CN107794272B - High-specificity CRISPR genome editing system - Google Patents
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Abstract
The invention provides a high-specificity CRISPR genome editing system, wherein a gRNA of a targeted Cas9 gene is introduced into the CRISPR/Cas9 genome editing system, and the gRNA of the targeted Cas9 gene can mediate the cutting of the Cas9 gene while (or after) the genome editing of a target cell is carried out, so that the expression time of the Cas9 gene is remarkably reduced, the off-target effect caused by the long-term expression of Cas9 in the CRISPR system is reduced, and the targeted specificity is increased.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a high-specificity CRISPR genome editing system.
Background
Genome Editing technology (Genome Editing) is a technology capable of performing site-specific modification on a target Genome so as to research and treat unknown functional genes. The artificial endonuclease-mediated genome editing technology is most commonly applied at present, has the advantages of getting rid of the dependence of the traditional technology on embryonic stem cells, having wide application range and having great potential in clinical application. The genome editing technology mediated by artificial endonuclease mainly comprises zinc finger nuclease technology (ZFNs), transcription activator like nuclease Technology (TALENs) and CRISPR/Cas technology.
ZFNs are the earliest developed general genome editing technology and can be used for implementing site-specific knockout and site-specific knockout mutation, but the development of ZFNs technology is limited by the disadvantages of large construction difficulty, high cost and the like. The TALENs technology is developed on the basis of ZFNs, and compared with the ZFNs technology, the TALENs technology has the advantages of high construction flexibility, low cost and the like. Unlike ZFNs and TALENs technologies, CRISPR/Cas technology has a unique DNA targeting mechanism, is more convenient and flexible to use, and is therefore developing as a major genome editing tool. Currently, CRISPR/Cas systems have been tested successfully in a variety of species, such as mice, zebrafish, drosophila, nematodes, and silkworms.
However, the off-target effect in genome editing has always prevented a wider range of applications of genome editing technology, and therefore, there is an urgent need in the art to develop genome editing technology with more target specificity.
Disclosure of Invention
The invention aims to provide a high-specificity genome editing system and application thereof.
In a first aspect of the present invention, there is provided a high specificity CRISPR genome editing system, wherein the CRISPR genome editing system comprises: a first gRNA, a second gRNA, and a Cas9 protein; wherein the first gRNA targets the genome of a target cell to be edited and the second gRNA targets the gene encoding the Cas9 protein.
In another preferred example, the second gRNA targets a gene sequence selected from the group consisting of: SEQ ID NO.3, and SEQ ID NO. 4.
In another preferred embodiment, the CRISPR genome editing system comprises a DNA construct represented by the following formula (I):
P1-R1-P2-R2-P3-C，(I)
in the formula (I), R1 is a first gRNA encoding gene, R2 is a second gRNA encoding gene, C is a Cas9 protein encoding gene, P1, P2 and P3 are optional promoter sequences, and "-" represents an optional connecting sequence.
In a second aspect of the invention, there is provided a DNA construct for CRISPR genome editing, said DNA construct having a structure represented by the following formula (I):
P1-R1-P2-R2-P3-C，(I)
in the formula (I), R1 is a first gRNA encoding gene, R2 is a second gRNA encoding gene, C is a Cas9 protein encoding gene, P1, P2 and P3 are optional promoter sequences, and "-" represents an optional connecting sequence.
In another preferred example, the second gRNA targets a gene sequence selected from the group consisting of: SEQ ID NO.3, and SEQ ID NO. 4.
In a third aspect of the invention, there is provided an expression vector expressing the CRISPR genome editing system of the first aspect of the invention; alternatively, the expression vector comprises a DNA construct according to the second aspect of the invention.
In a fourth aspect of the invention, there is provided a genetically engineered host cell comprising an expression vector according to the third aspect of the invention, or having integrated into its genome a DNA construct according to the second aspect of the invention.
In another preferred embodiment, the host cell is a eukaryotic cell, preferably an animal cell.
In a fifth aspect of the present invention, there is provided a genome editing method, comprising the steps of:
(1) providing a first genome editing system, and performing genome editing on a target gene by using the first genome editing system;
(2) after the genome editing occurs, decreasing the activity of the first genome editing system.
In another preferred example, in step (2), a second genome editing system is provided, which specifically targets the coding gene of the first genome editing system; and carrying out gene editing on the coding gene of the first genome editing system through the second genome editing system, thereby reducing the activity of the first genome editing system.
In another preferred example, the genome editing is genome editing inside a cell; preferably genome editing within the nucleus.
In another preferred example, the first genome editing system is an artificial endonuclease-mediated genome editing system; further, the second genome editing system specifically targets a gene encoding the artificial endonuclease of the first genome editing system.
In another preferred embodiment, the cell is selected from the group consisting of: animal cells, plant cells, and microbial cells.
In another preferred example, the first genome editing system and/or the second selected genome editing system is selected from the group consisting of: CRISPR-Cas genome editing systems, NgAgo-gDNA genome editing systems, zinc finger nuclease genome editing Systems (ZFNs), transcription activator-like nuclease genome Editing Systems (TALENs).
In another preferred example, the CRISPR-Cas genome editing system is a CRISPR-Cas9 genome editing system.
In another preferred example, the first genome editing system is a CRISPR-Cas9 genome editing system; in the step (2), a gRNA targeting the Cas9 gene of the CRISPR-Cas9 genome editing system is provided, and the Cas9 gene of the CRISPR-Cas9 genome editing system is cleaved under the action of the Cas9 protein of the CRISPR-Cas9 genome editing system and the gRNA targeting the Cas9 gene, thereby reducing the activity of the genome editing system.
In another preferred embodiment, the genome editing system is the high specificity CRISPR genome editing system according to the first aspect of the invention.
In a sixth aspect of the invention there is provided a use of a CRISPR genome editing system according to the first aspect of the invention, a DNA construct according to the second aspect of the invention, an expression vector according to the third aspect of the invention, or a host cell according to the first aspect of the invention in the manufacture of a medicament or a genome editing reagent.
In another preferred embodiment, the medicament is for genome editing of a genome in a target cell.
In a seventh aspect of the invention, there is provided a use of the CRISPR genome editing system of the first aspect of the invention, the DNA construct of the second aspect of the invention, the expression vector of the third aspect of the invention, or the host cell of the fourth aspect of the invention in the construction of a transgenic animal.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
Fig. 1 shows the principle of action of the self-control CRISPR genome editing system, in the conventional CRISPR genome editing system (left), a guide RNA (guide RNA, gRNA) for a targeted gene is promoted to be expressed under the transcription promoter of human U6. Cas9 gene is expressed under EFS promoter and translated into Cas9 protein. The gRNA and the Cas9 protein act simultaneously to cut the target gene; in a self-controlled CRISPR genome editing system (hereinafter referred to as a SeT CRISPR system), one gRNA for a targeted gene is initiated to be expressed under a human U6 transcription promoter; and the other gRNA aiming at the self Cas9 gene of the CRISPR system is simultaneously started to express under the transcription promoter of the mouse U6. Cas9 gene is expressed under EFS promoter and translated into Cas9 protein. The two gRNAs respectively act with Cas9 protein, and simultaneously cut the target cell genome and Cas9 sequence in the CRISPR system, so that the expression time of Cas9 gene in the system is remarkably reduced, the off-target effect caused by long-term expression of Cas9 in the CRISPR system is reduced, and the target specificity is increased.
FIG. 2, left panel, shows the results of T7E1 assay, showing significant cleavage activity of guide-2 and guide-3; the right panel shows immunoblot results showing that the expression of Cas9 protein was significantly down-regulated in experimental groups simultaneously expressing Cas9-gRNA-2 or Cas9-gRNA-3 compared to control group (expressing lentiCRISPR v2 vector alone) 96h after lentiviral infection.
Fig. 3, left panel, shows the T7E1 assay against the cellular P53 gene targeting site, showing that the common CRISPR lentiviral system (labeled "P53 gRNA") and the SeT CRISPR lentiviral system (labeled "P53 gRNA + Cas9 gRNA") have similar cleavage activity; the right panel shows the results of T7E1 detection of the Cas9 gene targeting site in the viral system, showing that Cas9 is cleaved simultaneously in the SeT CRISPR lentiviral system.
Fig. 4 shows that in the SeT CRISPR system, the expression level of Cas9 protein is significantly reduced compared with the common CRISPR system, and the expression time is significantly shortened.
Fig. 5 shows that in the conventional lentiCRISPR system, in the presence of one unpaired base between the targeted genomic sequence and the gRNA sequence, cleavage activity (i.e., off-target efficiency) was detected at both 4 days and 20 days when multiple grnas (carrying unpaired bases at different positions) were expressed, especially at 20 days, because Cas9 was expressed for a longer time, the off-target efficiency was more pronounced. In the SeTlentiCRISPR system, most of gRNA expressions result in obviously reduced cleavage activity compared with that of the common lentiCRISPR system.
Detailed Description
The inventor obtains a high-specificity CRISPR genome editing system through extensive and intensive research, and introduces a gRNA targeting a Cas9 gene into the CRISPR/Cas9 genome editing system, so that the gRNA targeting a Cas9 gene can mediate the cutting of the Cas9 gene while (or after) the genome editing of a target cell is carried out, thereby obviously reducing the expression time of the Cas9 gene, further reducing the off-target effect caused by the long-term expression of Cas9 in the CRISPR system and increasing the targeting specificity.
Before the present invention is described, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methodologies and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).
Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now exemplified.
CRISPR/Cas system
This system is an acquired immune system that is currently found in most bacteria and all archaea to destroy foreign plastids or bacteriophages and leave foreign gene fragments in their own genomes as "memory". The full name is a clustered regularly interspaced palindromic repeats/clustered regularly interspaced repeats clustered protein system (clustered regularly interspaced clustered short palindromic repeats/CRISPR-associated proteins).
Three different types of CRISPR/Cas systems have now been found, present in about 40% and 90% of sequenced bacteria and archaea. The second type of DNA is simple, and has Cas9 protein and guide rna (grna) as core components, and due to their DNA interference (DNAi) properties, they are currently actively used in genetic engineering as a genome editing tool, and as well as Zinc Finger Nucleases (ZFNs) and transcription activator like nucleases (TALENs), they generate double strand breaks of DNA in the genome to facilitate editing by using the non-homologous end joining (NHEJ) mechanism. The type II CRISPR/Cas is applied to the genome editing of mammalian cells and zebra fish through genetic engineering modification. The characteristics of simple design and easy operation are the most advantages. The future can be applied to various model creatures.
A cluster of genome repeats called CRISPR, i.e. clustered repeats in prokaryotic nucleomimetic DNA strands, was first described in a report on e.coli in 1987. In 2000, similar Repeats were found in other eubacteria and archaea and were named Short Spaced Repeats (SRSR). SRSR was renamed CRISPR in 2002. Wherein a part of the genes encode proteins of nuclease and helicase. These cognate proteins (CAS, CRISPR-associated proteins) and CRISPR constitute a CRISPR/CAS system.
CRISPR/Cas technology
The CRISPR/Cas technology, the CRISPR/Cas genome editing technology and the CRISPR/Cas genome editing method all refer to the genome editing technology for modifying a target gene by using the principle of a CRISPR/Cas system.
Cas9 protein
The core of CRISPR/Cas is the Cas9 protein and the guide rna (grna). The core technology of genome editing in different species by using a CRISPR/Cas system comprises the first step of heterologously expressing Cas9 protein with DNA (deoxyribonucleic acid) cutting enzyme activity in the species, and the second step of obtaining a gRNA and a target homologous sequence to guide Cas9 to a target for DNA cutting. In the second step, the specific operation method is well known to those skilled in the art.
Cas9 protein derived from Streptococcus pyogenes is a multidomain multifunctional Cas protein having a RuvC nuclease-like domain at the N-terminus and an HNH nuclease domain in the middle. The combination of the Cas9 protein and gRNA can realize the DNA cutting at a specific site, the CRISPR/Cas system recognition sequence derived from Streptococcus pyogenes is 23bp and can target 20bp, and the last 3-bit NGG sequence of the recognition site is called PAM (promoter ad jacent motif) sequence which is very important for the DNA cutting. At present, CRISPR/Cas systems of most eukaryotes (including silkworms, arabidopsis thaliana, yeasts, nematodes and the like) are originally derived from Streptococcus pyogenes, and Cas9 protein is humanized and modified.
Preferably, Cas9 provided by the present invention is derived from Streptococcus pyogenes (Streptococcus pyogenes). In a preferred embodiment of the invention, the amino acid sequence of the Cas9 protein is as follows:
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENI IHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD(SEQ ID NO.1)
to facilitate expression of the Cas9 gene in a host, the optimized Cas9 gene can also be constructed downstream of a strong constitutive promoter (e.g., pdc promoter (Li et al. microbial Cell sources 2012,11:84), but not limited thereto) and a strong inducible promoter (e.g., cbh1 promoter (Zou et al. microbial Cell sources 2012,11:21), but not limited thereto) when constructing an expression vector.
The DNA constructs of the invention include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand.
The invention also relates to variants of the above DNA constructs which encode a polypeptide having the same amino acid sequence as the invention or fragments, analogues and derivatives of the polypeptide. The variants of the DNA construct may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the polypeptide encoded thereby.
The nucleic acid fragments involved in the DNA constructs of the invention may be obtained by PCR amplification, recombinant methods or synthetic methods. Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods. In addition, the sequence can be synthesized by artificial synthesis, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.
The invention also relates to vectors comprising the DNA constructs of the invention, and to the use of the vectors of the invention to construct genetically engineered host cells.
The DNA construct of the present invention may be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors well known in the art. Any plasmid or vector may be used as long as it can replicate and is stable in the host. An important feature of expression vectors is that they generally contain an origin of replication, a promoter, a marker gene and translation control elements.
Methods well known to those skilled in the art can be used to construct the DNA constructs of the invention and expression vectors for appropriate transcription/translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to a suitable promoter in an expression vector to direct mRNA synthesis. Representative examples of promoters for use in the present invention are: lac or trp promoter of E.coli; a lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, LTRs of retrovirus, and other known promoters capable of controlling gene expression in prokaryotic or eukaryotic cells or viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
Furthermore, the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and Green Fluorescent Protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for E.coli. Vectors comprising the appropriate DNA sequences described above, together with appropriate promoter or control sequences, may be used to transform appropriate host cells to enable expression of the protein.
The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: escherichia coli, streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast; a plant cell; insect cells of Drosophila S2 or Sf 9; CHO, COS, 293 cells, or Bowes melanoma cells.
When the DNA construct of the present invention is expressed in higher eukaryotic cells, transcription will be enhanced if an enhancer sequence is inserted into the vector. Enhancers are cis-acting elements of DNA, usually about 300 base pairs, that act on a promoter to increase transcription of a gene. Examples include the SV40 enhancer at the late side of the replication origin at 100 to 270 bp, the polyoma enhancer at the late side of the replication origin, and adenovirus enhancers.
It will be clear to one of ordinary skill in the art how to select appropriate vectors, promoters, enhancers and host cells.
Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is prokaryotic, e.g., E.coli, competent cells capable of DNA uptake can be harvested after exponential growth phase using CaCl2Methods, the steps used are well known in the art. Another method is to use MgCl2. If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.
The main advantages of the invention are:
(1) the gene editing system can obviously reduce off-target effect and increase target specificity;
(2) the gene editing system is simple and convenient to use, other gene expressions except for a Cas9 expression system are not required, and a small molecule induction reagent is not required;
(3) can completely retain the high introduction efficiency and cell/tissue introduction specificity of the virus system.
The present invention will be described in further detail with reference to the following 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. Experimental procedures for conditions not specified in detail in the following examples are generally carried out under conventional conditions such as those described in molecular cloning, A laboratory Manual (Huang Petang et al, Beijing: scientific Press, 2002) by Sambrook. J, USA, or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight. The test materials and reagents used in the following examples are commercially available without specific reference.
Materials and methods
1. Experimental Material
1) Reagent
DMEM, fetal bovine serum, 0.25% Trypsin-EDTA from Thermo Fisher Scientific; penicillin-streptomycin (100X) solution was purchased from Biyuntian; t7 Endonuclease I was purchased from NEB; the genome DNA extraction kit and the DNA product rapid recovery kit are purchased from Tiangen company; ANTI-FLAG antibody was purchased from SIGMA
2) Cell line
293T cells and Huh7 cells were purchased from Shanghai Life sciences research institute cell banks and cultured adherent in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (100X).
3) Carrier
plentiCRISRP v2(Plasmid #52961) Plasmid and lentiviral packaging Plasmid: pMDLg/pRRE (Addgene #12251), pRSV-Rev (Addgene #12253), pMD2.G (Addgene #12259) were purchased from Addgene.
2. Method of producing a composite material
2.1 Lentiviral packaging
293T cells were transfected with Polyethyleneimine (PEI) method. Cells were plated at appropriate density on 10cm dishes one day before transfection, and transfected by PEI method after changing the medium to DMEM when the cells grew to approximately 80%. The PEI transfected lentivirus packaging plasmid is pMDLg/pRRE 6.53ug, pRSV-Rev 2.53ug, pMD2.G 3.51ug, core plasmid lentiCRISRP v 210 ug. 4-6 hours after transfection, the medium was changed to complete medium culture. And collecting the supernatant at 48h and 96h to obtain virus liquid.
2.2T7E1 detection
T7E1 detects a method of detecting CRISPR cleavage activity. The T7E1 endonuclease can recognize and cleave sites in the genome that are not perfectly paired. After genome extraction, a band with CRISPR target sites was amplified by PCR. After purification and recovery, the DNA fragments were randomly combined by heating at 95 ℃ for 5min and slowly cooling to room temperature. 500ng of the DNA fragment was cleaved with 1ul of T7EI endonuclease at 37 ℃ for 60 min. The cleavage was analyzed by 2% agarose gel electrophoresis detection. CRISPR cleavage activity was judged by comparing the intensity of cleaved and uncleaved electrophoretic bands.
Example 1 design and screening of high activity grnas against targeted Cas9
The experimental steps are as follows:
1) through sequence alignment, five high-activity gRNAs targeting Cas9 sequences are designed, and gRNA sequences are shown in Table 1.
2) Cloning five gRNAs into lentiCRISPR v2 vector (addendum #52961)
3) Separately packaging lentivirus and then infecting 293T cells
4) Respectively collecting cells 48h and 96h after infection, and respectively analyzing the Cas9 gene cutting condition of the cells through a T7E1 detection kit; and analyzing the expression condition of the Cas9 protein by immunoblotting reaction
Table 1 design of targeted gRNA sequences for Cas9, and primer sequence information for T7E1 detection
The experimental results are shown in FIG. 2, the left graph shows the T7E1 detection result, the band is not cut in the negative control group, the band is obviously cut in the Cas9guide-2 and Cas9guide-3 groups, and guide-2 (the target band is 243bp +423bp) and guide-3 (the target band is 298bp +368bp) have obvious cutting activity; the right panel shows immunoblot results showing that the expression of Cas9 protein was significantly down-regulated in experimental groups simultaneously expressing Cas9-gRNA-2 or Cas9-gRNA-3 compared to control group (expressing lentiCRISPR v2 vector alone) 96h after lentiviral infection. The results show that Cas9guide-2 and Cas9guide-3 have higher gene editing activity.
Example 2 construction of SeT CRISPR Lentiviral vectors
The experimental steps are as follows:
1) in a synthetic plasmid pmU6-gRNA (synthesized by Jinweizhi, the sequence of which is shown in SEQ ID NO.11), which contains the backbone sequence of a guide RNA targeting the genome of a target cell and a mouse U6 promoter, and has an EcoRI restriction enzyme cleavage site 5 'to the mouse U6 promoter and 3' to the gRNA backbone sequence;
the sequence of the synthetic plasmid pmU6-gRNA is as follows (SEQ ID NO. 11):
GAATTCAGATAGATCCGACGCCGCCATCTCTAGGCCCGCGCCGGCCCCCTCGCACAGACTTGTGGGAGAAGCTCGGCTACTCCCCTGCCCCGGTTAATTTGCATATAATATTTCCTAGTAACTATAGAGGCTTAATGTGCGATAAAAGACAGATAATCTGTTCTTTTTAATACTAGCTACATTTTACATGATAGGCTTGGATTTCTATAAGAGATACAAATACTAAATTATTATTTTAAAAAACAGCACAAAAGGAAACTCACCCTAACTGTAAAGTAATTGTGTGTTTTGAGACTATAAATATCCCTTGGAGAAAAGCCCACCTTGTCTTCGAGAAGACCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGTACTGAATTC
2) cloning gRNA-2 targeting Cas9 into pmU6-gRNA to obtain pm6-Cas9-gRNA
3) The m6-Cas9-gRNA fragment was ligated into the plenti-CRISPRv2 vector at the EcoRI cleavage site by EcoRI cleavage (see FIG. 1 for details)
Example 3 verification of whether there is a difference in gene targeting efficiency between the SeT CRISPR lentiviral system and the common CRISPR lentiviral system
The experimental steps are as follows:
1) taking a human-targeted P53 gene as an example, a common CRISPR lentiviral system (marked as 'P53 gRNA') and a SeT CRISPR lentiviral system (marked as 'P53 gRNA + Cas9 gRNA') are respectively constructed
2) Human hepatoma cell line Huh7 infected after respectively packaging lentivirus
3) Cells harvested at 4 days and 8 days are infected, and the cleavage conditions of the P53 and Cas9 targeting sites are detected by a T7E1 kit respectively
TABLE 2 Targeted gRNA sequences designed for P53, and primer sequence information for T7E1 detection
The experimental result is shown in fig. 3, the negative control lentiCRISPR v2 group band in the detection result of P53T7E1 is not cut, and the P53gRNA + Cas9gRNA group band are both obviously cut (the target band is 245bp +285 bp). In the Cas9T7E1 detection result, no band cleavage occurred in both P53gRNA and lentiCRISPR v2, and only the band of P53gRNA + Cas9gRNA group was cleaved (the target band was 243bp +423 bp). The result shows that the constructed SeT CRISPR lentiviral vector has high-efficiency gene editing capacity as the common CRISPR lentiviral vector, and the SeTCRISPR lentiviral vector can successfully cut the Cas9 gene transferred into cells.
Example 4 verification of whether expression time of Cas9 is significantly shortened in SeT CRISPR System
The experimental steps are as follows:
1) taking a human-targeted P53 gene as an example, a common CRISPR lentiviral system (marked as 'P53 gRNA') and a SeT CRISPR lentiviral system (marked as 'P53 gRNA + Cas9 gRNA') are respectively constructed
2) Human hepatoma cell line Huh7 infected after respectively packaging lentivirus
3) Cells were infected for 2 days, 4 days, 6 days and 8 days, and expression level of Cas9 protein in cells was detected by immunoblotting reaction
The experimental results are shown in fig. 4, and the results show that the content of the Cas9 protein is not obviously changed after 2 days, as the P53gRNA group and the lentiCRISPR v2 group. And the content of the Cas9 protein of the P53gRNA + Cas9gRNA group is obviously reduced from 2 days later. Indicating that the expression time of Cas9 is significantly shortened in the SeT CRISPR system.
Example 5 verification of whether the off-target Rate is significantly reduced in the SeT CRISPR System
1) Aiming at a targeting sequence of a P53 gene, a plurality of guideRNAs are designed, and an unpaired nucleotide is introduced at different positions so as to systematically study the cutting efficiency of a CRISPR system when one unpaired nucleotide exists between the sequence on a gRNA and a targeted genome sequence. The specific gRNA sequences are shown in Table three.
2) Human hepatoma cell line Huh7 infected after respectively packaging lentivirus
3) Cells harvested after 4 days and 20 days of infection, respectively, were cleaved in different CRISPR systems and expressing different grnas against the P53 targeting site by T7E1 kit
TABLE 3gRNA sequence information carrying one unpaired nucleotide
As shown in fig. 5, the editing efficiency of the regular CRISPR group is significantly higher in the mismatched group from day 4, and the editing efficiency of the mismatched group is more significantly higher in the 20 th day. While certain editing efficiency can be observed in the mismatch group from day 4 to day 20 in the SeT CRISPR group, the editing efficiency in the mismatch group also increases slowly, but the editing efficiency in the mismatch group is obviously reduced compared with that in the common CRISPR. The result shows that the off-target rate of the SeT CRISPR plasmid is obviously reduced compared with the common CRISPR.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Claims (9)
1. A highly specific CRISPR genome editing system, comprising: a first gRNA, a second gRNA, and a Cas9 protein; wherein the first gRNA targets the genome of a target cell to be edited and the second gRNA targets the gene encoding the Cas9 protein, the CRISPR genome editing system comprising a DNA construct of formula (I):
P1-R1-P2-R2-P3-C ，(I)
in the formula (I), R1 is a first gRNA encoding gene, R2 is a second gRNA encoding gene, C is a Cas9 protein encoding gene, P1, P2 and P3 are optional promoter sequences, and "-" represents an optional connecting sequence; the second gRNA targets a gene sequence selected from the group consisting of: SEQ ID NO. 3.
2. A DNA construct for CRISPR genome editing, said DNA construct having a structure represented by the following formula (I):
P1-R1-P2-R2-P3-C ，(I)
in formula (I), R1 is a first gRNA-encoding gene that targets the genome of a target cell to be edited; r2 is a second gRNA encoding gene targeting the encoding gene of the Cas9 protein; c is a Cas9 protein encoding gene, P1, P2, P3 are optional promoter sequences, "-" denotes an optional linker sequence; the second gRNA targets a gene sequence selected from the group consisting of: SEQ ID NO. 3.
3. An expression vector expressing the CRISPR genome editing system of claim 1; alternatively, the expression vector comprises the DNA construct of claim 2.
4. A genetically engineered host cell comprising the expression vector of claim 3, or having the DNA construct of claim 2 integrated into its genome.
5. The genetically engineered host cell of claim 4, wherein the host cell is a eukaryotic cell.
6. The genetically engineered host cell of claim 4, wherein the host cell is an animal cell.
7. A method of genome editing for non-diagnostic and non-therapeutic purposes by introducing the expression vector of claim 3 into a cell to be edited.
8. Use of the CRISPR genome editing system of claim 1, the DNA construct of claim 2, the expression vector of claim 3, or the host cell of claim 4 in the preparation of a genome editing reagent.
9. Use of the CRISPR genome editing system of claim 1, the DNA construct of claim 2, the expression vector of claim 3, or the host cell of claim 4 in the construction of a transgenic animal.
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| US9840699B2 (en) | 2013-12-12 | 2017-12-12 | President And Fellows Of Harvard College | Methods for nucleic acid editing |
| EP3177718B1 (en) | 2014-07-30 | 2022-03-16 | President and Fellows of Harvard College | Cas9 proteins including ligand-dependent inteins |
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