CN108285905B - Method for inhibiting gene expression level in eukaryotic cell based on CRISPR-Cas13a and application thereof - Google Patents
Method for inhibiting gene expression level in eukaryotic cell based on CRISPR-Cas13a and application thereof Download PDFInfo
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Abstract
Relates to the field of genetic engineering, and particularly discloses a method for inhibiting gene expression level in a eukaryotic cell based on CRISPR-Cas13a and application thereof. Forming a Cas13 a-guide RNA complex with a Cas13a protein by designing and synthesizing a guide RNA complementary to a certain sequence in a target gene mRNA; in eukaryotic cells, the complex binds to the target gene mRNA with complementary sequence and activates RNA degradation activity of Cas13a protein under the guidance of guide RNA, thereby causing degradation and expression level inhibition of the target gene mRNA. In addition, the method can efficiently and specifically inhibit the expression level of the single-base mutant oncogene by screening the position of the guide RNA and artificially adding a mismatch, while not affecting the wild-type gene expression. Through the design of guide RNA, the method can be used for gene expression silencing in eukaryotic cells and the cancer gene expression specificity inhibition of single base mutation, and has the characteristics of high specificity, stability, accuracy, simplicity, convenience and rapidness.
Description
Field of the method
The invention relates to the field of genetic engineering, in particular to a method for inhibiting gene expression level in eukaryotic cells based on CRISPR-Cas13a and application thereof.
Background method
With the development of medicine, it is increasingly recognized that abnormal expression or mutation of genes is the key to the development of many diseases, especially in malignant tumors, where a large number of oncogene mutations exist, the most abundant of which are single base mutations. Aiming at some mutant pathogenic genes, one can design and synthesize small molecular inhibitors to specifically inhibit the functions of the mutant proteins; however, there are still some mutant genes that lack sites at the protein level at which inhibitors can be designed, and thus, specific inhibition at the gene expression level is a key issue in current medical development.
Currently, the most prevalent gene expression suppression tool is still the RNA interference technology that has been developed for about 20 years. RNA interference (RNAi), also known as post-transcriptional gene silencing (PTGS), refers to the introduction of specific homologous double-stranded RNA (dsrna) into cells to render the target gene non-expressed or reduced in expression level. However, this technique has disadvantages in specificity, has an obvious off-target effect, and cannot recognize a single-base mutated oncogene, and thus, development of a novel gene expression inhibition technique is urgently required.
In 1987, scientists found some Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) in the genome of bacteria; the CRISPR-associated protein (Cas) gene was then found in the vicinity of these repeats. With the development of genomics and bioinformatics, scientists noticed that these CRISPR sequences are complementary to DNA sequences of many viruses or phages, and it is gradually recognized that Cas protein can cleave and destroy these complementary gene sequences with the help of these CRISPR sequences, and these results indicate that CRISPR-Cas system is a specific defense mechanism for prokaryotes to specifically cope with foreign invaders such as viruses. With the discovery of a large number of different CRISPR-Cas systems, according to the composition structure of the final gene cleavage effect complex, the CRISPR-Cas systems are divided into 2 families, the first family comprises 1 type and 3 type Cass, and the cleavage effect complex needs a plurality of Cas proteins to participate in the composition; the second family includes type 2 Cas, the effector complex of which only needs one Cas protein to make up and is therefore of more interest. For type 2 Cas, only one Cas protein and one guide RNA are needed to allow precise cleavage and editing of the targeted gene sequence.
Currently, type 2 Cas mainly includes Cas9, Cas12a (also referred to as Cpf1), Cas13a (once referred to as C2C 2). Unlike Cas9 and Cas12a, which use DNA as the targeting nucleic acid, Cas13a uses RNA as the targeting nucleic acid sequence.
Disclosure of Invention
The invention aims to provide a method for inhibiting gene expression level in eukaryotic cells based on CRISPR-Cas13 a. Forming a Cas13 a-guide RNA complex with a Cas13a protein by designing and synthesizing a guide RNA complementary to a certain sequence in a target gene mRNA; in eukaryotic cells, the complex binds to the target gene mRNA with complementary sequence and activates RNA degradation activity of Cas13a protein under the guidance of guide RNA, thereby causing degradation and expression level inhibition of the target gene mRNA. In addition, the technology can efficiently and specifically inhibit the expression level of the single-base mutant oncogene by position screening of guide RNA and artificially adding mismatch, while not affecting the wild-type gene expression.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for inhibiting gene expression level in eukaryotic cells based on CRISPR-Cas13 a.
The effector that ultimately exerts gene expression inhibition function of this method is Cas13 a-guide RNA complex.
The Cas13a protein in the invention can be from different bacteria, such as Leptotrichia shahii (LshCas13a), Leptotrichia buccalis (LtuCas 13a), Leptotrichia wadei (LwCas13a) and the like, and is preferably LwCas13 a.
The guide RNA comprises an anchor sequence and a guide sequence complementary to a target gene, and has a structure of 5 '-anchor sequence-guide sequence-3', wherein the anchor sequence is related to a bacterial source of a Cas13a protein and is divided into 5'-GGCCACCCCAAUAUCGAAGGGGACUAAAAC-3' aiming at LshCas13a, 5'-GACCACCCCAAAAAUGAAGGGGACUAAAAC-3' aiming at LkuCas 13a and 5'-GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAAC-3' aiming at LwCas13 a; the guide sequence is complementary to a sequence in a certain section of the target gene mRNA, and has a length of 20 to 28 bases, preferably 28.
According to the invention, the Cas13 a-guide RNA complex can be formed in advance and then transfected into eukaryotic cells in a protein purification and in-vitro transcription mode; after the expression of exogenous Cas13a and guide RNA is realized in eukaryotic cells by means of introduction of exogenous gene plasmids, Cas13 a-guide RNA complexes are directly formed in the cells. Preferably, the complex is formed in advance and then transfected into a eukaryotic cell.
Secondly, the eukaryotic cell gene expression inhibition method based on CRISPR-Cas13a is used for the specific inhibition of the single-base mutation oncogene expression.
In the application, the high-efficiency specific inhibition of the oncogene with single base mutation can be realized by the precise design of the guide RNA: the optimization of the gene expression inhibition efficiency can be realized by screening the position of the guide RNA; by artificially adding mismatch, the gene expression inhibition specificity can be optimized.
When the position selection of the guide RNA is carried out, the guide sequence in the guide RNA covers a single-base mutation site, and the guide sequence has 28 bases at most, so that the position selection is carried out at most at 28 positions; the guide RNAs at different positions have different gene expression inhibition levels, and the guide RNA with the highest inhibition efficiency can be screened.
When artificial addition mismatch is carried out, the position of the artificially added mismatch is different from the position of a single base mutation, and the number of the addition mismatch is usually 1-2, so that the number of the mismatch between the guide sequence and the wild-type gene in the guide RNA is 2-3 (comprising 1-2 persons of addition mismatch and 1 single base mutation); the number of mismatches can obviously influence the gene expression inhibition efficiency of the Cas13 a-guide RNA complex, thereby realizing that the Cas13 a-guide RNA complex can only specifically inhibit the oncogene expression with single base mutation, and simultaneously does not influence the wild-type gene expression.
The invention has the beneficial effects that:
the invention discloses a method for inhibiting gene expression level in eukaryotic cells based on CRISPR-Cas13a, which has the main mechanism that a Cas13 a-guide RNA compound is formed with Cas13a protein by designing and synthesizing a guide RNA which is complementary with a certain sequence in target gene mRNA; in eukaryotic cells, the complex binds to the target gene mRNA with complementary sequence and activates RNA degradation activity of Cas13a protein under the guidance of guide RNA, thereby causing degradation and expression level inhibition of the target gene mRNA.
The CRISPR-Cas13 a-based gene expression inhibition method disclosed by the invention has completely different biological mechanisms compared with the conventional small interfering RNA technology, and the maximum inhibition efficiency is close to 90%. Compared with the small interfering RNA which needs double-stranded RNA, the design and synthesis of the guide RNA are simpler and more convenient, and only one single-stranded guide RNA is needed.
The invention also discloses the application of the CRISPR-Cas13 a-based eukaryotic cell gene expression inhibition method in the single base mutation cancer gene expression specificity inhibition. In the application, the method can efficiently and specifically inhibit the expression level of the single-base mutant oncogene by position screening of guide RNA and artificial mismatch addition, and simultaneously does not affect the wild-type gene expression. This is of great importance for the treatment of tumors with single base mutations, especially when the mutated oncogenes cannot be designed with effective inhibitors at the protein level, such as KRAS mutations.
The inventor selects KRAS wild type cell HEK293T and KRAS-G12D mutant pancreatic cancer cell AsPC1 as models, and interferes the KRAS gene expression level in the two cells according to the gene expression inhibition method disclosed by the invention, and the result shows that the method can inhibit over 90% of KRAS-G12D gene expression by accurate position screening of guide RNA, and meanwhile, the expression of the wild type KRAS gene can not be obviously influenced by artificially adding mismatch.
Drawings
Fig. 1 is a detection of inhibition efficiency of a gene expression inhibition method based on CRISPR-Cas13 a. The KRAS-G12D gene in the KRAS-G12D mutant pancreatic cancer cell AsPC1 was used as a model. The results show that the position of the guide sequence in the guide RNA can obviously influence the inhibition efficiency of the method, and the method can inhibit the expression of the KRAS-G12D gene by 94 percent at most.
Fig. 2 is a specific optimization of the gene expression inhibition method based on CRISPR-Cas13 a. By selecting KRAS wild type cell HEK293T and KRAS-G12D mutant pancreatic cancer cell AsPC1 as models and artificially adding a mismatch mode, the method can inhibit about 70% of KRAS-G12D gene expression without affecting wild type KRAS gene expression.
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 may be made by 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
This example is used to test the efficiency of inhibition of gene expression by the methods of the invention.
In the example, the KRAS-G12D gene in the KRAS-G12D mutant pancreatic cancer cell AsPC1 was selected as a target gene for detection.
Firstly, guide RNAs with different guide sequence positions are synthesized by an in vitro transcription method, and specific sequences are shown in Table 1.
Secondly, the Lw.Cas13a protein was purified by expression in vitro using a bacterial expression system.
Finally, the Cas13 a-guide RNA complex was transfected into AsPC1 cells using the protein-RNA complex transfection kit from Invitrogen; after 24 hours, total RNA was extracted from the cells, and the expression level of KRAS-G12D gene was detected by using a reverse transcription-Realtime PCR kit from TAKARA, the PCR primer sequences were as follows:
KRAS gene upstream primer: 5'-GGAGAGAGGCCTGCTGAAAA-3', respectively;
KRAS gene downstream primer: 5'-CCCTCCCCAGTCCTCATGTA-3', respectively;
control gene beta-actin upstream primer: 5'-GGGAAATCGTGCGTGACAT-3', respectively;
control gene beta-actin upstream primer: 5'-TCAGGCAGCTCGTAGCTCTT-3' are provided.
The experimental results show that the position of the guide sequence in the guide RNA can obviously influence the inhibition efficiency of the method compared with the control group, and the method can inhibit the KRAS-G12D gene expression by 94 percent at most.
Example 2
This example is used to test the specificity of the method of the invention for inhibition of gene expression.
In the embodiment, a KRAS wild-type cell HEK293T and a KRAS-G12D mutant pancreatic cancer cell AsPC1 are selected as models for detection.
Firstly, different guide RNAs were synthesized by in vitro transcription, and the specific sequences are shown in Table 2, in which guide RNAs had guide sequences in which a mismatched base different from the mutation site was artificially added at different positions.
Secondly, the Lw.Cas13a protein was purified by expression in vitro using a bacterial expression system.
Finally, the Cas13 a-guide RNA complex was transfected into AsPC1 cells using the protein-RNA complex transfection kit from Invitrogen; after 24 hours, total RNA was extracted from the cells, and the expression level of KRAS-G12D gene was detected by using a reverse transcription-Realtime PCR kit from TAKARA, the PCR primer sequences were as follows:
KRAS gene upstream primer: 5'-GGAGAGAGGCCTGCTGAAAA-3', respectively;
KRAS gene downstream primer: 5'-CCCTCCCCAGTCCTCATGTA-3', respectively;
control gene beta-actin upstream primer: 5'-GGGAAATCGTGCGTGACAT-3', respectively;
control gene beta-actin upstream primer: 5'-TCAGGCAGCTCGTAGCTCTT-3' are provided.
The experimental result shows that the gene expression inhibition efficiency of the guide RNA can be influenced by artificially adding mismatch, and the method can inhibit about 70% of KRAS-G12D gene expression without influencing the wild KRAS gene expression through precise design and screening.
Table 1 shows guide RNAs for different position guide sequences of KRAS-G12D gene
Table 2 shows guide RNAs against KRAS-G12D gene with artificial mismatches at different positions
It should be understood that the method schemes for carrying out the equal proportion enlargement or reduction of the used amount of the reagents or raw materials in the above embodiments are substantially the same as the above embodiments.
Although the invention has been described in detail hereinabove with respect to specific embodiments thereof, it will be apparent to those skilled in the art that many modifications and improvements can 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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<210> 48
<211> 65
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
ggauuuagac uaccccaaaa acgaagggga cuaaaacgca cucuugccua cgccaucagc 60
ucgaa 65
<210> 49
<211> 65
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
ggauuuagac uaccccaaaa acgaagggga cuaaaacgca cucuugccua cgccaucagc 60
uccau 65
Claims (6)
- The application of the CRISPR-Cas13a system in preparing an oncogene expression level inhibitor is characterized in that a Cas13 a-guide RNA complex is formed with a Cas13a protein by designing and synthesizing a guide RNA which is complementary to a certain sequence in a target gene mRNA; in eukaryotic cells, the complex binds to the target gene mRNA with complementary sequence and activates RNA degradation activity of Cas13a protein under the guidance of guide RNA, thereby causing degradation and expression level inhibition of the target gene mRNA;by means of position screening of guide RNA and artificial addition of 1-2 mismatched bases different from mutation sites, the method can inhibit the expression level of cancer gene with single base mutation effectively and specifically without affecting wild gene expression.
- 2. Use according to claim 1, wherein the Cas13a protein used is from the bacteria Leptotrichia shahii, Leptotrichia buccallis or Leptotrichia wadei.
- 3. Use according to claim 1 or 2, wherein the guide RNA used comprises an anchor sequence related to the bacterial origin of the Cas13a protein and a guide sequence complementary to the gene of interest, the guide sequence being complementary to a sequence in the mRNA of the gene of interest and being 20-28 bases in length.
- 4. Use according to claim 1 or 2, characterized in that the Cas13 a-guide RNA complex can be transfected into eukaryotic cells after its pre-formation by means of protein purification and in vitro transcription; after the expression of exogenous Cas13a and guide RNA is realized in eukaryotic cells by means of introduction of exogenous gene plasmids, Cas13 a-guide RNA complexes are directly formed in the cells.
- 5. Use according to claim 1 or 2, wherein in the positional selection of the guide RNA the guide sequence in the guide RNA should cover a single base mutation site, with a maximum of 28 positions selected; the level of suppressor gene expression differs between positions of the guide RNA.
- 6. The use according to claim 1 or 2, characterized in that the artificially added mismatch site should be different from the single base mutation site, the number of addition mismatches is 1-2, so that the number of mismatches between the guide sequence and the wild type gene in the guide RNA is 2-3, including 1-2 person addition mismatches and 1 single base mutation, to achieve that the Cas13 a-guide RNA complex can specifically inhibit the oncogene expression of only the single base mutation without affecting the wild type gene expression.
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| CN111041049A (en) * | 2019-12-04 | 2020-04-21 | 江苏大学 | Preparation method and application of CRISPR-Cas13a system based on near-infrared light control |
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| CN113528515B (en) * | 2020-04-22 | 2023-05-05 | 中国科学院分子植物科学卓越创新中心 | CRISPR-Cas13a based technology for interfering and blocking reverse transcription transposition of viruses |
| CN113528514B (en) * | 2020-04-22 | 2023-05-05 | 中国科学院分子植物科学卓越创新中心 | CRISPR-Cas12a based technology for interfering and blocking reverse transcription transposition of viruses |
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| Omar O等.RNA targeting with CRISPR-Cas13.《Nature》.2017,第550卷 * |
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