresses the frame shift mutation into cells; cloning a frame shift mutant reporter gene containing a gene editing target to the downstream of a eukaryotic expression vector promoter and the upstream of a terminator;
(3) transferring the endonuclease expression element or endonuclease into the cell;
(4) and detecting the expression level of the reporter gene in the cell.
The method for constructing the vector for expressing the frame shift mutant reporter gene in the step (1) comprises the following steps:
1) inserting a DNA sequence, namely a gene editing target sequence, into the downstream of an ATG (initiator of gene) of a reporter gene by using a DNA mutation technology and using restriction endonuclease; the reporter gene is one of a fluorescent protein reporter gene and a catalytic enzyme reporter gene; the number of bases of the inserted DNA sequence is not equal to a multiple of 3;
2) obtaining the reporter gene containing the frame shift mutation of the gene editing target point.
The invention relates to a method for detecting the cutting efficiency of a gene editing target spot, which comprises the following steps: constructing a vector expressing the frame shift mutated reporter gene, and transferring the vector expressing the frame shift mutated reporter gene into animal cells.
The carrier for constructing the reporter gene for expressing the frameshift mutation is composed of a target point detection carrier of the frameshift mutation reporter gene and a gene editing target point sequence, and the gene editing target point sequence is inserted into the N end of the target point detection carrier of the frameshift mutation reporter gene.
The carrier expressing the frame shift mutant reporter gene is transferred into animal cells, when endonuclease successfully cuts a target spot, the cells repair the mutant reporter gene, and the target spot cutting efficiency is obtained by detecting the activity of the reporter gene.
The fluorescent protein reporter gene is EGFP or RFP; the catalytic enzyme type reporter gene is firefly luciferase or renilla luciferase; the endonuclease expression element or the endonuclease is a fluorescent protein reporter gene and a biological enzyme reporter gene, and the fluorescent protein reporter gene is an EGFP reporter gene and the biological enzyme reporter gene is a firefly luciferase reporter gene.
The gene editing target sequence inserted in the step (2) has:
1) comprises the complete sequence of one or more gene editing targets;
2) the number of bases of the inserted DNA sequence containing the gene editing target is a multiple of 3.
The cell includes mammalian cell and insect cell.
The mammalian cell is human embryonic kidney cell HEK293 and derived cells thereof, and human cervical cancer cell Hela; the insect cell is insect cell SF 9.
The method for transferring the reporter gene containing the frame shift mutation of the gene editing target point into the cell comprises the following steps: chemical transfection method, electric shock transfection method, gene gun transfection method, virus transfection method or their combination.
The endonucleases are Zinc Finger Nucleases (ZFNs), transcription activator-like-factor-mediated nucleases (TA L ENs), and CRISPR/Cas9 nuclease having:
1) can recognize a DNA sequence, namely a gene editing target sequence, and the gene editing target sequence can be manually designed and changed;
2) can cut DNA at fixed points.
The endonuclease is transferred into cells in the form of one of a nuclease expression vector, a nuclease expression virus, nuclease coding RNA, a nuclease protein and a nuclease/RNA complex.
The steps (2) and (3) are carried out simultaneously.
The code-shift mutation EGFP gene sequence for detecting the cutting efficiency of the gene editing target point is shown as SEQ ID NO: 1 is shown.
SEQ ID NO：1
atgccaccatgggccaccatggctcgagctcaagcttcgaattctgcagtcgacggtaccgcgggcccgggatcctagggataacagggtaatagaagcggtgggtctggcggcggaggatctgcggccgcatgtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaa
The frame shift mutant firefly gene sequence for detecting the gene editing target cutting efficiency is represented by SEQ ID NO: 2, respectively.
SEQ ID NO：2
atgagaagatgccaaaaacattaagaagggcccagcgccattctacccactcgaagacgggaccgccggcgagcagctgcacaaagccatgaagcgctacgccctggtgcccggcaccatcgcctttaccgacgcacatatcgaggtggacattacctacgccgagtacttcgagatgagcgttcggctggcagaagctatgaagcgctatgggctgaatacaaaccatcggatcgtggtgtgcagcgagaatagcttgcagttcttcatgcccgtgttgggtgccctgttcatcggtgtggctgtggccccagctaacgacatctacaacgagcgcgagctgctgaacagcatgggcatcagccagcccaccgtcgtattcgtgagcaagaaagggctgcaaaagatcctcaacgtgcaaaagaagctaccgatcatacaaaagatcatcatcatggatagcaagaccgactaccagggcttccaaagcatgtacaccttcgtgacttcccatttgccacccggcttcaacgagtacgacttcgtgcccgagagcttcgaccgggacaaaaccatcgccctgatcatgaacagtagtggcagtaccggattgcccaagggcgtagccctaccgcaccgcaccgcttgtgtccgattcagtcatgcccgcgaccccatcttcggcaaccagatcatccccgacaccgctatcctcagcgtggtgccatttcaccacggcttcggcatgttcaccacgctgggctacttgatctgcggctttcgggtcgtgctcatgtaccgcttcgaggaggagctattcttgcgcagcttgcaagactataagattcaatctgccctgctggtgcccacactatttagcttcttcgctaagagcactctcatcgacaagtacgacctaagcaacttgcacgagatcgccagcggcggggcgccgctcagcaaggaggtaggtgaggccgtggccaaacgcttccacctaccaggcatccgccagggctacggcctgacagaaacaaccagcgccattctgatcacccccgaaggggacgacaagcctggcgcagtaggcaaggtggtgcccttcttcgaggctaaggtggtggacttggacaccggtaagacactgggtgtgaaccagcgcggcgagctgtgcgtccgtggccccatgatcatgagcggctacgttaacaaccccgaggctacaaacgctctcatcgacaaggacggctggctgcacagcggcgacatcgcctactgggacgaggacgagcacttcttcatcgtggaccggctgaagagcctgatcaaatacaagggctaccaggtagccccagccgaactggagagcatcctgctgcaacaccccaacatcttcgacgccggggtcgccggcctgcccgacgacgatgccggcgagctgcccgccgcagtcgtcgtgctggaacacggtaaaaccatgaccgagaaggagatcgtggactatgtggccagccaggttacaaccgccaagaagctgcgcggtggtgttgtgttcgtggacgaggtgcctaaaggactgaccggcaagttggacgcccgcaagatccgcgagattctcattaaggccaagaagggcggcaagatcgccgtgtaa
The two expression vectors disclosed by the invention are applied to detection of CRISPR/Cas9 target points.
The method for detecting the gene editing target cutting efficiency has the beneficial effects that: the method is sensitive and visual in detection and can quantify; the target point cutting occurs in cells, is very similar to the reaction conditions of actual gene editing, and can reflect the target point cutting effect in the actual gene editing.
Drawings
FIG. 1 is a schematic diagram of the detection of nuclease cleavage efficiency in accordance with the present invention;
FIG. 2 is a schematic structural diagram of a detection vector pTYNE constructed by the present invention;
FIG. 3 is a graph showing the results of the test in example 3;
FIG. 4 is a schematic diagram of the inserted sequence of the pTYNE-53 vector after the construction of example 4 is completed;
FIG. 5 is a graph showing the results of the test in example 4.
Detailed Description
Example 1
The invention discloses a method for constructing a carrier of a frame shift mutant EGFP reporter gene, which comprises the following steps as shown in figure 2:
(1) constructing a carrier of a frame-shift mutation EGFP reporter gene, taking green fluorescent protein as a frame-shift mutation reporter gene carrier pTYNE of the reporter gene, inserting the frame-shift mutation EGFP reporter gene into the downstream of an initiation codon ATG of the frame-shift mutation reporter gene carrier pTYNE of the reporter gene by using restriction endonucleases XbaI and NotI, and cutting a synthesized product to obtain the carrier of the frame-shift mutation EGFP reporter gene;
(2) transferring the carrier of the code shift mutation EGFP reporter gene constructed in the step (1) into eukaryotic cells; the EGFP reporter gene containing the frame shift mutation is cloned between a CMV promoter and an SV40 terminator in a eukaryotic expression vector.
Example 2
The invention relates to a method for detecting the cutting efficiency of a gene editing target spot, which comprises the following steps as shown in figures 1 and 2:
(1) constructing 2 spCas9 and sgRNA co-expression vectors, using green fluorescent protein as a frame shift mutation reporter gene vector pTYNE of a reporter gene, and inserting spCas9 and a sgRNA reporter gene into the downstream of an initiation codon ATG of the frame shift mutation reporter gene vector pTYNE of the reporter gene by using restriction endonuclease, wherein one expression can cut positive sgRNA of the reporter gene vector pTYNE; another negative sgRNA whose expression failed to cleave the reporter vector pTYNE; the method comprises the following steps:
1) synthesizing negative and positive sgRNA expression frames in vitro respectively;
2) the EcoRV linearized spCas9 and sgRNA co-expression vector spCas9gRNA 1;
3) recombining a positive sgRNA expression frame to the downstream of a U6 promoter on a spCas9gRNA1 vector to obtain a positive vector spCas 9-sgRNA-P;
4) recombining a positive sgRNA expression frame to the downstream of a U6 promoter on a spCas9gRNA1 vector to obtain a negative vector spCas 9-sgRNA-N;
(2) adopting a frameshift mutation reporter gene vector pTYNE of a reporter gene to co-transfect HEK293T cells with spCas9-sgRNA-P and spCas9-sgRNA-N respectively, and verifying the reporter function of the pTYNE vector on the cutting of Cas9 protein;
the method comprises the following specific steps:
1) HEK293T cells were plated into 24-well plates 24 hours prior to transfection, while 2 wells were prepared;
2) 2 plasmid dilutions were prepared according to the following system;
| group A | Group B |
| pTYNE 0.4ug | pTYNE 0.4ug |
| pCas9-N 0.4ug | pCas9-P 0.4ug |
| DMEM Medium X ul | DMEM Medium X ul |
| Total volume of 50ul | Total volume of 50ul |
3) Preparation of transfection reagent dilutions:
Polyfect-V transfection reagent 3.2ul
DMEM medium 96.8ul
4) Mixing the plasmid diluent and the transfection reagent diluent, respectively, adding 50ul of the transfection reagent diluent into the two sets of plasmid diluents, mixing, and incubating at room temperature for 15 min;
5) adding the transfection solution to HEK293T cells;
6) EGFP fluorescence expression was detected after 48 hours. And (3) detection results: as shown in fig. 3.
Example 3
The method for detecting the gene editing target cutting efficiency provided by the invention is shown in figures 1 and 2, and the pTYNE reporter gene vector is used for verifying the human TP53 gene knockout target, and the realization steps are as follows:
(1) designing two targets according to the 4 th exon sequence of the human TP 53;
wherein the target point 1: acctgccctgtgcagctgtggg, respectively; target 2: ttgattccacacccccgcccgg, respectively;
(2) constructing a pTYNE-TP53 verification vector; the fourth exon part sequence of TP53 was ligated to pTYNE vector via XhoI and HindIII cleavage sites, the cleavage efficiency of the target was verified by pTYNE vector, and the constructed vector was named pTYNE-TP 53; schematic diagram of pTYNE-TP53 vector insertion sequence after construction (shown in FIG. 4);
3. constructing a target point 1 and a target point 2 into a spCas9/gRNA1 vector to obtain a Cas9, a gRNA co-expression vector spCas9/gRNA1-P531 and a spCas9/gRNA1-P532 for a TP53 gene;
4. verifying the target by pTYNE-TP 53;
5. HEK293T cells were plated into 24-well plates 24 hours prior to transfection; preparing 3 holes simultaneously; 2 plasmid dilutions were prepared according to the following system:
| group 1 (negative control) | Group 2 (target one) | Group 3 (target two) |
| pTYNE-53 0.4ug | pTYNE-53 0.4ug | pTYNE-53 0.4ug |
| 0.4ug of pCas9/gRNA1 empty vector | pCas9/gRNA1-P531 0.4ug | pCas9/gRNA1-P532 0.4ug |
| DMEM Medium X ul | DMEM Medium X ul | DMEM Medium X ul |
| Total volume of 50ul | Total volume of 50ul | Total volume of 50ul |
6. Preparation of transfection reagent dilutions:
Polyfect-V transfection reagent 4.8ul
DMEM medium 145.2ul
Mixing the plasmid diluent and the transfection reagent diluent, respectively, adding 50ul of the transfection reagent diluent into the two sets of plasmid diluents, mixing, and incubating at room temperature for 15 min;
adding the transfection solution to HEK293T cells;
detecting EGFP fluorescence expression after 48 hours;
7. the experimental results were verified as in fig. 5.
As shown in FIG. 5, the obtained detection result shows that both the target point I and the target point II have effects, and the effect of the target point I is better than that of the target point II.
Claims (10)
1. A method for detecting the cutting efficiency of a gene editing target spot is characterized in that: the detection method comprises the following steps:
(1) constructing a carrier for expressing the frame shift mutated reporter gene, namely constructing the frame shift mutated reporter gene containing a gene editing target;
(2) transferring the carrier of the reporter gene which is constructed in the step (1) and expresses the frame shift mutation into cells; cloning a frame shift mutant reporter gene containing a gene editing target to the downstream of a eukaryotic expression vector promoter and the upstream of a terminator;
(3) transferring the endonuclease expression element or endonuclease into the cell;
(4) and detecting the expression level of the reporter gene in the cell.
2. The method of claim 1, wherein the efficiency of gene editing target cleavage is measured by: the method for constructing the vector for expressing the frame shift mutant reporter gene in the step (1) comprises the following steps:
1) inserting a DNA sequence, namely a gene editing target sequence, into the downstream of an ATG (initiator of gene) of a reporter gene by using a DNA mutation technology and using restriction endonuclease; the reporter gene is one of a fluorescent protein reporter gene and a catalytic enzyme reporter gene; the number of bases of the inserted DNA sequence is not equal to a multiple of 3;
2) obtaining the reporter gene containing the frame shift mutation of the gene editing target point.
3. The method of claim 1, wherein the efficiency of gene editing target cleavage is measured by: the fluorescent protein reporter gene is EGFP or RFP; the catalytic enzyme type reporter gene is firefly luciferase or renilla luciferase; the endonuclease expression element or the endonuclease is a fluorescent protein reporter gene and a biological enzyme reporter gene, the fluorescent protein reporter gene is an EGFP reporter gene, and the biological enzyme reporter gene is a firefly luciferase reporter gene.
4. The method of claim 2, wherein the efficiency of gene editing target cleavage is measured by: the gene editing target sequence inserted in the step 1) has:
1) comprises the complete sequence of one or more gene editing targets;
2) the number of bases of the inserted DNA sequence containing the gene editing target is a multiple of 3.
5. The method of claim 1, wherein the efficiency of gene editing target cleavage is measured by: the cell includes mammalian cell and insect cell.
6. The method of claim 1, wherein the efficiency of gene editing target cleavage is measured by: the mammalian cell is human embryonic kidney cell HEK293 and derived cells thereof, and human cervical cancer cell Hela; the insect cell is insect cell SF 9.
7. The method of claim 1, wherein the efficiency of gene editing target cleavage is measured by: the method for transferring the reporter gene containing the frame shift mutation of the gene editing target point into the cell comprises the following steps: chemical transfection method, electric shock transfection method, gene gun transfection method, virus transfection method or their combination.
8. The method for detecting the cleavage efficiency of a gene editing target as claimed in claim 1, wherein the endonuclease is Zinc Finger Nucleases (ZFNs), transcription activator-like-factor-mediated nucleases (TA L ENs), and CRISPR/Cas9 nuclease having:
1) can recognize a DNA sequence, namely a gene editing target sequence, and the gene editing target sequence can be manually designed and changed;
2) can cut DNA at fixed points.
9. The method of claim 7, wherein the efficiency of gene editing target cleavage is measured by: the endonuclease is transferred into cells in the form of one of a nuclease expression vector, a nuclease expression virus, nuclease coding RNA, a nuclease protein and a nuclease/RNA complex.
10. The method of claim 1, wherein the efficiency of gene editing target cleavage is measured by: the steps (2) and (3) are carried out simultaneously.
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